CN1954073B - Modified Bouganin proteins, cytotoxins and methods and uses thereof - Google Patents

Abstract

Modified forms of bouganin protein are provided having biological activity and a reduced propensity to activate human T cells as compared to the non-modified bouganin protein. Also provided are T-cell epitope peptides of bouganin, and modified T-cell epitope peptides of bouganin, which have a reduced propensity to activate human T cells as compared to the non-modified T-cell epitope peptide. Also provided are cytotoxins that comprise a ligand which binds to cancer cells, and is attached to modified bouganin proteins. Also provided are methods of inhibiting or destroying mammalian cancer cells using the cytotoxins and pharmaceutical compositions for treating human cancer.

Description

Modified bouganin protein, cytotoxin, method and use thereof

field of invention

The present invention relates to modified bouganin proteins and cytotoxins comprising the modified proteins useful as cancer therapeutics. Specifically, the immunogenicity of bouganin toxin is reduced by removing or altering T-cell epitopes. the

Background of the invention

There are many instances where the efficacy of a therapeutic protein is limited by an undesired immune response to the therapeutic protein. Several mouse monoclonal antibodies have shown potential as therapeutic agents in many human disease settings, but they were ineffective in specific cases by inducing a pronounced human anti-mouse antibody (HAMA) response [Schroff, RW et al. (1985) Cancer Res. 45 :879-885; Shawler, DL et al. (1985) J. Immunol. 135 :1530-1535]. For monoclonal antibodies, a number of techniques have been developed in an attempt to reduce the HAMA response [WO89/09622; EP0239400; EP0438310, WO91/06667]. These recombinant DNA methods generally reduce the mouse genetic information in the final antibody construct while increasing the human genetic information in the final construct. Nevertheless, in some cases the resulting "humanized" antibodies elicited an immune response in the patient [Issacs JD (1990) Sem. Immunol. 2 :449,456; Rebello, PR et al. (1999) Transplantation 68 :1417-1420].

Key to the induction of an immune response is the presence within the protein of peptides capable of stimulating T-cell activity through presentation on class II MHC molecules, so-called T-cell epitopes. Such T-cell epitopes are generally any sequence of amino acid residues capable of binding an MHC class II molecule. "T-cell epitope" implies an epitope that is recognized by the T-cell receptor (TCR) when it binds to an MHC molecule, and which, at least in theory, is capable of promoting the activation of these T cells by binding to the TCR T-cell response. the

MHC class II molecules are a group of highly polymorphic proteins that play an important role in helper T-cell selection and activation. Human leukocyte antigen group DR (HLA-DR) is the major isoform of this group of proteins; however, isoforms HLA-DQ and HLA-DP perform similar functions. In the human population, individuals carry two to four DR alleles, two DQ and two DP alleles. The structures of many DR molecules have been elucidated, and they appear as open-ended peptide-binding grooves with numerous hydrophobic pockets that bind to protein hydrophobic residues (pocket residues) [Brown et al. (1993) Nature 364 :33; Stern et al. (1994) Nature 368 :215]. The polymorphisms used to distinguish allotype class II molecules contribute to the extreme diversity of different peptide-binding surfaces within the peptide-binding groove and, at the population level, ensure maximum flexibility in recognizing heterologous proteins and initiating immune responses against pathogenic organisms Spend.

The immune response to therapeutic proteins occurs through the MHC class II peptide presentation pathway. At this point the foreign protein is ingested and processed and presented together with DR, DQ or DP class II MHC molecules. MHC class II molecules are expressed by specialized antigen-presenting cells (APCs), such as macrophages and dendritic cells. Binding of MHC class II peptide complexes by cognate T-cell receptors on the T-cell surface, as well as cross-binding to other specific co-receptors such as the CD4 molecule, induces an activated state in T-cells. This activation leads to the release of cytokines and further activates other lymphocytes such as B cells to produce antibodies or T-killer cells to form a whole-cell immune response. the

As recognized in WO98/52976, WO00/34317, WO02/069232, WO02/079232 and WO02/079415, epitope removal is the first step in T-cell epitope identification. In these teachings, predicted T-cell epitopes are removed by making judicious amino acid substitutions in the protein of interest. In addition to computer techniques, there are a number of methods for determining the ability of synthetic peptides to bind MHC class II molecules. An exemplary method utilizes a B-cell line of defined MHC allotype as a source of MHC class II binding surfaces and can be applied to MHC class II ligand identification [Marshall KW et al. (1994) J. Immunol. 152 :4946 -4956; O'Sullivan et al. (1990) J. Immunol. 145 :1799-1808; Robadey C. et al. (1997) J. Immunol 159 :3238-3246]. However, such techniques are not suitable for screening multiple potential epitopes from the extremely diverse MHC allotypes, nor do they validate the ability of a binding peptide to function as a T-cell epitope.

The technology of developing soluble complexes of recombinant MHC molecules and synthetic peptides is also gradually being adopted