WO2016187571A2 - Her2 immunotoxins and methods of using the same - Google Patents

Abstract

The present invention relates to methods for preventing or treating cancer using an immunotoxin comprising (a) a ligand that binds to a protein on the cancer cell and; (b) a toxin that is cytotoxic to the cancer cell. In a specific embodiment, the invention is directed to the prevention or treatment of cancer using a recombinant immunotoxin comprising an anti-HER2/neu antibody or antibody fragment that is fused to a deimmunized bouganin toxin. Also encompassed by the invention are combination therapy methods, including the use of reduced dosages of chemotherapeutic agents, for the prevention or treatment of cancer. Also encompassed by the invention are formulations and methods for direct administration of the recombinant immunotoxin to the carcinoma, for the prevention or treatment of cancer.

Description

HER2 IMMUNOTOXINS AND METHODS OF USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No.

62/164,444, filed May 20, 2015, and U.S. Provisional Application Serial No. 62/278,905, filed January 14, 2016, each of 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-032_01WO_ST25.txt. The text file is about 98.4 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 immunotoxins recognizing a human epidermal growth factor receptor 2 (HER2/neu) and methods for the prevention or treatment of cancer by administering to patients in need thereof, said immunotoxin which binds to HER2/neu are over expressed on the surface of cancer cells.

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 directed to these antigens have been used in immunotherapy of several types of cancer.

[0007] One antigen found to be over expressed on breast cancer cells is HER2 (also known as neu or ErbB2). HER2 is human epidermal growth factor receptor 2, a 185 kDa transmembrane glycoprotein with tyrosine kinase activity belonging to the family of human epidermal growth factor receptors which include HER1 to HER4. HER2 is over expressed in 25-30% of human breast cancer, and high expression correlates with poor prognosis for the disease.

[0008] A murine antibody, 4D5, was found to inhibit growth of HER2 over expressing cell lines in vitro and to also have a cytotoxic effect on HER2-expressing human breast tumor xenografts in athymic mice. The 4D5 anti-HER2 antibody was subsequently humanized to decrease its immunogenicity in humans. The humanized antibody, trastuzumab, has been tested in clinical trials and is approved for treatment of patients with metastatic breast cancer whose tumors over express the HER2 protein and who had received one or more prior chemotherapy regimens.

[0009] The first generation antibody-drug conjugate Kadcyla^® (ado-trastuzumab emtansine) was recently approved and is currently marketed for the treatment of metastatic breast cancer in patients who had previously received trastuzumab and a taxane. However, owing to its potential for systemic toxicity, Kadcyla^® is not approved for first line treatment, and patients taking Kadcyla^® must be closely monitored for such systemic toxicity.

[0010] Thus there is still considerable need for the development of effective tumor- specific therapies with enhanced tumor killing and reduced risk of systemic toxicity.

SUMMARY OF THE INVENTION

[0011] The present invention relates to novel immunotoxins, which are both effective in tumor targeting and killing and show reduced systemic toxicity, 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 the cancer cells. Where desired, the immunotoxin may be coadministered, concurrently administered, and/or sequentially administered with one or more other anti-cancer agents, and/or in conjunction with radiation or surgery.

[0012] In one aspect, the invention contemplates an immunotoxin comprising: (a) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin. In one embodiment, the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti- HER2/neu antibody fragment. In a further 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.

[0013] 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.

[0014] 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.

[0015] 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 one embodiment, the deimmunized bouganin toxin is linked to the heavy chain variable region by a linker encoded by an amino acid sequence of SEQ ID NO: 17. In another embodiment, the deimmunized bouganin toxin is linked to the light chain variable region by a linker encoded by an amino acid sequence of SEQ ID NO: 17. [0016] 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.

[0017] In one embodiment, the deimmunized bouganin toxin is linked to the anti-

HER2/neu binding protein by a linker encoded by an amino acid sequence chosen from SEQ ID NOs: 17, 32-36, 62 and 63. In some embodiments, the deimmunized bouganin toxin is linked to the anti-HER2/neu binding protein by a linker encoded by an amino acid sequence set forth in SEQ ID NO: 62. In another embodiment, the deimmunized bouganin toxin is encoded by an amino acid sequence selected from SEQ ID NOs: 12, 58, 59, 60, 61. In some embodiments, the deimmunized bouganin toxin is encoded by an amino acid sequence set forth in SEQ ID NO: 12.

[0018] In one 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 another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 64. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 66. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 68. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 70. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 72. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 74.

[0019] 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) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin. In one embodiment, the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment. In a further 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. [0020] In one embodiment, the cancer is breast, ovarian, gastric, lung (non small cell lung cancer, NSCLC) or pancreatic. In a particular embodiment, the cancer is metastatic breast cancer.

[0021] 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 delivered systemically. In another embodiment, the direct administration is delivered intravenously.

[0022] 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. In another embodiment, the method includes treating a patient with cancer after the patient has failed to respond to a small molecule drug or a small molecule drug conjugate. In some embodiments, the method includes treating a patient with cancer after the patient has failed to respond to an antibody drug conjugate (ADC). In one embodiment, the method includes treating a patient with metastatic breast cancer after the patient has failed to respond fully to trastuzumab and/or a taxane.

[0023] The invention also relates to a method for enhancing the activity of an anticancer agent comprising administering to an animal in need thereof an anti-cancer agent and an effective amount of an immunotoxin, wherein said immunotoxin comprises: (a) an anti- HER2/neu binding protein and; (b) a deimmunized bouganin toxin. In one embodiment, the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment. In a further 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.

[0024] The invention also relates to a method for overcoming mechanisms of resistance affecting the efficacy of small molecule drugs or small molecule drug conjugates comprising administering an effective amount of an immunotoxin to a subj ect in need thereof, wherein said immunotoxin comprises: (a) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin. In one embodiment, the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment. In a further 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.

[0025] The invention also relates to a kit for treating or preventing cancer comprising an effective amount of an immunotoxin comprising: (a) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin, and directions for the use thereof to treat the cancer. In one embodiment, the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment. In a further 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.

[0026] The invention also related to an expression vector comprising an immunotoxin comprising: (a) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin. In one embodiment, the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment. In a further 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.

[0027] 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 THE DRAWINGS

[0028] FIG. 1 shows the PelB-DeBouganin-F-AvP07-17-(V_H-V_L-C6.5)-His nucleotide (SEQ ID NO: 22) and amino acid (SEQ ID NO: 23) sequences.

[0029] FIG. 2 shows the PelB-DeBouganin-F-AvP07-17-(V_H-V_L-C6.5) nucleotide

(SEQ ID NO: 24) and amino acid (SEQ ID NO: 25) sequences.

[0030] FIG. 3 shows the PelB-AvP07-17-(V_H-V_L-C6.5)-F-deBouganin-His nucleotide

(SEQ ID NO: 26) and amino acid (SEQ ID NO: 27) sequences.

[0031] FIG. 4 shows the PelB-AvP07-17-(V_H-V_L-C6.5)-F-deBouganin nucleotide

(SEQ ID NO: 28) and amino acid (SEQ ID NO: 29) sequences.

[0032] FIG. 5 shows the PelB-deBouganin-F-AvP07-17-(V_L-V_H-C6.5) nucleotide

(SEQ ID NO: 30) and amino acid (SEQ ID NO: 31) sequences.

[0033] FIG. 6 A and FIG. 6B show small scale expression of AvP07-17 diabody and

AvP07-17/deBouganin fusion proteins. FIG. 6A) Schematic representation of AvP07-17-His, His-AvP07-17, deBouganin-AvP07-17-His and His-AvP07-17-deBouganin constructs. FIG. 6B) Induced supernatants of two independent deBouganin-AvP07-17-His clones (lane 1 and 2), two independent His-AvP07-17-deBouganin clones (lane 3 and 4), AvP07-17-His (lane 5), His-AvP07-17 (lane 6) were loaded under reducing conditions on an SDS-PAGE gel and immunoblotted with an anti-Histidine antibody followed by a goat anti-mouse antibody labelled with HRP. The arrows indicate single chain AvP07-17 and AvP07-17/debouganin fusion proteins migrating approximately at 28 and 56 kDa, respectively.

[0034] FIG. 7A-7C show cytotoxic activities of fusion proteins against SkBr3 and

MCF-7 cells measured by MTS assay. Concentrations ranging from 0.01 to 10 nM of deBouganin-AvP07-17-His (FIG. 7A), His-AvP07-17-deBouganin (FIG. 7B) or AvP07-17- deBouganin-His (FIG. 7C) were incubated with Her-2 positive SkBr3 cells (black squares) or Her-2 negative MCF-7 cells (white squares). After 5 days incubation, cell viability was measured and IC5₀ determined. The graphs are a representative example of at least two independent experiments.

[0035] FIG. 8 shows deBouganin-AvP07-17-His human serum stability.

DeBouganin-AvP07-17-His at 0 hour (lane 1), at 24 hours (lane 2), at 48 hours (lane 3), at 72 hours (lane 4) and at 96 hours (lane 5) were immunoblotted with anti-bouganin (rabbit polyclonal) followed by a goat anti-rabbit labeled with HRP. Lanes C and S correspond to 200 ng of purified protein and human serum only, respectively. Arrow indicates full length fusion protein.

[0036] FIG. 9A and FIG. 9B show a Western blot analysis of purified deBouganin-

AvP07-17 fusion proteins with and without His tag. FIG. 9A) Purified deBouganin-AvP07- 17-His (lane 1) and deBouganin-AvP07-17 (lane 2) under non-reducing conditions were resolved on a SDS PAGE gel and stained with Coomassie. FIG. 9B) The same samples as in FIG. 9A were immunoblotted with an anti-deBouganin mouse monoclonal antibody followed by a goat anti-mouse antibody labelled with HRP. For Western blot analyses 200 ng of sample were loaded, and for Coomassie staining 2 μg of sample were loaded.

[0037] FIG. 10 shows cytotoxic activities of deBouganin-AvP07-17 fusion proteins with and without a His tag against SkBr3 measured by MTS assay. Concentrations ranging from 0.01 to 10 nM deBouganin-AvP07-17-His (white squares) or deBouganin-AvP07-17 (black squares) were incubated with Her-2 positive SkBr3 cells. After 5 days incubation, cell viability was measured and IC₅₀ determined.

[0038] FIG. 11 shows the binding reactivity measured with deBouganin-AvP07-17 against a panel of breast cancer cell lines. DeBouganin-AvP07-17 binding reactivity at 0.1 μg/mL (blue), 0.5 μg/mL (grey) and 1 μg/mL (dark blue) was measured using anti- deBouganin antibody.

[0039] FIG. 12 shows the binding reactivity of deBouganin-AvP07-17 pre-incubated in mouse or human serum against SkBr3 cells over time. DeBouganin-AvP07-17 was pre- incubated in mouse (black) or human (grey) serum up to 72 hours and the binding reactivity at 0.1 μg/mL was measured against SkBr3 cells using anti-deBouganin antibody. The percentage reactivity against T=0 time point was determined and is represented here.

[0040] FIG. 13 shows a mammosphere assay: the ability of BT474 to form mammospheres was assayed in the presence of increasing concentrations of deBouganin- AvP07-17 (blue) and T-DMl (green) or media alone (grey).

[0041] FIG. 14A-14C show representative images of 7 day old mammospheres treated at 10 nM as indicated. All images were taken at xlO magnification.

[0042] FIG. 15 shows Trastuzumab-deBouganin (T-deBouganin) potency vs. T-DMl potency against Her2 3+ cancer cell lines.

[0043] FIG. 16A-16C show the potency of Herceptin-deBouganin (Herc-deB, T-deB) compared to T-DMl and/or Herceptin (Trastuzumab). FIG. 16A shows the viability of Her2 positive and Her2 negative cells treated with InM Herc-deB, T-DMl or Herceptin. FIG. 16B compares the potency of Herc-deB vs. T-DMl on HCC1419 Her2 3+ cells. FIG. 16C compares the potency of Herc-deB vs. T-DMl on HCC1569 Her2 3+ cells.

[0044] FIG. 17 shows the expression levels of anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 proteins by Western blot in various Her2 3+ cancer cell lines.

[0045] FIG. 18 shows the potency of Herc-deB vs. T-DMl in the presence or absence of MK571, an MRP (multidrug resistance protein) pump inhibitor.

[0046] FIG. 19 shows the potency of Herc-deB vs. T-DMl in the presence or absence of heregulin, a soluble secreted growth factor.

[0047] FIG. 20 A and FIG. 20B show the results of a tumorosphere assay. FIG. 20 A shows the ability of BT474 to form tumorospheres in the presence of increasing concentrations of Herc-deB (blue) and T-DMl (green) or media alone (grey). FIG. 20B shows representative images of tumorospheres treated at 10 nM as indicated.

[0048] FIG. 21 A and FIG. 2 IB show antitumor activity of Herc-deB and T-DMl in a

BT-474 xenograft model. FIG. 21A shows the median tumor volume (in mm³) over days as a function of dosing with Herc-deB or T-DMl . FIG. 21B shows the % survival over days as a function of dosing with Herc-deB or T-DMl .

[0049] FIG. 22A and FIG. 22B show the potency of DeBouganin-C6.5-diabody compared to T-DMl and Herceptin. FIG. 22A shows the viability of Her2 positive and Her2 negative cells treated with DeBouganin-C6.5-diabody, T-DMl or Herceptin. FIG. 22B compares the potency of DeBouganin-C6.5-diabody vs. Herceptin on HCC1419 Her2 3+ cells. [0050] FIG. 23 shows the percentage of viable BT-474 or ZR-75-30 cells as a measure of potency of VB7-756, T-DMl, T-MMAE and Lapatinib in the presence or absence of heregulin, a soluble secreted growth factor.

[0051] FIG. 24 shows the potency of VB7-756 vs. T-DMl and T-MMAE against BT-

474 cells. BT-474 cells were treated with 10 nM VB7-756, T-MMAE or T-DMl under adherent conditions for 5 days. Surviving cells were washed and plated under adherent conditions. Cell viability was measured after 5 days.

[0052] FIG. 25A-25B shows the potency of VB7-756 against HCC1419 cells that have evaded T-DMl or T-MMAE cytotoxicity. HCC1419 cells were pre-treated with 10 nM T-MMAE or T-DMl under adherent conditions for 5 days. Surviving cells were washed and plated under adherent conditions. Cells were then treated with VB7-756, T-MMAE or T- DM1, and cell viability was measured after 5 days. FIG. 25A shows MTS curves of VB7-756 (filled circles, blue line), T-DMl (open circles, red line), and T-MMAE (inverted triangle, green line) against HCC1419 cells that have evaded treatment with T-DMl . FIG. 25B shows MTS curves of VB7-756 (filled circles, blue line), T-DMl (open circles, red line), and T- MMAE (inverted triangle, green line) against HCC1419 cells that have evaded treatment with T-MMAE.

[0053] FIG. 26 shows representative images of tumorospheres from HCC1419 cells treated with 10 nM VB7-756, T-DMl or T-MMAE and subsequently incubated under tumorosphere forming conditions. NT = no treatment.

[0054] FIG. 27 shows representative images of tumorospheres from T-DMl or T-

MMAE treated HCC1419 cells subsequently incubated under tumorosphere forming conditions with 10 nM VB7-756, T-DMl or T-MMAE. NT = no treatment.

DEFINITIONS

[0055] As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to an "immunotoxin" is a reference to one or more immunotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0056] As used herein, 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%.

[0057] As used herein, the term "animal," "patient," or "subject" includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals. [0058] As used herein, the term "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0059] As used herein, the term "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.

[0060] As used herein, 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.

[0061] As used herein, 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.

[0062] As used herein, "deimmunized" refers to a molecule that lacks or elicits reduced immune response when compared to the wild type counterpart. 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. 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, 62 and 63.

[0063] As used herein, the term "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 regime 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.

[0064] As used herein, 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. For instance, as described below, the invention encompasses recombinant proteins that are comprised of a functional portion of a HER2/neu binding protein and a toxin. Methods of making these fusion proteins are known in the art and are described for instance in WO93/07286.

[0065] As used herein, the term "functionally equivalent fragment" of a HER2/neu binding protein 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. 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.

[0066] 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.

[0067] 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 G₄S 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 incorporated herein 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 incorporated herein by reference in its entirety. [0068] 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-(VH-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-l 7" 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-Vn 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: 62), 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: 65 (nucleotide sequence) and by SEQ ID NO: 64 (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: 63), 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: 67 (nucleotide sequence) and by SEQ ID NO: 66 (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: 69 (nucleotide sequence) and by SEQ ID NO: 68 (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: 62), 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: 71 (nucleotide sequence) and by SEQ ID NO: 70 (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: 63), 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: 73 (nucleotide sequence) and by SEQ ID NO: 72 (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 (VH) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 75 (nucleotide sequence) and by SEQ ID NO: 74 (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.

[0069] 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, each of which is herein incorporated by reference in its 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 each of which is herein incorporated by reference in its entirety) chemically conjugated to deimmunized Bouganin. As used herein, the term "T-DM1" refers to trastuzumab conjugated to maytansinoid, a microtubule- disrupting agent.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] 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.

[0074] As used herein, the phrase "humanized antibody or antibody fragment" means that the antibody or fragment comprises human framework regions. The humanization of antibodies from non-human species has been well described in the literature. See for example EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997).

[0075] As used herein, the term "immunologic activity" of HER2/neu binding protein refers to the ability to specifically bind a human epidermal growth factor receptor 2. 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."

[0076] As used herein, 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.

[0077] As used herein, the term "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.

[0078] As used herein, the term "immunotoxin" comprises (1) a binding protein attached to (2) a toxin. The terms "immunotoxin" and "immunoconjugate" are used interchangeably herein.

[0079] As used herein, 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.

[0080] As used herein, 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.

[0081] As used herein, 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-L1), 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.

[0082] As used herein, 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.

[0083] As used herein, 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.

[0084] As used herein, 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.

[0085] 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.

[0086] As used herein, 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.

[0087] As used herein, the phrase "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.

[0088] As used herein, "physiologic conditions" for antibody binding reflect but do not necessarily exactly duplicate the conditions in which a human epidermal growth factor receptor 2-binding polypeptide would encounter a human epidermal growth factor receptor 2 molecule in vivo. Binding under physiologic conditions should be reasonably predictive that binding in vivo will occur.

[0089] As used herein, the phrase "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. [0090] As used herein, the phrase "reduced dose" refers to a dose that is below the normally administered and/or recommended dose. The normally administered dose of a cancer therapeutic can be found in reference materials well known in the art such as, for example, the latest edition of the Physician's Desk Reference.

[0091] As used herein, the term "reduced propensity to elicit an immune response" means that the deimmunized bouganin toxin is less immunogenic than non-deimmunized bouganin toxin.

[0092] As used herein, the term "reduced propensity to activate human T-cells" means the deimmunized bouganin toxin has a reduced propensity to activate human T-cells as compared to the non-modified bouganin toxin. One of skill in the art can test whether or not a modified bouganin toxin has a reduced propensity to activate T-cells using assays known in the art including assessing the stimulation index of potential immunogenic peptides of the toxin.

[0093] As used herein, the term "resistance" or "drug resistance" refers to a phenomenon that results when diseases become tolerant to pharmaceutical treatments. For example, in cancer, drug resistance constitutes a lack of response or a reduction in response to drug-induced tumor growth inhibition. Resistance may be inherent in a subpopulation of heterogeneous cancer cells or be acquired as a cellular response to drug exposure. Mechanisms of drug resistance may include, but are not limited to, epigenetics, drug inactivation, drug efflux, alterations in the drug target, activation of prosurvival pathways, DNA damage repair, epithelial-mesenchymal transition and ineffective induction of cell death. Resistance may be to a small molecule drug or a small molecule drug conjugate such as an antibody drug conjugate (ADC). Without being bound by any one theory, one possible cause of drug resistance is the presence of MDR pumps on the surface of cancer cells that actively move chemotherapy from inside the cell to the outside. MDR pumps are comprised of 48 members divided into seven subfamilies which have different substrate affinity. Studies have shown that P-glycoprotein (ABCB 1 or MDR1), multidrug resistance associated- protein 1 (MRP1 or ABCC1) and breast cancer resistance protein (BCRP1 or ABCG2) represent the majority of overexpressed MDR pumps in cultured tumor cells. P-glycoprotein expression provides the strongest resistance to a variety of small molecule drugs including taxanes, vinca alkaloids, anthracyclines and epipodophyllotoxins. Cancer cells produce high amounts of these pumps, such as p-glycoprotein, in order to protect themselves from chemotherapeutics. Medications to inhibit the function of p-glycoprotein are undergoing investigation, but due to toxicities and interactions with anti-cancer drugs their development has been difficult. Another mechanism of resistance is gene amplification, a process in which multiple copies of a gene are produced by cancer cells. Drugs that reduce the expression of genes involved in replication aim to inhibit the proliferation of cancer cells. However, in cancer cells that contain more copies of the replication gene(s), the drug cannot prevent all expression of the gene(s) and thus, the cancer cells can restore their proliferative ability. Cancer cells can also cause defects in the cellular pathways of apoptosis. As most chemotherapy drugs kill cancer cells in this manner, defective apoptosis allows survival of these cells, making them resistant. Many chemotherapy drugs also cause DNA damage, which can be repaired by enzymes in the cell that carry out DNA repair. Upregulation of these genes can overcome the DNA damage and prevent the induction of apoptosis. Mutations in drug target proteins, such as tubulin, can occur which prevent drugs from binding to the target proteins, leading to resistance to these types of drugs. Drugs used in chemotherapy can induce cell stress, which can kill a cancer cell; however, under certain conditions, cell stress can induce changes in gene expression that enables resistance to several types of drugs.

[0094] As used herein, 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. 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. [0095] A "signal peptide" or "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. Signal peptides 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 signal peptide is not present in the secreted protein, only during protein production. "Signal peptide" and "leader sequence" are used interchangeably herein. In one embodiment, the leader sequence comprises PelB (pectate lyase B) shown in SEQ ID NO: 21.

[0096] As used herein, the term "small molecule drug" refers to a low molecular weight (about less than 900 daltons) organic compound that may help regulate a biological process. Small molecule drugs can rapidly diffuse across cell membranes to act intracellularly. Small molecule drugs can be tyrosine kinase inhibitors, serine threonine kinase inhibitors, cell cycle kinase inhibitors, apoptosis inducers, angiogenesis inhibitors, microtubule disruption compounds, gene expression modulators, signal transduction inhibitors, hormone production regulators, DNA alkylating agents, antimetabolites, DNA intercalating agents, DNA cross-linking agents, among others. "Small molecule drug conjugates" comprise a targeting ligand, a linker and a drug payload.

[0097] As used herein, 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.

[0098] As used herein, the term "therapeutic" means an agent utilized to discourage, combat, ameliorate, prevent or improve an unwanted condition, disease or symptom of a patient.

[0099] As used herein, 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. [00100] As used herein, the phrase "treating cancer" refers to inhibition of cancer cell replication, 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.

[00101] As used herein, the term "V region" or "V domain" of a HER2/neu antibody or antibody fragment refers to the variable region or domain of a HER2/neu light chain or the variable region or domain of a HER2/neu heavy chain, either alone or in combination. These V regions are depicted in SEQ ID NOS: 1-4.

[00102] As used herein, the term "variant" refers to any pharmaceutically acceptable derivative, analogue, or fragment of an immunotoxin, an antibody or antibody fragment, a toxin (e.g., bouganin 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 human epidermal growth factor receptor 2 (HER2/neu)-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.

[00103] A variant immunotoxin having a variation of the human epidermal growth factor receptor 2 (HER2/neu)-binding portion of the reference immunotoxin competes with the binding of an anti-HER2/neu 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-HER2/neu reference antibody is bound to HER2/neu, 10 percent of these bound reference antibodies is displaced when equilibrium is reached with an equivalent concentration of the variant anti-HER2/neu immunotoxin. As a non-limiting example, competition between antibodies, or between an antibody and an immunotoxin, is measured by binding labeled anti-HER2/neu reference antibody to HER2/neu on the surface of cells or to an HER2/neu-coated solid substrate, such that virtually all HER2/neu sites are bound by the antibody, contacting these antibody-antigen complexes with unlabeled test anti-HER2/neu antibody or unlabeled test immunotoxin, and measuring the amount of labeled antibody displaced from HER2/neu binding sites, wherein the amount of freed, labeled antibody indicates the amount of competition that has occurred.

[00104] As used herein, 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.

DETAILED DESCRIPTION OF THE INVENTION

[00105] 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.

[00106] One 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 ER2/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.

Binding proteins

[00107] The present invention contemplates immunotoxins comprised of: (a) an anti-

HER2/neu binding protein and; (b) a deimmunized bouganin toxin.

[00108] In one embodiment, the HER2/neu antibody fragment includes Fab, Fab',

F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, immunoglobulin scaffolds, multimers, and any combination thereof 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 (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).

[00109] 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. 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. The preparation of humanized antibodies is described in EP-B 10 239400. 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. Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain). 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.

[00110] 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.

[00111] 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 a 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 (Gly₄-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.

[00112] In one embodiment, the immunotoxin comprising an anti-HER2/neu binding protein attached to a deimmunized bouganin toxin comprises an anti-HER2/neu antibody fragment. 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 particular embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 2. 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 particular embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 4.

[00113] In one embodiment, the antibody fragment is a diabody. In one embodiment, the anti-HER2/neu diabody is comprised of a heavy chain variable region and a light chain variable region. 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.

[00114] 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.

[00115] 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.

[00116] In one embodiment, the immunotoxin comprising an anti-HER2/neu binding protein attached to a deimmunized bouganin toxin comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment. In some embodiments, the deimmunized bouganin toxin is encoded by an amino acid sequence set forth in SEQ ID NO: 12. In another embodiment, a deimmunized bouganin toxin is linked to an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment by a furin protease sensitive linker. In a further embodiment, the wild-type furin protease sensitive linker sequence is shown in SEQ ID NO: 17. In a particular embodiment, the furin protease sensitive linker is deimmunized. In another embodiment, the deimmunized furin protease sensitive linker is represented by SEQ ID NOs: 32-36, 62 and 63. In some embodiments, the deimmunized bouganin toxin is linked to the anti-HER2/neu binding protein by a linker encoded by an amino acid sequence set forth in SEQ ID NO: 62. 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.

[00117] 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.

[00118] 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

[00119] 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 some embodiments, the toxin may be deBouganin as shown in SEQ ID NO: 12 (amino acid sequence) and SEQ ID NO: 11 (nucleotide sequence). In another embodiment, the toxin may be deBouganin encoded by an amino acid sequence selected from SEQ ID NOs: 58, 59, 60, 61.

[00120] In other nonlimiting embodiments, the toxin comprises an agent that acts to disrupt DNA. Thus, toxins may comprise, 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.

[00121] 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.

[00122] In other nonlimiting embodiments, the toxin portion of an immunotoxin of the invention may comprise 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.

[00123] In other nonlimiting embodiments, the toxin portion of an immunotoxin of the invention may comprise 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.

[00124] 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.

[00125] In other nonlimiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an topoisomerase II inhibitor including, without limitation, doxorubicin, amonafide, anthrapyrazole derivative, pyrazoloacridine, bisantrene HC1, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril, Ν,Ν-dibenzyl daunomycin, oxanthrazole, and rubidazone.

[00126] In other nonlimiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an RNA or DNA antimetabolite including, without limitation, L- alanosine, 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.

[00127] 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.

[00128] In some embodiments, the immunotoxin is a humanized antibody fragment that binds to human epidermal growth factor receptor 2 (HER2/neu) linked to modified bouganin, wherein 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) is encoded by an amino acid sequence selected from SEQ ID NOs: 12, 58, 59, 60, 61. Once bound, the immunotoxin is internalized and the deBouganin kills cells or blocks the protein synthesis, thereby leading to cell death. Importantly, cells which do not widely express HER2/neu, and therefore cannot internalize the immunotoxin, are protected from the potential side-effects of the toxin.

[00129] In some embodiments, the immunotoxin may be a diabody attached to modified bouganin protein. 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. 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. 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. 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. [00130] 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 another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 64. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 66. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 68. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 70. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 72. In another embodiment, the immunotoxin comprises an amino acid sequence set forth in SEQ ID NO: 74.

[00131] In some embodiments, the immunotoxin may be a Fab attached to modified bouganin protein. In some embodiments, the Fab 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.

[00132] In some embodiments, the immunotoxin may be a ScFv of anti-HER2/neu antibody attached to deimmunized Bouganin. In some embodiments, the ScFv 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: 2. In some embodiments, the ScFv 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. In some embodiments, the ScFv 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. In some embodiments, the ScFv 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. [00133] 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.

[00134] 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-hydroxysuccinimide and sulfo-m maleimidobenzoyl-N- hy droxy succinimi de.

[00135] 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. [00136] 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.

[00137] In one embodiment, the cleavable linker fuses an anti-HER2/neu 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, 62 and 63 (amino acid sequences) and by SEQ ID NOs: 38-47 (nucleotide sequences).

[00138] 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 wild-type 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 wild-type 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 wild-type 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 wild-type 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 wild-type 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, 62 and 63. 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 encoded by the amino acid sequence set forth in SEQ ID NO: 62. 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, 62 and 63. 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, 62 and 63. 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, 62 and 63. In one embodiment, the anti- HER2/neu diabody comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10.

[00139] 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, 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 shown in SEQ ID NOs: 64, 66, 68, 70, 72 or 74. 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, 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, or a variant of an immunotoxin comprised of an amino acid sequence shown in SEQ ID NOs: 64, 66, 68, 70, 72 or 74. 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 shown in SEQ ID NOs: 64, 66, 68, 70, 72 or 74, 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 shown in SEQ ID NOs: 64, 66, 68, 70, 72 or 74, 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, 64, 66, 68, 70, 72 or 74, or may comprise a different portion of the same toxin or a different toxin.

[00140] 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. The affinity of anti-HER2/neu antibody for the plSS™¹¹² extracellular domain is less than or equal to K_D=1 nM according to the method of Friguet (Friguet et al, 1985 J. Immunol. Methods, 77:305-319; see US 5821337). Consequently, the present invention includes immunotoxins having a dissociation constant (K_D) of less than or equal to 1 nM.

[00141] 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. [00142] 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-s canning 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.

[00143] 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.

[00144] Furthermore, the skilled artisan would appreciate that increased avidity may compensate for lower binding affinity. The avidity of an immunotoxin for HER2/neu is a measure of the strength of the HER2/neu-binding protein's binding of HER2/neu, which can have multiple binding sites. The functional binding strength between HER2/neu and the HER2/neu-binding protein 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 HER2/neu-binding sites and HER2/neu 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.

[00145] 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.

[00146] 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.

[00147] 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 cancer cells; and a toxin or variant thereof. Such immunoglobulin polypeptides can be redesigned to affect their binding characteristics to a target a tumor associated molecule, or to improve their physical characteristics, for example.

[00148] 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 cancer cells; and a toxin or variant thereof. Such non-immunoglobulin polypeptide can be designed to bind to a target tumor associated molecule. Moreover, a 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.

[00149] 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.

[00150] 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.

[00151] In other non-limiting 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 encoded by an amino acid sequence selected from SEQ ID NOs: 12, 58, 59, 60, 61.

Deimmunized Bouganin toxin

[00152] A variety of toxins may be used to design a HER2/neu-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.

[00153] 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 is encoded by an amino acid sequence selected from SEQ ID NOs: 58, 59, 60, 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 and in PCT/CA2014/050950, each of which is incorporated herein by reference in its entirety.

[00154] 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.

[00155] In some embodiments, the modified bouganin peptide is modified at one or more T-cell epitopes in the bouganin protein sequence.

[00156] The term "T-cell epitope" means an amino acid sequence which is able to bind 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.

[00157] 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.

[00158] 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.

[00159] 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 binding groove.

[00160] 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 ligands by any suitable means.

[00161] 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.

[00162] 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.

Methods of Use

[00163] Disclosed herein are methods of using immunotoxins described herein. The present invention contemplates methods of treating or preventing cancer comprising administering an effective amount of said immunotoxins to a subject in need thereof.

[00164] 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 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 comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 27. 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 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, a method of treating or preventing cancer in a subject in need thereof may involve administering a therapeutically effective amount of or an immunotoxin comprised of an amino acid sequence shown in SEQ ID NOs: 64, 66, 68, 70, 72 or 74. 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.

[00165] 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 cancer, pancreatic cancer, and prostate cancer. Cancers originating from any epithelial cell may also be targeted by these immunotoxins. In a preferred embodiment, the cancer is breast or ovarian cancer.

[00166] In some embodiments, the antibodies and immunotoxins of the invention may be used to treat a patient with cancer after the patient has failed to respond or has responded poorly to a small molecule drug or a small molecule drug conjugate.

[00167] In preferred non-limiting embodiments, the cancer is amenable to treatment by direct administration of the 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 immunotoxin.

[00168] 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.

[00169] 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.

[00170] The immunotoxins disclosed herein may also be used to detect or monitor cancer 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, 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; 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 blots 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 other 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 a complex, 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; measuring the amount of the complex in the test sample; and normalizing the results against a control.

[00171] In another embodiment, a method of detecting or monitoring cancer in a subject may involve administering to the subject 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; and detecting the immunotoxin. In a further embodiment, a method of detecting or monitoring cancer in a subject may involve administering to the subject 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; and detecting the immunotoxin.

[00172] 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 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; and detecting the immunotoxin by in vivo imaging. The immunotoxin may be an anti- HER2/neu antibody or antibody fragment attached to a deimmunized Bouganin. In some embodiments, a method of imaging a tumor in a subject may involve administering to the subject 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; and detecting the immunotoxin by in vivo imaging. The immunotoxin may further include an effector molecule.

[00173] 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.

[00174] 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 immunotoxin after resection to measure the completeness of tumor resection.

[00175] In certain embodiments, the immunotoxins 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 ¹⁸F, ⁶⁷Ga, ^81mKr, ⁸²Rb, ^99mTc, ^mIn, ¹² I, ^mI, ¹ Xe, ²⁰¹T1 and ⁹⁰Y. Preferably, the antibodies as described herein are labeled with ¹⁸F, ^{12 /1 m}In, ⁹⁰Y or ^99mTc.

[00176] The immunotoxins 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.

[00177] In another preferred embodiment, the immunotoxins 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 immunotoxins of the present invention may be labeled with any suitable contrast agent. Preferred contrast agents include contrast agents for medical imaging. Preferably, the immunotoxins 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.

[00178] 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.

[00179] 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.

[00180] 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 nonlimiting embodiment, the other cancer therapeutic comprises another immunotoxin comprised of anti-HER2/neu binding protein. In another embodiment, the other cancer therapeutic comprises another immunotoxin comprised of anti-HER2/neu antibody or antibody fragment. In another nonlimiting 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 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.

[00181] 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.

[00182] 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^®).

[00183] 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.

[00184] 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.

[00185] Pharmaceutical compositions for combination therapy may also include, without limitation, antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin), asparaginase, Bacillus and Guerin, procaine, tetracaine, lidocaine, propranolol, anti-mitotic agents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, antihistaminic agents, anti-nausea agents, etc.

[00186] 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 cancer therapeutic having clinically significant efficacy. Such efficacy of the reduced dose of the other cancer therapeutic 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 cancer therapeutics.

[00187] 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 cancer therapeutics for relatively short duration and/or in fewer treatment cycles.

[00188] Thus, in accordance with the present invention, combination therapies comprising an immunotoxin and another cancer therapeutic 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 cancer therapeutic, 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.

[00189] In a preferred embodiment, the invention provides methods for treating and/or ameliorating the clinical condition of patients suffering from breast cancer or ovarian cancer. Accordingly, the invention provides methods for (i) decreasing the tumor size, growth rate, invasiveness, malignancy grade, and/or risk of recurrence, (ii) prolonging the disease-free interval following treatment, and/or (iii) improving symptoms of the cancer and/or affected function in a patient, comprising administering to the patient an effective amount of an immunotoxin. Clinical improvement may be subjectively or objectively determined, for example by evaluating the size of the tumor, whether the tumor has spread to lymph nodes and other parts of the body, tumor histology, and other indices known to the clinical arts.

[00190] 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, urinalysis, CT scan, and pharmacokinetic analysis. Furthermore, synergistic effects of a combination therapy comprising the immunotoxin and another cancer therapeutic may be determined by comparative studies with patients undergoing monotherapy.

[00191] 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.

[00192] Moreover, the effective dose of a specific immunotoxin construct may depend on additional factors, including the type of cancer, the size of the tumor, the stage of the cancer, the immunotoxin's toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.

[00193] In one embodiment, the effective dose by direct administration of 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 mi crograms/tumor/ day .

[00194] In other embodiments, the immunotoxin administration is at a dosage of about

0.01 mg/kg/dose to about 2000 mg/kg/dose. [00195] In another embodiment, the effective dose of 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.

[00196] In another embodiment, the immunotoxin is administered intratumorally 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 cm³ and 500 microgram per cm³ tumor or a dose sufficient to reach about between 14 picomole and 7 nanomole per cm³ 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 cm³, a target dose of 14 picomoles (1 microgram per cm³), 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.

[00197] 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 cancer therapeutics 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).

[00198] For example, in one embodiment, the additional cancer therapeutic comprises dacarbazine at a dose ranging from approximately 200 to 4000 mg/m²/cycle. In a preferred embodiment, the dose ranges from 700 to 1000 mg/m²/cycle.

[00199] In another embodiment, the additional cancer therapeutic comprises fludarabine at a dose ranging from approximately 25 to 50 mg/m²/cycle.

[00200] In another embodiment, the additional cancer therapeutic comprises cytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to 2000 mg/m²/cycle.

[00201] In another embodiment, the additional cancer therapeutic comprises docetaxel at a dose ranging from approximately 1.5 to 7.5 mg/kg/cycle. [00202] In another embodiment, the additional cancer therapeutic comprises paclitaxel at a dose ranging from approximately 5 to 15 mg/kg/cycle.

[00203] In yet another embodiment, the additional cancer therapeutic comprises cisplatin at a dose ranging from approximately 5 to 20 mg/kg/cycle.

[00204] In yet another embodiment, the additional cancer therapeutic comprises 5- fluorouracil at a dose ranging from approximately 5 to 20 mg/kg/cycle.

[00205] In yet another embodiment, the additional cancer therapeutic comprises doxorubicin at a dose ranging from approximately 2 to 8 mg/kg/cycle.

[00206] In yet another embodiment, the additional cancer therapeutic comprises epipodophyllotoxin at a dose ranging from approximately 40 to 160 mg/kg/cycle.

[00207] In yet another embodiment, the additional cancer therapeutic comprises cyclophosphamide at a dose ranging from approximately 50 to 200 mg/kg/cycle.

[00208] In yet another embodiment, the additional cancer therapeutic comprises irinotecan at a dose ranging from approximately 50 to 75, 75 to 100, 100 to 125, or 125 to

150 mg/m²/cycle.

[00209] In yet another embodiment, the cancer therapeutic comprises vinblastine at a dose ranging from approximately 3.7 to 5.4, 5.5 to 7.4, 7.5 to 11 , or 1 1 to 18.5 mg/m²/cycle.

[00210] In yet another embodiment, the additional cancer therapeutic comprises vincristine at a dose ranging from approximately 0.7 to 1.4, or 1.5 to 2 mg/m²/cycle.

[00211] In yet another embodiment, the additional cancer therapeutic comprises methotrexate at a dose ranging from approximately 3.3 to 5, 5 to 10, 10 to 100, or 100 to 1000 mg/m²/cycle.

[00212] 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. [00213] 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/m²/cycle.

[00214] 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/m²/cycle.

[00215] 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.

[00216] 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/m²/cycle. In another embodiment, the additional cancer therapeutic comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.), at a dose ranging from approximately 5 to 50 mg/m²/cycle.

[00217] 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.

[00218] 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/m²/Cycle.

[00219] 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/m²/cycle.

[00220] 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/m²/cycle.

[00221] 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/m²/cycle.

[00222] 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/m²/cycle. [00223] 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.

[00224] In another embodiment, the additional cancer therapeutic comprises 5- fluorouracil 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.

[00225] 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.

[00226] 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/m²/cycle.

[00227] 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.

[00228] 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/m²/cycle.

[00229] 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/m²/cycle.

[00230] 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.

[00231] 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 (Mai one et al., 1990, Cancer Res. 51 : 2077-2083), a 14-amino acid peptide corresponding to a fragment of collagen I (Tolma et al, 1993, J. Cell Biol. 122: 497-511), a 19 amino acid peptide corresponding to a fragment of Thrombospondin I (Tolsma et al, 1993, J. Cell Biol. 122: 497-511), a 20-amino acid peptide corresponding to a fragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57: 1329-1334), and a variant thereof, including a pharmaceutically acceptable salt thereof.

[00232] 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.

[00233] In another embodiment, an immunotoxin is administered in combination with one or more cytokines which include, without limitation, a lymphokine, 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.

[00234] 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-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins, and mutated, tumor-specific antigens.

[00235] 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).

[00236] In yet another embodiment, an immunotoxin is administered in association with a gene therapy program to treat or prevent cancer.

[00237] 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.

[00238] 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.

[00239] 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

[00240] 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 immunotoxins are administered intravenously as a bolus or by continuous infusion over a period of time. In other embodiments, the immunotoxins may be administered directly to the cancer site.

[00241] 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 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.

[00242] 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.

[00243] 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).

[00244] 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.

[00245] 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.

[00246] In another embodiment, a pharmaceutical composition comprises an immunotoxin and one or more additional anticancer agent, optionally in a pharmaceutically acceptable carrier.

[00247] 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.

[00248] 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.

[00249] 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.

[00250] 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 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.

[00251] For oral administration, the immunotoxins can be formulated readily by combining these immunotoxins 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.

[00252] 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 immunotoxin doses.

[00253] 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.

[00254] For buccal administration, the compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.

[00255] 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., dichlorodifluoromethane, 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 and a suitable powder base such as lactose or starch.

[00256] 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.

[00257] 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.

[00258] 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.

[00259] 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.

[00260] 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.

[00261] 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.

[00262] 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.

[00263] 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

[00264] 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.

[00265] 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 US2008/019451 1, and US2007/0292918, both of which are incorporated herein for all purposes.

[00266] 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 vectors are compatible with the one or more host cells 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.

[00267] 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.

[00268] 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.

[00269] 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.

[00270] 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.

[00271] 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.

[00272] 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)). [00273] 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).

[00274] 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).

[00275] 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).

[00276] 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. [00277] 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).

[00278] 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

[00279] 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). 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 VH and VL regions of SEQ ID NO: 64. In another embodiment, the antibody or antibody fragment comprises VH and VL regions of SEQ ID NO: 66. In another embodiment, the antibody or antibody fragment comprises VH and VL regions of SEQ ID NO: 68. In another embodiment, the antibody or antibody fragment comprises VH and VL regions of SEQ ID NO: 70. In another embodiment, the antibody or antibody fragment comprises VH and VL regions of SEQ ID NO: 72. In another embodiment, the antibody or antibody fragment comprises VH and VL regions of SEQ ID NO: 74.

[00280] In one embodiment, the antibody or antibody fragment comprises the VH region shown in SEQ ID NO: 2 and the V_L region shown in SEQ ID NO: 4.

[00281] The present application also includes the use of the novel nucleic acid sequences for the preparation of antibodies or antibody fragments and methods thereof.

[00282] 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. [00283] 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.

[00284] 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, 62 and 63. The linker sequences can be used in the preparation of immunotoxins.

[00285] 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

[00286] 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

[00287] The following examples are intended to illustrate but not limit the disclosure.

EXAMPLE 1

CONSTRUCTION, EXPRESSION AND ASSAYS FOR BIOLOGICAL ACTIVITY OF AVP07-17 DIABODIES

AND AVP07- 17-DEBOUGANIN FUSION PROTEINS

[00288] AvP07-17 is an anti-HER2/neu diabody engineered with the C6.5 anti-HER2 scFv (in VH-VL orientation) with a short G₄S linker between the V domains. The high tumor uptake and high tumor to blood ratio make diabodies an attractive strategy for an immunotoxin targeted therapy.

[00289] AvP07-17 (V_H-V_L) was genetically linked to deBouganin (SEQ ID NO: 12) at either the N-terminal or C-terminal end via a furin protease cleavable linker (SEQ ID NO: 17) and the corresponding fusion constructs placed under the control of the arabinose promoter into the pING3302 vector. To facilitate the initial purification process, a Histidine (His) tag (SEQ ID NO: 19) was also included. The constructs were grown and induced using high cell density fermentation in 2L or 15L vessels using the Xoma expression system. DeBouganin-AvP07-17-His (SEQ ID NO: 23) and AvP07-17-deBouganin-His (SEQ ID NO: 27) were both purified at over 90% purity. A deBouganin-C6.5 diabody with a VL-VH orientation (SEQ ID NO: 31) was also engineered, purified and tested. However this design was shown to be unstable after storage for few days at 4°C or -20°C. With an IC5₀ of 75 pM against SkBr3, deBouganin-AvP07-17(V_H-V_L)-His (SEQ ID NO: 23) was therefore selected as the optimal orientation.

[00290] Due to the clearance of His-tagged proteins by the liver, corresponding versions without a His-tag would be preferred for preclinical studies. Thus, deBouganin- AvP07-17 (SEQ ID NO: 25) was engineered and a new purification process developed. This process yielded over 95% pure material for deBouganin-AvP07-17. DeBouganin-AvP07-17 biological activity was similar to the His version. Furthermore, deBouganin-AvP07-17 potency against a large panel of Her2 3+ tumor cell lines ranged from double digit pM to sub- nM.

[00291] Finally, the deBouganin-AvP07-17 cytotoxicity towards cancer stem cells

(CSC) was tested using the mammosphere assay and compared to T-DM1 (Ado-trastuzumab Emtansine, trastuzumab linked to the microtubule-disrupting agent maytansinoid).

[00292] DeBouganin-AvP07-17 prevented mammosphere formation at concentration similar to the IC5₀ obtained by an MTS assay, demonstrating that a deBouganin payload is effective against CSC. On the other hand, T-DM1 only had a marginal effect.

Molecular Engineering and Small-Scale Expression

A) AvP07-17 constructs

[00293] His-AvP07-17 and AvP07-17-His constructs were generated by Splice Overlapping Extension PCR method, SOE-PCR. The fragments were cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing. The pCR 2.1 plasmid containing the correct insert was digested with EcoRI-XhoI and ligated into the pING3302 plasmid pre- digested with EcoRI-XhoI. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 0.9 kB insert was then used to transform E. coli E104 and selected colonies grown for small-scale expression.

B) AvP07-17-deBouganin fusion constructs

[00294] DeBouganin- AVP07-17(VH-V_l), AvP07-17-deBouganin(V_H-V_L) and deBouganin- AVP07-17(VL-VH) fusion constructs with or without a His tag were engineered in two steps. First, the AvP07-17 inserts created by SOE-PCR were cloned into the pCR2.1 vector and transformed into 10F E. coli cells for sequencing. The pCR 2.1 plasmids containing the correct insert were digested with enzyme restrictions and ligated with the deBouganin insert into the pING3302 plasmid. Chemically competent 10F E. coli cells were transformed with the ligation reaction and the DNA plasmid from a clone with the complete insert was used to transform E. coli El 04.

C) Small-Scale Expression

[00295] Transformed E104 cells containing either the AvP07-17, AvP07-17-His,

AvP07-17-deBouganin, deBouganin-AvP07-17(V_H-V_L) or deBouganin- AvP07-17(V_L-V_H) were inoculated into 5 mL 2-YT 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.

Western Blot Analysis

[00296] The level of expression was estimated by Western blot analysis. Briefly, 16 μΐ. 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 His-AvP07-17, AvP07-17-His, His- AvP07-17-deBouganin and deBouganin-AvP07-17-His proteins were detected using an anti- His antibody (1/1000) overnight at 4°C followed by a goat anti-mouse antibody coupled to HRP(1/1000) 1 hour at room temperature. DeBouganin- AvP07-17(V_H-V_L) and (V_L-V_H) fusion proteins were detected using an anti-deBouganin antibody (1/1000) overnight at 4°C followed by a goat anti -rabbit antibody coupled to HRP (1/1000) 1 hour at room temperature. The membrane was developed using DAB to determine the level of expression.

Fermentation

[00297] Fed-batch fermentation was performed in a 20 L CHEMAP fermenter using

Glycerol minimum medium (GMM). Then, 150 mL of the seed culture (grown in 500 mL of GMM containing 25 μg/mL of tetracycline and supplemented with trace element D, calcium chloride, nicotinic acid and thiamine (at 28°C) was used to inoculate a 20 L CHEMAP bioreactor containing 15 L of GMM media with supplement elements as described previously. The temperature was set at 28°C and the pH maintained at 7.0 with the addition of a 50% ammonium hydroxide solution via the pH control loop throughout the entire fermentation. The agitation rate was set at 300 rpm with airflow of 3 standard liters per minutes (slpm) and incremented successively at 600 rpm and 6 slpm and then at 1000 rpm and 10 slpm to maintain the dissolved oxygen above 41% during the batch phase. When the carbon source of the batch media was exhausted, the dissolved oxygen increased above 90% which triggers the addition of feed 1 solution (50% glycerol solution). Then, the dissolved oxygen (DO) setpoint was set at 41% and the feeding was based on a cascade control of the DO reading. At an optical density of 50, the culture was induced by switching to feed 2 solution (50% glycerol + 30 g/L arabinose solution) and the induction was carried out for 30 hours under the same control as the feed 1.

Purification

A) Purification process for molecules with a His tag

[00298] At 30 hours post induction, the culture supernatant was harvested by centrifugation at 8000 rpm for 30 min, followed by microfiltration and 10-fold concentration and finally diafiltration for 5 diavolumes against 20 mM sodium phosphate buffer, pH 7.0. Purification was carried out using Ni-charged Chelating-sepharose as primary capture, followed by a cation exchange step using SP-sepharose column followed by a size exclusion column. Briefly, the supernatant with 20 mM imidazole added and pH adjusted to 7.0 was applied onto a Ni²⁺ charged chelating column equilibrated with 20 mM imidazole in 20 mM sodium phosphate, 150 mM NaCl pH 7.0. The column was washed with 20 mM sodium phosphate, 150 mM NaCl pH 7.0 containing 50 mM imidazole, pH 7.0 until A280 absorbance baselined. Bound AvP07-17-deBouganin was subsequently eluted with 250 mM Imidazole in 20 mM sodium phosphate, 150 mM NaCl pH 7.5. The Ni²⁺ eluate was then diluted 5-fold with 20 mM sodium phosphate pH 6.0 buffer and applied onto an SP-sepharose column previously equilibrated in 20 mM sodium phosphate, 50 mM NaCl pH 6.0 ± 0.1. The SP-sepharose column was then washed with equilibration buffer until UV absorbance baselined and bound AVP07-17 eluted with 20 mM sodium phosphate, 300 mM NaCl pH 7.5. This eluate was then applied onto a 500 mL sephacryl S-200 size exclusion column that was equilibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.5. The eluting peaks monitored by A280 were fractionated in 10 mL fractions and analyzed by SE-HPLC and SDS-PAGE.

B) Purification process for molecules without a His tag [00299] At 30 hours post induction, the culture supernatant was harvested by centrifugation at 8000 rpm for 30 min, followed by microfiltration and 10-fold concentration and finally diafiltration for 5 diavolumes against 20 mM sodium phosphate buffer, pH 7.0. Purification was carried out as follows. Briefly, the concentrated diafiltered supernatant at pH 7.0 was applied onto a CM sepharose column equilibrated with 20 mM sodium phosphate, 25 mM NaCl pH 7.0. After washing to UV baseline with equilibration buffer, deBouganin- AVP07-17 was eluted in 20 mM sodium phosphate, 150 mM NaCl pH 7.5. The CM eluate was then diluted 5-fold with 20 mM sodium phosphate buffer and pH adjusted to 7.5, then applied onto a Q-sepharose column previously equilibrated in 20 mM sodium phosphate, 50 mM NaCl pH 7.5 and flow-through containing the product was collected. The pH of Q- sepharose flow-through was adjusted to pH 6.0 and applied directly onto an SP-sepharose column previously equilibrated in Q-sepharose equilibration buffer. This buffer was also used to wash the column to UV baseline after sample loading, and bound deBouganin-AVP07-17 eluted with 20 mM sodium phosphate, 300 mM NaCl pH 7.5. This eluate was then applied onto a 500 mL sephacryl S200 size exclusion column that was equilibrated with PBS pH 7.4. The eluting peaks monitored by A280 were fractionated in 10 mL fractions and analyzed by SE-HPLC and SDS-PAGE.

[00300] To remove a 50 kDa contaminant, the fractions with deBouganin-AvP07-17 were pooled and diluted with 20 mM sodium phosphate to achieve a NaCl concentration of 100 mM. The pH was then adjusted to pH 7.0 and flowed through an SP-sepharose column that was previously equilibrated with 20 mM sodium phosphate, 100 mM NaCl buffer, pH 7.0. The column was then washed to baseline and the wash collected and pooled with the flow-through. At this point, the flow-through containing deBouganin-AVP07-17 was concentrated by ultrafiltration using a 10 kDa membrane.

Biological activity

A) Flow cytometry

[00301] Human breast cancer SkBr3 and MCF-7 cells were grown in their respective media as per ATCC protocols. Cells were harvested at 30% to 40% confluence with viability greater than 90%. SK-BR-3 was used as a positive cell line and MCF-7 as a negative cell line for Her-2 antigen expression.

[00302] Flow cytometry was used to demonstrate that the purified proteins retain binding specificity using an antigen-positive cell line SkBr3 and an antigen-negative cell line, MCF-7. Binding was detected using either a mouse anti-His or a rabbit anti-deBouganin antibody. Briefly, immunotoxins to be tested were incubated with 0.25 x 10 tumor cells for 1.5 hours on ice. After washing, cell surface bound reactivity was detected with either mouse anti-His or rabbit anti-deBouganin (1/100) after incubation for an hour on ice. The cells were washed and incubated with FITC -conjugated anti-rabbit IgG for 30 minutes on ice. Subsequently, the cells were washed, resuspended in PBS 5% FCS containing propidium iodide for assessment of diabody binding by flow cytometry.

[00303] Human breast cancer SkBr3, BT474, HCC2218, HCC1419, MDA-MB-453, MDA-MB-361, T47D and MCF-7 cells were grown in their respective media as per ATCC protocols. Cells were harvested at 30% to 40% confluence with viability greater than 90%. DeBouganin-AvP07-17 was incubated with 0.25xl0⁶ tumor cells for 1.5 hours on ice. After washing, cell surface bound reactivity was detected with rabbit anti-deBouganin (1/100) by incubating for an hour on ice. The cells were then washed and incubated with FITC- conjugated anti-rabbit IgG for 30 minutes on ice. Subsequently, the cells were washed, resuspended in PBS 5% FCS containing propidium iodide for assessment of diabody binding by flow cytometry.

B) MTS assay

[00304] The cytotoxicities of the fusion proteins were measured by MTS assay.

Briefly, SkBr3 and MCF-7 cells were seeded at 1000 cells per well and incubated at 37°C for 3 hours. Subsequently, equimolar concentrations of the fusion proteins were added to the cells and after 5 days, the cell viability was determined. AvP07-17-C-His, N-His-AvP07-17 or deBouganin at equimolar concentrations were used as controls.

[00305] The cytotoxicities of deBouganin- AvP07-l 7 were also measured as follows.

Briefly, SkBr3, BT474, HCC2218, HCC1419, MDA-MB-453, MDA-MB-361, T47D, MCF- 7, Calu-3, NCI-N87, OE-19, AU565, HCC1569, HCC1954 and HCC202 cells were seeded at 5000 cells per well and incubated at 37°C for 3 hours. Subsequently, equimolar concentrations of the proteins were added to the cells and after 5 days, the cell viability was determined. Equimolar concentrations of free deBouganin and free MMAE drug were used as controls.

C) Serum stability

[00306] To ensure stability, deBouganin-AvP07-17 was incubated at 37°C in mouse and human serum. After 24, 48 and 72 hours, an aliquot was taken and the integrity of the fusion protein analyzed by Western blot using the rabbit anti-deBouganin as a probe. The biological activity was also measured by flow cytometry using rabbit anti-deBouganin followed by anti-rabbit-FITC for detection. D) Cancer stem cell assay

[00307] To assess mammosphere forming efficiency, BT474 cells were trypsinized, placed in mammosphere media (DMEM/F12, 2% B27 supplement, 20 ng/ml rEGF, 0.5 μg/ml hydroxy cortisone, 0.5 μg/ml insulin) and resuspended as single cells using a 25 gauge needle syringe prior to being counted on a hematocytometer. Cells were plated on ultra low attachment six well plates at a density of 1000 cells/cm². T-DM1 or deBouganin-AvP07-17 were diluted at 0.1, 1 and 10 nM in mammosphere media and added at the time of plating. BT474 cells were cultured for 7 days without replenishing the media. At this time, all mammospheres over 50 μιτι in diameter were counted using an inverted microscope fitted with a graticule. Each well was counted twice independently. Results are representative of two independent experiments.

EXAMPLE 2

RESULTS ON MOLECULAR ENGINEERING, EXPRESSION AND BIOLOGICAL ACTIVITY OF AVP07- 17

DIABODIES AND AVP07-17-DEBOUGANIN FUSION PROTEINS

1) Molecular engineering, expression and biological activity of AvP07-17 diabodies and deBouganin fusion molecules

[00308] To demonstrate that diabodies can be expressed as soluble proteins in E. coli using L-arabinose as an inducer, two constructs encoding for AvP07-17 were engineered in the pING3302 plasmid and transformed into E. coli El 04 cells. A His tag was placed at either the N-terminus or C-terminus in order to determine the optimal orientation for purification (FIG. 6A). Small-scale expression was performed as described in Example 1. Soluble expression was determined by analyzing supernatant samples. As expected, bands at approximately 28 kDa were observed in both samples (FIG. 6B, lanes 5 and 6). While the AvP07-17 diabodies have a molecular weight of 56 kDa, intact diabodies cannot be resolved as the interaction between heavy and light chains dissociates under SDS-PAGE conditions.

[00309] Following the expression of soluble AvP07-17 diabody, two AvP07-17- deBouganin fusion constructs were engineered with deBouganin located at either the N- terminus or C-terminus of AVP07-17 in order to determine the optimal orientation of deBouganin. A peptidic furin linker is present between AvP07-17 and deBouganin. The constructs are schematically represented in FIG. 6A. Soluble expression was determined by analyzing supernatant samples. As expected, a band at approximately 50 kDa corresponding to the molecular weight of a single chain is detected for both AvP07-deBouganin fusion proteins (FIG. 6B, lanes 1 to 4). Similarly to the diabody alone, intact diabody fusion proteins with a theoretical molecular weight of 112.5 kDa cannot be detected by SDS-PAGE as the interaction between heavy and light chains dissociates under these conditions.

[00310] In order to assess the potency of the fusion molecules, the cytotoxicities of the fusion molecules were determined against Her-2 positive SkBr3 and Her-2 negative MCF-7 cells as described in Example 1. As shown in Figure 7, the cytotoxicity of deBouganin- AvP07-17-His (FIG. 7A, IC₅₀=0.075 nM) was approximately 13-fold higher than that of His- AvP07-17-deBouganin against SkBr3 cells (FIG. 7B, IC₅₀=lnM). No cytotoxic effect was observed against MCF-7 cells at up to 10 nM. Of note, the His-AvP07-17-deBouganin sample was only 60 % pure, possibly explaining the lesser cytotoxicity observed for this sample.

[00311] In order to address the lack of purity observed for His-AvP07-17-deBouganin, a new construct was engineered where the Histidine tag was relocated from the N- to the C- terminus. As shown in FIG. 7C, AvP07-17-deBouganin-His has an IC5₀ of 300 pM.

[00312] Overall these results indicate that N-terminal deBouganin is the optimal orientation for AvP07-17 deBouganin fusion immunotoxins as this conformation translates into the highest potency (Table 1).

[00313] Table 1: IC5₀ of fusion molecules against SkBr3 cells

2) Human serum stability

[00314] To ensure the stability of proteins, deBouganin-AvP07-17-His was incubated in human serum at 37°C to a maximum of 96 hours. The stability of the proteins was assessed by Western blot analysis. As seen in FIG. 8, the analysis reveals some truncated bands. However, the majority of the sample remains intact.

3) Purification and testing of deBouganin- AvP 07-17 without a His tag

[00315] A deBouganin-AvP07-17 fusion molecule without any His tag was engineered, expressed and purified as described in Example 1. Western blot analysis of the purified product, using anti-deBouganin antibody, revealed a band just under 49 kDa as expected for samples resolved under non-reduced conditions (FIG. 9B, lane 2). Analysis of the samples by SDS-PAGE separation and Coomassie staining indicated the sample is over 90% pure (FIG. 9A, lane 2). This deBouganin-AvP07-17 diabody fusion molecule without any His tag is also known as VB7-756.

[00316] DeBouganin-AvP07-17 fusion cytotoxicity was determined against Her-2 positive SkBr3 as described in Example 1. As shown in FIG. 10, the cytotoxicity of deBouganin-AVP07-17 was comparable to its counterpart with a His tag (35 pM vs. 50 pM).

4) Purification and testing of deBouganin-AvF '0 ⁷ -17 diabody in VL-VH orientation

[00317] In order to determine the optimal diabody orientation (VH-VL VS. VL-VH), a deBouganin-VL-VH AvP07-17 diabody was engineered as described in Example 1. As previously shown, in the context of the deBouganin-AvP07-17(Vn-VL), deBouganin located at the N-terminus of the VH chain was preferred as this design has a significantly better expression and better potency compared to the construct where deBouganin is located at the C-terminus of the VL chain. With these results in mind, only one deBouganin- VL-VH fusion diabody was designed where deBouganin was genetically fused to the N-terminus of the VL chain.

[00318] Small scale expression showed slightly higher expression levels for deBouganin-VL-Vn compared to deBouganin- VH-VL (data not shown). DeBouganin- VL-VH was subsequently successfully fermented, purified and its binding reactivity and potency tested. DeBouganin- VL-VH had a 25% higher binding affinity against SkBr3 cells compared to that of deBouganin-AvP07-17 (data not shown). In addition, deBouganin- VL-VH potency outperformed that of deBouganin- AvP07-l 7 (VH-VL) for six breast cancer cell lines tested. However, analysis of samples stored at 4°C and -20°C by HPLC revealed that deBouganin- VL-VH aggregates under both conditions. This aggregation likely contributes to the higher binding reactivity and potency observed. Overall the data suggest that deBouganin-C6.5 diabody in VL-VH orientation is unstable.

5) Biological Characterization of deBouganin-AvP07-17 (VB7-756)

A) deBouganin-AvP07-17 binding reactivity against breast cancer cell lines

[00319] The binding reactivity of deBouganin- AvP07-l 7 against breast cancer cell lines was evaluated by flow cytometry at 1 μg/mL, 0.5 μg/mL and 0.1 μg/mL. As shown in FIG. 11, deBouganin-AvP07-17 binding affinity is cell line specific, likely reflecting differences in Her-2 expression. The ranking order observed positively correlates with reported Her-2 expression. For example, BT474 and SkBr3 cells have a higher binding reactivity than MDA-MB-453 and MDA-MB-361. BT474 and SkBr3 cells have been reported to express 1 million Her-2 receptors per cell, while MDA-MB-453 and MDA-MB- 361 have been shown to express 100 thousand receptors per cell.

B) deBouganin- AvP07-l 7 cytotoxicities against breast cancer cell lines

[00320] The cytotoxicities of deBouganin-AvP07-17 was determined against a panel of breast cancer cell lines with disparate Her-2 expression levels as described in Example 1. DeBouganin-AvP07-17 has a cytotoxic effect in all high (3+) and moderate (2+) Her-2 expressing cell lines tested with an IC5₀ in the sub-nanomolar range (Tables 2 and 3). DeBouganin-AvP07-17 is not cytotoxic against cell lines with low (1+) Her-2 expression. Free deBouganin consistently has a decreased potency of at least 3 logs as compared to deB ouganin- AvP 07-17.

[00321] Table 2: deBouganin- AvP07-l 7 IC5₀ values in nM against a panel of breast cancer cell lines.

Numbers in parenthesis indicate standard error.

[00322] The potency of deBouganin-AvP07-17 was further evaluated against additional Her-2 positive breast and non-breast carcinoma cell lines as well as the Her-2 negative MDA-MB-231 cell line. DeBouganin-AvP07-17 is selectively cytotoxic only against Her-2 expressing cell lines. As previously seen, its IC50 IS in the subnanomolar range.

[00323] Table 3: deBouganin-AvP07-17 IC5₀ values in nM against a panel of breast and non-breast cancer cell lines.

Numbers in parenthesis indicate standard error.

6) deBouganin-AvP07-l 7 diabody (VB 7-756) stability

[00324] To measure the thermo-stability, deBouganin-AvP07-17 was incubated in

PBS, mouse or human serum over a period of 3 days at 37°C. Flow cytometry analysis showed that deBouganin-AvP07-17 is stable in PBS at 37°C up to 3 days (data not shown). However, a decreased binding reactivity is observed for samples incubated in mouse and human serum over time. At 24, 48 and 72 hours, a 17%, 40% and 47% decreased binding reactivity was observed in mouse serum (FIG. 12). In addition, Western Blot analysis shows that deBouganin-AvP07-17 is partially cleaved when incubated in mouse serum at 48 and 72 hours (data not shown). In human serum, at 24, 48 and 72 hours, a 17%, 21% and 28% decreased binding reactivity is observed (Figure 12). In vitro serum stability studies suggest that deBouganin-AvP07-17 is more stable in human as compared to mouse serum. Nonetheless, 80% of the material is active after 24 hours in mouse serum.

7) deBouganin-AvP07-17 (VB7-756) inhibits BT474 mammosphere formation

[00325] Recent research has shown the existence of a side population of cells that possess the ability of self-renewal and are thought to be responsible for tumor initiation and development. These cells, termed cancer stem cells, have been demonstrated to be relatively resistant to chemotherapeutics. In order to assess the effect of deBouganin-AvP07-17 on cancer stem cells, BT474 tumorosphere forming efficiency was evaluated upon treatment with deBouganin-AvP07-17. The effect of T-DM1 was also assessed at equimolar concentrations. As shown in FIG. 13, treatment with 0.1 nM, 1 nM and 10 nM deBouganin- AvP07-17 reduced mammosphere forming efficiency by 85%, 97% and 99%, respectively, as compared to non-treated sample. This inhibition is significantly greater than equimolar T- DM1 treatments that inhibit tumorosphere formation by 14%, 37% and 50.7%. Overall these results indicate that deBouganin-AvP07-17 is significantly more effective at inhibiting mammosphere formation (FIG. 14A-14C).

[00326] The data presented demonstrates the feasibility of purifying biologically active

AvP07-17 diabodies and AvP07-17-deBouganin fusion immunotoxins using microbial fermentation. When genetically fused to deBouganin, both orientations at either N or C terminal end of AvP07-17 were engineered and tested. In addition a deBouganin-C6.5 diabody fusion in the VL-VH orientation was also engineered. However, this design was shown to be unstable. With an IC5₀ of 75 pM against SkBr3 cells, deBouganin- AvP07-l 7- His (SEQ ID NO: 23) was selected as the optimal orientation. A panel of breast cancer cell lines was evaluated for binding by flow cytometry using deBouganin- AvP07-l 7. The rank order binding reactivity positively correlates with reported Her-2 expression. The IC5₀S against Her2 3+ positive cell lines were in the double digit pM for most of the cell lines tested and found similar to deBouganin chemically conjugated to Herceptin with a DAR (drug— antibody ratio) of approximately 2. This data demonstrated that a deBouganin anti-Her2 diabody fusion construct can reproduce the potency of Herceptin chemically linked to deBouganin.

[00327] For ease of purification, deBouganin-AvP07-17 was created with a His tag.

However, the recovery of the diabody and diabody fusion protein was poor. The analysis of the Coomassie gel and Western blot suggested that the His tag may be clipped during either fermentation or purification. In addition, recent data showed that proteins with a His tag are prone to be cleared by the liver leading to their degradation which could affect PK and yield liver toxicity. Therefore, a final deBouganin- AvP07-l 7 with no His tag (SEQ ID NO: 25, VB7-756) was engineered, purified using a new process and the biological characteristics found to be identical to the His version. Of note, the purity of the materials was higher than 95% and the yield should easily satisfy the PK and efficacy studies.

[00328] The cancer stem cells hypothesis advocates the existence of a side population of cells within a tumor that possess properties of self-renewal essential for tumor initiation and development. Cancer stem cells have also been shown to possess resistance to chemotherapeutics. While equimolar T-DM1 treatments only showed mimimal reduction in BT474 tumorosphere formation, significant reductions were observed for deBouganin- AvP07-17 with almost complete inhibition at 10 nM. DeBouganin-AvP07-17 was demonstrated to be potent in vitro against cancer stem cells, suggesting that deBouganin as a payload is not susceptible to CSC mechanism of resistance.

EXAMPLE 3

COMPARISON OF HERCEPTIN-DEBOUGANIN AND T-DMl

Materials and Methods

Cell Culture

[00329] All tumor cell lines (American Type Culture Collection, Manassa, VA) and

OE-19 (Sigma, St. Louis, MO) were cultured in their respective media as per the provider's instructions in a humidified incubator at 37°C in the presence of 5% carbon dioxide.

Antibodies, toxins and immunotoxins

[00330] Trastuzumab (Herceptin^®) antibody is as described in US 5821337, incorporated by reference in its entirety herein. Trastuzumab-deBouganin (T-deB) immunotoxin was prepared by chemically conjugating trastuzumab and purified deBouganin containing a C-terminal His tag. Conjugation was accomplished as described by Bolognesi et al. (Bolognesi A, Polito L, Tazzari PL, et al. In vitro anti-tumour activity of anti-CD80 and anti-CD86 immunotoxins containing type 1 ribosome-inactivating proteins. British Journal of Haematology. 2000;110:351-361). Briefly, trastuzumab and deBouganin solutions were exchanged to 50 mM sodium borate, pH 9.0 at a concentration of 3.85 mg/mL and 5.6 mg/mL, respectively. 2-iminothiolane was added to a final concentration of 0.6 mM for trastuzumab and 1.0 mM for deBouganin and incubated at 28°C for 60 min. Glycine was added to a final concentration of 200 mM and the mixture incubated at room temperature for 15 min, followed by the addition of 5,5'-dithiobis(2-nitrobenzoic acid) to a final concentration of 2.5 mM. After 10 min at room temperature, protein was separated from free reagents by gel filtration on PD-10 desalting columns (GE Healthcare, Uppsala, Sweden). The sulihydryl-deBouganin was reduced with 50 mM of 2-mercaptoethanol and passed through a PD-10 desalting column. Modified trastuzumab and reduced modified deBouganin were combined at a molar ratio of 1 trastuzumab to 16 deBouganin and incubated for 18 hours at room temperature. Free deBouganin and trastuzumab were removed by Protein G affinity chromatography and IMAC columns, respectively. The resulting purified T-deB conjugate was formulated in 20 mM NaHPO4/150 mM NaCl pH 7.5, filtered sterilized, and the protein concentration estimated using the micro BCA kit (Thermo Fisher Scientific, Waltham, MA). The purity and identity of the T-deB conjugate was confirmed by SEC- HPLC and Western blot, respectively, and the in vitro biological activity tested as described below. To determine the drug to antibody ratio, the trastuzumab-deBouganin conjugate was reduced with 2-mercaptoethanol and analyzed by SEC-HPLC. The number of deBouganin molecules per trastuzumab antibody in the reduced conjugate was interpolated from a standard curve generated from the HPLC profiles for trastuzumab and deBouganin combined in 1 : 1, 1 :2 and 1 :3 molar equivalents of trastuzumab: deBouganin.

Cell-surface reactivity

[00331] The reactivity of trastuzumab, T-DM1 and T-deB against tumor cells was determined by flow cytometry using a FACS Calibur (BD Biosciences, Mississauga, Ontario). Briefly, 2 x 10⁵ cells were incubated with antibody for 2 hours on ice. After washing away unbound material, bound antibody was detected using a fluorescein isothiocyanate (FITC) labeled goat anti-human H&L chain antibody (Pierce, Rockland, IL). Cells were analyzed on a FACS Calibur following propidium iodide (Molecular Probes, Eugene, OR) staining. To determine the functional affinity of antibody binding, the K_D was calculated by the Lineweaver-Burk method of plotting the inverse of the median fluorescence as a function of the inverse of the antibody concentration. The K_D was determined as follows 1/F=l/Fmax + (K_D/Fmax)(l/[Ab]) where F corresponds to the background subtracted median fluorescence and Fmax was calculated from the plot. In vitro protein translation inhibition assay

[00332] The biological activity of T-deB was assessed using the TnT quick coupled transcription/translation system (Promega, Madison, WI). Briefly, the incorporation of biotinylated lysine tRNA into a protein template was measured in the presence of increasing concentration of deBouganin or T-deB and revealed by Western blot using streptavidin-HRP. The inhibition level was then compared to a control sample without deBouganin.

Potency

[00333] The potency was measured by an MTS assay (Promega, Madison, WI).

Briefly, tumor cells were seeded at 5000 cells per well in a 96-well plate and allowed to adhere for 3 hours at 37°C. Conjugated antibodies or free drugs were added to the cells over a range of concentrations and incubated for 5 days. The IC50 was interpolated from the resulting plot. For combination studies, MK571 and heregulin were purchased from Sigma and ABT-737 from Selleckchem (Houston, TX). The maximal concentration of inhibitor that had no effect on cell proliferation on its own was used for the potency assays (ABT-737: 0.25 μΜ for HCC1419, 0.075 μΜ for HCC1569; MK571 : 30 μΜ for HCC1419 and HCC1569). The inhibitor was held at a fixed concentration in combination with a range of concentrations of the conjugated antibodies. For the heregulin assays a fixed concentration of 2 nM was used in combination with a range of concentrations of the conjugated antibodies. All treated cells were incubated for 5 days and the IC5₀ interpolated from the resulting plot.

[00334] The cytotoxicity of taxol, doxorubicin and VB6-845 was assessed with an

MTS assay using P-glycoprotein-positive NCI-H69-LX4, HCT-15 and DLD-1 cells and P- glycoprotein-negative NCI-H69 and SW-480 cells. After 5 days, the IC5₀ was determined.

Inhibition of Tumor osphere formation

[00335] To assess tumorosphere forming efficiency, BT-474 cells were trypsinized, placed in mammosphere media (DMEM/F12 (Life Technologies, Burlington, ON), 2% B27 supplement (Life Technologies), 20 ng/mL recombinant epidermal growth factor (Sigma), 0.5 μg/mL hydrocortisone (Stem Cell Technologies, Vancouver, BC), 5 μg/mL insulin (Sigma)) and resuspended as single cells using a 25 gauge needle. Cells were plated in ultra- low attachment six well plates at a density of 10,000 cells/well and T-DM1 or T-deB added at the time of plating. After 10 days, all tumorospheres greater than 50 μιτι in diameter were counted using an inverted microscope fitted with a graticule. Each well was counted twice independently. Results are representative of two independent experiments. In vivo efficacy studies

[00336] Female CB.17 SCID mice were implanted with 1 mm³ BT-474 tumor fragments subcutaneously in the flank. Animals were assigned to 4 treatment groups (n=6) when tumor volumes reached an average size of 100 mm³. Group 1 mice were vehicle treated controls. Groups 2 and 3 were treated with either 1 mg/kg or 2 mg/kg T-deB administered by intraperitoneal (i.p.) injection. Animals in Groups 2 and 3 received either 4 doses of 1 mg/kg of T-deB on Days 1, 6, 16 and 21 (Group 2) or 2 doses of 2 mg/kg T-deB on Days 1 and 21 (Group 3).

[00337] For T-DM1, dosing was performed by intravenous (i.v.) injection of 1.5 mg/kg on Days 1 and 21 for a total dose of 3 mg/kg (molar equivalent to 4 mg/kg of T-deB). Dosing volumes for i.p. and i.v. injections were 10 mL/kg and 5 mL/kg, respectively, scaled to the body weight of each animal. Animals were monitored for tumor size twice weekly using caliper measurement and study endpoint was a tumor volume of 1000mm³ or d42. Toxicity was defined as a weight loss of >20% of total starting body weight.

Statistical Analysis

[00338] Differences were tested using the Student t-test and a p-value of <0.05 was considered statistically significant.

Results

Generation and characterization of T-deB conjugate

[00339] To evaluate the deBouganin payload in the context of a full length IgG, deBouganin was randomly conjugated to trastuzumab via chemically inserted sulfhydryl groups to generate trastuzumab-deBouganin (T-deB). SEC-HPLC analysis demonstrated that 100% of the final product was conjugated with an average DAR of 1.9 deBouganin molecules per trastuzumab. In a rabbit reticulocyte assay, the T-deB conjugate inhibited protein synthesis at levels comparable to unconjugated deBouganin suggesting that its activity was unaffected by conjugation.

[00340] Cell surface binding and functional affinity of T-deB were assessed by flow cytometry against a range of breast cell lines and compared to trastuzumab and T-DM1. The calculated binding affinity of T-deB was virtually the same as trastuzumab with K_D values of 3.80 x 10^"9 M and 3.19 x 10^"9 M, respectively. T-deB is highly potent against Her 2 over expressing cell lines

[00341] Once the activity and binding selectivity was demonstrated, T-deB potency was assessed against a panel of tumor cell lines expressing various levels of Her2 and compared to T-DM1. T-deB was more effective than T-DM1 at killing Her2 3+ cancer cell lines (FIG. 15). As seen in Table 4, T-deB exhibited greater potency than T-DM1 against 6/7 of the Her2 3+ breast cancer cell lines, of which three were significantly better, and 2/3 of the non-breast cell lines with IC5₀ values in the subnanomolar range. T-deB showed nM range killing of the Her2 2+ cell lines and IC5₀ values exceeding 10 nM for all Her2 1+ and Her2 negative cell lines. In contrast, no clear association between Her2 expression and T-DM1 potency was observed; four of the Her2 3+ cell lines were less sensitive than two of the Her2 2+ cell lines.

[00342] Table 4: Trastuzumab-deB and T-DM1 IC5₀ values in nM against a panel of breast and non-breast cancer cell lines.

Numbers in parenthesis indicate standard error. [00343] T-deB was more effective than either T-DMl or Herceptin^® at killing Her2 positive cell lines (FIG. 16A). At 100 nM of T-DMl, 50% of HCC1419 and HCC1569 cells were still viable whereas 80% killing was measured with only 10 nM of T-deB (FIG. 16B and FIG. 16C). Against the cell lines with 1+ Her2 expression levels, T-deB did not show any appreciable cytotoxicity whereas T-DMl was slightly cytotoxic against T47D cells (Table 4). With the exception of BT-474 cells, trastuzumab did not display any marked cytotoxicity against any of the cell lines tested. Overall, the IC5₀ of T-deB was less variable amongst the different Her2 3+ tumor cells as all values were within one log, whereas the IC5₀ values observed with T-DMl varied over a 3 log range.

[00344] The targeting index, defined as the IC5₀ of the free drug divided by the IC5₀ of the respective ADC (antibody-drug conjugate), was calculated by determining the cytotoxicity of DM1 and deBouganin against the intermediate and high Her2 expressing cell lines. For 10 out of 12 cell lines, T-deB potency was 2000-fold over free deBouganin. In contrast T-DMl showed a much lower targeting index with a range of 1.1 to 101 -fold with half of the cell lines lower than 20-fold (Table 5).

[00345] Table 5: Targeting Index (TI) for deBouganin and DM1

indicates IC₅₀ values generated with free drug. Values derived from a minimum 2 representative experiments with 3 replicates per dilution.

bTargeting Index, Ή. TI is the Free Drug IC₅₀ expressed as a function of the ADC IC₅₀; the higher the value, the greater the differential in IC₅₀ between free drug and the targeted ADC.

Numbers in parenthesis indicate standard error.

Anti-apoptotic Bcl-2 family members modulate T-DMl, but not T-deB, cytotoxicity

[00346] In order to investigate whether Bcl-2 family members account for the lower sensitivity of several of the Her2 expressing cell lines to T-DMl compared to T-deB, the expression levels of three anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 proteins were examined by Western blot. BT-474, HCC1419 and Calu-3 cells showed increased expression of Bcl-xL compared to the levels observed in T-DMl sensitive SK-BR-3 cells (FIG. 17). Calu-3 cells also expressed increased levels of Bcl-2, while none of the cell lines showed any appreciable increase in Mcl-l expression. HCC202 and HCC1569 cells did not show increased levels of any members of the pro-survival Bcl-2 family of proteins examined. To investigate whether up-regulated anti-apoptotic Bcl-2 proteins may account for the lower sensitivity of several of the Her2 expressing cell lines to T-DMl compared to T-deB, potency was tested in the presence of ABT-737, a Bcl-2 family inhibitor. HCC1419 and HCC1569 cells showed a 3.6- fold and 7.6-fold increase in sensitivity to T-DMl, respectively, in the presence of ABT-737 (Table 6). Sensitivity to T-DMl against SK-BR-3, BT-474, HCC202 and Calu-3 cells was unaffected by ABT-737. In contrast, Bcl-2 inhibition did not affect T-deB induced cytotoxicity in any of the cell lines tested (Table 6). These results suggest that altered expression of pro-survival Bcl-2 family members is one mechanism by which cancer cells may evade T-DMl-induced cytotoxicity, whereas T-deB potency is not affected by Bcl-2- mediated resistance.

[00347] Table 6: Herc-deB and T-DMl IC5₀ values in nM against breast cancer cell lines HCC1419 and HCC1569 in the absence or presence of the Bcl-2 family inhibitor ABT- 737.

Numbers in parenthesis indicate standard error.

T-DMl, but not T-deB, potency is affected by multidrug resistance efflux pumps

[00348] To evaluate the role of efflux pumps in resistance to T-DMl, a multidrug resistance protein (MRP) inhibitor, MK571, was tested in combination with T-DMl and T- deB. Inhibition of MRP pumps resulted in a 3.3-fold and 4.7-fold increase in T-DMl potency against HCC1419 and HCC1569 cells, respectively (FIG. 18). No increased sensitivity to T-DMl in the presence of MK571 was observed for any of the other cell lines tested. As expected, MRP inhibition had no effect on T-deB induced cytotoxicity against any of the cell lines tested, suggesting that deBouganin is not affected by multidrug transporters. In contrast, these results suggest that active efflux of DM1 once released inside the cell may contribute to the lower sensitivity of HCC1419 and HCC1569 cells to T-DM1.

VB6-845 andPgP-1 glycoprotein

[00349] When treated with taxol or doxorubicin, NCI-H69-LX4 cells were more resistant than the NCI-H69 parental cells. In contrast, VB6-845 (anti-EpCAM Fab- deBouganin, described in US 8263744, incorporated by reference herein) was more potent against NCI-H69-LX4, demonstrating that deBouganin cytotoxicity was not altered by overexpressed P-glycoprotein.

[00350] Table 7: VB6-845, doxorubicin and taxol IC50 values in nM against various cell lines.

ean o n epen ent expe ments

MDR (multidrug resistance) status confirmed by rhodamine efflux

[00351] To confirm this data, DLD-1 and HCT-15 colon cell lines overexpressing PgP-

1 were incubated with VB6-845 and the potency compare to SW-480. VB6-845 potency was not affected by the overexpression of PgP-1. As controls, cells were incubated with small molecule drugs. Taxol and doxorubicin potencies were altered against DLD-1 and HCT-15 and in comparison to SW-480.

Heregulin stimulation does not affect T-deB cytotoxicity

[00352] T-DM1 -mediated cytotoxicity can be inhibited by heregulin. Thus the effect of heregulin stimulation on T-deB potency was also examined. T-deB potency remained virtually unchanged for all three cell lines tested in the presence of heregulin (Table 8). In contrast, the presence of heregulin reduced the potency of T-DMl 3.6-fold against Calu-3 cells and even more dramatically for BT-474 and ZR-75-30 cells leading to an IC5₀ greater than 10 nM (Table 8 and FIG. 19). Thus, T-deB killing is not reduced by heregulin stimulation.

[00353] Table 8: Herc-deB and T-DMl IC₅₀ values in nM against various cell lines in the absence or presence of heregulin

T-deB is highly potent against cancer stem cells (CSCs)

[00354] CSCs are relatively resistant to cell cycle dependent chemotherapeutics. In order to assess the effect of T-deB and T-DMl on cancer cells with CSC properties, BT-474 tumorosphere forming efficiency was evaluated upon treatment with T-deB and T-DMl . As shown in Figure 20A, treatment with 0.1 nM, 1 nM and 10 nM Herc-deB reduced tumorosphere forming efficiency by 73%, 92% and 100%, respectively as compared to the non-treated cells. Furthermore, no tumorospheres were obtained after the re-culture of the contents of the 10 nM of Herc-deB well in the absence of drug. This inhibition was significantly better than equimolar T-DMl treatments that inhibited tumorosphere formation by only 14%, 25% and 39% (FIG. 20A). Of note, tumorosphere inhibition by T-DMl did not improve with a concentration of 100 nM. Overall these results indicate that unlike T-DMl, Herc-deB is more potent and effective at inhibiting tumorosphere formation (FIG. 20B).

Antitumor activity of T-deB and T-DMl in a BT-474 xenograft model

[00355] SCID mice bearing established BT-474 xenografts were treated with a total dose of either 4 mg/kg of T-deB or an equimolar dose of T-DMl (3 mg/kg). The T-deB conjugate displayed superior anti-tumor activity compared to T-DMl, with tumors being of a smaller size at all time points beginning one week after the initial dosing (Figure 21A). The superior potency of T-deB was further highlighted by fractionating the 4 mg/kg total dose over 4 doses of 1 mg/kg within the same time period resulting in an even more pronounced tumor growth delay (TGD) with the majority of tumors remaining below 200 mm³ throughout the study (FIG. 21A). In contrast, most of the T-DMl treated mice exceeded this size within one week.

[00356] The TGD resulted in an increased survival time for both T-deB treated groups with only 1/6 mice in Group 2 reaching tumor end point by day 42 with a survival rate of 83% (FIG. 21B). In contrast, 4/6 mice in the TDM-1 treated group reached the 1000 mm³ endpoint volume by day 28. No significant toxicity as indicated by weight loss was observed for any of the treatment groups over the duration of the study.

EXAMPLE 4

COMPARISON OF DEBOUGANIN-C6.5 -DIABODY (VB7-756) WITH HERCEPTIN-DEBOUGANIN AND

T-DMl

T-deB is highly potent against Her 2 over expressing cell lines compared to Herc-deB and T- DM1

[00357] DeBouganin-C6.5-diabody (VB7-756, in a VH-VL orientation) was assessed against a panel of tumor cell lines expressing various levels of Her2 and compared to Herc- deB and T-DMl. As seen in Table 9, DeBouganin-C6.5-diabody exhibited comparable potency to Herc-deB with sub nanomolar killing against all Her2 3+ breast cancer cell lines. DeBouganin-C6.5-diabody potency was comparable to or significantly better than T-DMl potency against these same cell lines (Table 9).

[00358] Table 9: DeBouganin-C6.5-diabody (VB7-756), Herc-deB and T-DMl IC₅₀ values in nM against a panel of cell lines.

MDA-MB-453 2+ 0.225 (0.085) 0.210 (0.080) 0.440 (0.060)

MCF7 1 + >10 >10 >10

T47D 1 + >10 >10 8.000 (2.000)

MDA-MB-231 0 >10 >10 >10

Numbers in parenthesis indicate standard error.

[00359] DeBouganin-C6.5-diabody (VB7-756) was significantly more effective than

T-DMl or Herceptin at killing Her2 positive cancer cell lines (FIG. 22 A). The potency of DeBouganin-C6.5-diabody against HCC1419 cells was greater than that of Herceptin, with an IC₅₀ of 0.2 nM for the diabody versus 0.89 nM for Herceptin (FIG. 22B).

EXAMPLE 5

COMPARISON OF DEBOUGANIN-C6.5 -DIABODY (VB7-756) WITH T-MMAE AND T-DMl

VB7-756 and T-MMAE potency against carcinoma cell lines

[00360] VB7-756 and T-MMAE (Trastuzumab linked to the antimitotic drug monomethyl auristatin E) cytotoxicities were tested against a panel of cancer cells with disparate Her2+ expression. As seen in Table 10, VB7-756 and T-MMAE were potent against all high (3+) and moderate (2+) Her2+ cell lines with IC5₀S in subnanomolar to nanomolar ranges.

[00361] Table 10: DeBouganin-C6.5-diabody (VB7-756) and T-MMAE IC₅₀ values in nM against carcinoma cell lines

dilution. Values in parentheses indicate standard error (SE).

aVB7-756 IC₅₀ significantly better than T-MMAE (p<0.05).

bT-MMAE IC₅₀ significantly better than VB7-756 (p<0.05).

[00362] The targeting index of deBouganin was compared to that of MMAE. Targeting

Index (TI) is the free drug IC50 expressed as a function of the ADC IC50. Therefore, the higher the value, the greater the differential in IC5₀ between free drug and the targeted ADC. A 3 log difference between targeted and non-specific killing was observed for VB7-756 against all cell lines (Table 11).

[00363] Table 11: Comparison of targeting index (TI) for deBouganin vs. MMAE

BT-474 3748.3 10

HCC202 8037.0 3.0

HCC1419 2222.0 2.2

HCC1954 4016.4 3.0

HCC2218 7647.1 1 .5

SK-BR-3 6501.5 7.4

MDA-MB-361 3719.0 10.7 MDA-MB-453 2200.0 1 .2

Calu-3 2377.0 4.3

NCI-N87 19318.2 6.6

OE-19 19230.8 8.5

IC5₀ Values derived from a minimum 2 representative experiments with 3 replicates per dilution. 3 log difference between targeted and non-specific killing indicated in bold.

VB7-756 potency in the presence of heregulin

[00364] Her2/Her3-positive BT-474 and ZR-75-30 cells were treated with VB7-756, T-MMAE, T-DMl or Lapatinib, a known tyrosine kinase inhibitor that binds Her2 and EGFR receptors. As seen in Table 12, VB7-756, T-MMAE, T-DMl and Lapatinib were potent against both cell lines with IC5₀S in subnanomolar to nanomolar ranges. However, in the presence of 20 nM of heregulin which promotes Her2/Her3 dimerization, T-MMAE, T-DMl and Lapatinib potency was significantly inhibited, whereas VB7-756 potency remained unchanged. Moreover, the number of viable cells at 10 nM of T-MMAE, T-DMl and 1000 nM of Lapatinib in the presence of heregulin was also significantly increased (p<0.05) (FIG. 23).

[00365] Table 12: T-DMl, T-MMAE, Lapatinib and VB7-756 IC₅₀ values in nM against two cell lines in the absence or presence of heregulin

IC5₀ va ues are t e mean o a m n mum o 2 representat ve exper ments w t 3 rep cates per dilution. Values in parentheses indicate the SE.

VB7-756 potency against BT-474 tumor cells evading T-MMAE and T-DMl killing

[00366] As seen in FIG. 23, between 20 to 40 % of BT-474 tumor cells are still alive after T-DMl and T-MMAE treatment. Therefore, tumor cells that have evaded T-DMl or T- MMAE killing were collected, reseeded and treated with VB7-756, T-DMl and T-MMAE. BT-474 cells were treated with 10 nM VB7-756, T-MMAE or T-DMl under adherent conditions for 5 days. Surviving cells were washed and plated under adherent conditions. Cell viability was measured after 5 days (FIG. 24).

[00367] BT-474 cells were treated with 10 nM T-MMAE or T-DMl under adherent conditions for 5 days. Surviving cells were washed and plated under adherent conditions. Cells were treated with VB7-756, T-MMAE or T-DMl and cell viability was measured after 5 days. No IC5₀ was measured with T-DMl and T-MMAE against BT-474 that escaped T- DM1 killing (Table 13). Similarly, T-MMAE resistant BT-474 cells were not killed by T- MMAE or T-DMl (Table 14). In contrast, VB7-756 was potent against BT-474 tumor cells evading T-DMl and T-MMAE killing (Tables 13 and 14).

[00368] Table 13: VB7-756, T-DMl and T-MMAE IC₅₀ values against BT-474 cells that evaded treatment with T-DMl

[00369] Table 14: VB7-756, T-DMl and T-MMAE IC₅₀ values against BT-474 cells that evaded treatment with T-MMAE

VB7-756 potency against tumorosphere forming BT-474 cells evading T-MMAE and T-DMl killing [00370] The ability of BT-474 cells surviving VB7-756, T-DMl or T-MMAE treatment to form tumorospheres was evaluated. BT-474 cells were treated with 10 nM VB7- 756, T-MMAE or T-DMl under adherent conditions for 5 days. Cells surviving treatment were placed under tumorosphere forming conditions. As seen in Table 15, VB7-756 at a concentration of 10 nM was sufficient to completely abolish tumorosphere formation. However, no inhibition was obtained with T-MMAE and T-DMl . Of note, the content of the wells treated with VB7-756 were collected and reseeded into tumorosphere media. After 7 days incubation, no tumorospheres were present demonstrating the cytotoxic effect of deBouganin against a tumorosphere initiating cell population.

[00371] Table 15: Percent tumorosphere forming efficiency of BT-474 cells that evaded treatment with VB7-756, T-DMl or T-MMAE

Values are the mean of 3 representative experiments. Values in parentheses indicate the SE.

[00372] Moreover, the effect of VB7-756, T-DMl and T-MMAE on tumorosphere initiating cells evading T-DMl or T-MMAE cytotoxicity was also assessed. BT-474 cells were treated with 10 nM T-MMAE or T-DMl under adherent conditions for 5 days. Cells surviving treatment were placed under tumorosphere forming conditions and treated with 100 nM VB7-756, T-MMAE or T-DMl As seen in Tables 16 and 17, VB7-756 prevented tumorosphere formation while T-DMl and T-MMAE had only a partial inhibition.

[00373] Table 16: Percent tumorosphere forming efficiency of BT-474 cells that evaded treatment with T-DMl and then treated with VB7-756, T-MMAE or T-DMl

[00374] Table 17: Percent tumorosphere forming efficiency of BT-474 cells that

VB7-756 potency against HCC1419 tumor cells evading T-MMAE and T-DMl killing

[00375] HCC1419 cells were pre-treated with 10 nM T-MMAE or T-DMl under adherent conditions for 5 days. Surviving cells were washed and plated under adherent conditions. Cells were then treated with VB7-756, T-MMAE or T-DMl, and cell viability was measured after 5 days.

[00376] No IC₅₀ was measured with T-DMl and T-MMAE against HCC1419 tumor cells that had escaped T-DMl or T-MMAE killing (Table 18), indicating that surviving cells are resistant to subsequent treatment with T-DMl or T-MMAE. In contrast, VB7-756 was potent against HCC1419 tumor cells evading T-DMl and T-MMAE killing (Table 18), demonstrating that VB7-756 can overcome mechanisms of resistance to which T-DMl and T-MMAE are susceptible. The potency of VB7-756 versus T-DMl or T-MMAE against HCC1419 tumor cells that had escaped T-DMl or T-MMAE killing was confirmed in MTS assays (Figure 25A-25B).

[00377] Table 18: VB7-756, T-DMl and T-MMAE IC₅₀ values against HCC1419 cells that evaded treatment with T-DMl or with T-MMAE

IC₅₀ (nM)

VB7-756 T-DM1 T-MMAE

HCC1419 0.155 (0.015) 1 .900 (0.900) 5.705 (5.095)

HCC1419 treated with T-DM1 0.19 (0.01 ) >10 >10

HCC1419 treated with T-MMAE 0.17 (0.05) >10 >10

Values derived from a minimum of 3 experiments with 3 replicates per dilution. Values in parentheses indicate the SE. VB7-756, T-DMl and T-MMAE potency against tumorosphere forming BT-474 and HCC1419 cells

[00378] The ability of BT-474 and HCC 1419 cells treated with VB7-756, T-DMl and

T-MMAE to form tumorospheres was evaluated. BT-474 or HCC 1419 cells were treated with 10 nM VB7-756, T-DMl or T-MMAE under adherent conditions for 5 days. Cells surviving treatment were placed under tumorosphere forming conditions. As seen in Table 19 and FIG. 26, VB7-756 was more effective than T-DMl or T-MMAE in preventing tumorosphere formation.

[00379] Table 19: Percent tumorosphere forming efficiency of BT-474 or HCC1419 cells treated with VB7-756, T-DMl or T-MMAE

Treatment BT474 HCC1419

VB7-756 0 2.9 (2.9)

T-DM 1 91 .7 (4.8) 91 .3 (1 1 .7)

T-MMAE 87.8 (5.8) 90.4 (13.6)

Values derived from 2 representative experiments. Tumorosphere forming efficiency expressed as % relative to NT (no treatment) control. Values in parentheses indicate the SE.

[00380] VB7-756 potency against tumorosphere forming BT-474 and HCC1419 cells evading T-DMl and T-MMAE killing

[00381] BT-474 and HCC1419 cells were treated with 10 nM T-MMAE or T-DMl under adherent conditions for 5 days. Cells surviving treatment were placed under tumorosphere forming conditions and treated with 10 nM VB7-756, T-MMAE or T-DMl . As seen in Table 20 and FIG. 27, VB7-756 is more effective than T-DMl or T-MMAE in preventing tumorosphere formation in T-DMl or T-MMAE treated cells.

[00382] Table 20: Percent tumorosphere forming efficiency of BT-474 or HCC1419 cells that evaded treatment with T-DMl or T-MMAE and then treated with VB7-756, T-DMl or T-MMAE

BT-474 HCC141 9

T-DM1 T-MMAE T-DM1 T-MMAE

Treatment

treated treated treated treated VB7-756 0.14 (0.14) 0.6 (0.6) 2.1 (0.4) 1 .8 (0.5)

T-DM1 42.8 (29.4) 72.6 (6.9) 110.6 (2.1) 83.6 (10.1)

T-MMAE 60.1 (28.0) 70.6 (8.1) 114 (6.1) 90.9 (12.9)

Values derived from 2 representative experiments. Tumorosphere forming efficiency expressed as % relative to NT (no treatment) control. Values in parentheses indicate the SE.

[00383] Overall these results demonstrate that deBouganin's distinct mechanism of action could overcome mechanisms of resistance affecting the efficacy of small molecule drugs such as DM1 or MMAE.

Claims

What is claimed is:

1. An immunotoxin comprising: (a) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin.

2. The immunotoxin of claim 1 , wherein the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment.

3. The immunotoxin of claim 2, wherein 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.

4. The immunotoxin of claim 2, wherein the anti-HER2/neu antibody or the anti- HER2/neu antibody fragment comprises a heavy chain variable region.

5. The immunotoxin of claim 4, wherein the heavy chain variable region comprises an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence of SEQ ID NO: 2.

6. The immunotoxin of claim 4, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 2.

7. The immunotoxin of claim 2, wherein the anti-HER2/neu antibody or the anti- HER2/neu antibody fragment comprises a light chain variable region.

8. The immunotoxin of claim 7, wherein the light chain variable region comprises an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence of SEQ ID NO: 4.

9. The immunotoxin of claim 7, wherein the light chain variable region comprises an amino acid sequence of SEQ ID NO: 4.

10. The immunotoxin of any one of claims 2 to 9, wherein 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.

11. The immunotoxin of claim 10, wherein the anti-HER2/neu antibody fragment is a diabody.

12. The immunotoxin of claim 10, wherein the anti-HER2/neu antibody fragment is a scFv.

13. The immunotoxin of claim 10, wherein the anti-HER2/neu antibody fragment is an Fab.

14. The immunotoxin of any one of claims 1 to 13, wherein the deimmunized bouganin toxin is linked to the anti-HER2/neu binding protein by a linker comprising an amino acid sequence chosen from SEQ ID NOs: 17, 32-36, 62 and 63.

15. The immunotoxin of claim 11, wherein the diabody is comprised of a heavy chain variable region and a light chain variable region.

16. The immunotoxin of claim 15, wherein the heavy chain variable region and the light chain variable region are linked by a linker.

17. The immunotoxin of claim 16, wherein the linker comprises an amino acid sequence of SEQ ID NO: 15.

18. The immunotoxin of claim 11 , wherein the deimmunized bouganin toxin is linked to the heavy chain variable region by a linker comprising an amino acid sequence of SEQ ID NO: 17.

19. The immunotoxin of claim 11, wherein the deimmunized bouganin toxin is linked to the light chain variable region by a linker comprising an amino acid sequence of SEQ ID NO: 17.

20. The immunotoxin of any one of claims 1 to 19, wherein the deimmunized bouganin toxin comprises an amino acid sequence selected from SEQ ID NOs: 12, 58, 59, 60 and 61.

21. The immunotoxin of claim 11, wherein the immunotoxin comprises amino acids 23- 535 of the amino acid sequence shown in SEQ ID NO: 23.

22. The immunotoxin of claim 11, wherein the immunotoxin comprises amino acids 23- 529 of SEQ ID NO: 25.

23. The immunotoxin of claim 11, wherein the immunotoxin comprises amino acids 23- 535 of SEQ ID NO: 27.

24. The immunotoxin of claim 11, wherein the immunotoxin comprises amino acids 23- 529 of SEQ ID NO: 29.

25. The immunotoxin of claim 11, wherein the immunotoxin comprises amino acids 23- 529 of SEQ ID NO: 31.

26. 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) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin.

27. The method of claim 26, wherein the anti-HER2/neu binding protein comprises an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment.

28. The method of claim 27, wherein 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.

29. The method of claim 27, wherein the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises a heavy chain variable region.

30. The method of claim 29, wherein the heavy chain variable region comprises an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence of SEQ ID NO: 2.

31. The method of claim 29, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 2.

32. The method of claim 27, wherein the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises a light chain variable region.

33. The method of claim 32, wherein the light chain variable region comprises an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence of SEQ ID NO: 4.

34. The method of claim 32, wherein the light chain variable region comprises an amino acid sequence of SEQ ID NO: 4.

35. The method of any one of claims 27 to 34, wherein 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.

36. The method of claim 35, wherein the anti-HER2/neu antibody fragment is a diabody.

37. The method of claim 35, wherein the anti-HER2/neu antibody fragment is a scFv.

38. The method of claim 35, wherein the anti-HER2/neu antibody fragment is an Fab.

39. The method of any one of claims 26 to 38, wherein the deimmunized bouganin toxin is linked to the anti-HER2/neu binding protein by a linker comprises an amino acid sequence selected from SEQ ID NOs: 17, 32-36, 62 and 63.

40. The method of claim 36, wherein the diabody is comprised of a heavy chain variable region and a light chain variable region.

41. The method of claim 40, wherein the heavy chain variable region and the light chain variable region are linked by a linker.

42. The method of claim 41, wherein the linke rcomprises an amino acid sequence of SEQ ID NO: 15.

43. The method of claim 36, wherein the deimmunized bouganin toxin is linked to the heavy chain variable region by a linker comprises an amino acid sequence of SEQ ID NO: 17.

44. The method of claim 36, wherein the deimmunized bouganin toxin is linked to the light chain variable region by a linker comprising an amino acid sequence of SEQ ID NO: 17.

45. The method of any one of claims 26 to 44, wherein the deimmunized bouganin toxin comprises by an amino acid sequence selected from SEQ ID NOs: 12, 58, 59, 60 and 61.

46. The method of claim 36, wherein the immunotoxin comprises amino acids 23-535 of the amino acid sequence of SEQ ID NO: 23.

47. The method of claim 36, wherein the immunotoxin comprises amino acids 23-529 of the amino acid sequence of SEQ ID NO: 25.

48. The method of claim 36, wherein the immunotoxin comprises amino acids 23-535 of the amino acid sequence of SEQ ID NO: 27.

49. The method of claim 36, wherein the immunotoxin comprises amino acids 23-529 of the amino acid sequence of SEQ ID NO: 29.

50. The method of claim 36, wherein the immunotoxin comprises amino acids 23-529 of the amino acid sequence of SEQ ID NO: 31.

51. The method of claim 26, wherein the cancer is breast, ovarian, gastric, lung (non small cell lung cancer, NSCLC) or pancreatic.

52. The method of claim 26, wherein the immunotoxin is administered directly to the cancer site.

53. The method of claim 26, wherein the direct administration is intratumoral, intravesicular or peritumoral.

54. The method of claim 26, wherein the direct administration is systemic.

55. The method of claim 54, wherein the systemic administration is intravenous.

56. The method of any one of claims 26-55 additionally comprising the administration of one or more further cancer therapeutics for simultaneous, separate or sequential treatment or prevention of cancer.

57. A method for enhancing the activity of an anti-cancer agent comprising administering to a subject in need thereof an anti-cancer agent and an effective amount of an immunotoxin of any one of claims 1-25.

58. A kit for treating or preventing cancer comprising an effective amount of an immunotoxin comprising: (a) an anti-HER2/neu binding protein and; (b) a deimmunized bouganin toxin, and directions for the use thereof to treat the cancer.

59. An expression vector comprising the immunotoxin of any of claims 1-25.

60. The immunotoxin of claim 11, wherein the deimmunized bouganin toxin is linked to the heavy chain variable region by a linker comprises an amino acid sequence of SEQ ID NO: 62.

61. The immunotoxin of claim 11, wherein the immunotoxin comprises an amino acid sequence of SEQ ID NO: 64.

62. The immunotoxin of claim 11, wherein the immunotoxin comprises an amino acid sequence of SEQ ID NO: 70.

63. The method of claim 36, wherein the deimmunized bouganin toxin is linked to the heavy chain variable region by a linker comprising an amino acid sequence of SEQ ID NO: 62.

64. The method of claim 36, wherein the immunotoxin comprises an amino acid sequence of SEQ ID NO: 64.

65. The method of claim 36, wherein the immunotoxin comprises an amino acid sequence of SEQ ID NO: 70.

66. The method of any one of claims 63-65 additionally comprising the administration of one or more further cancer therapeutics for simultaneous, separate or sequential treatment or prevention of cancer.

67. A method for enhancing the activity of an anti-cancer agent comprising administering to a subject in need thereof an anti-cancer agent and an effective amount of an immunotoxin of any one of claims 60-62.

68. An expression vector comprising the immunotoxin of any of claims 60-62.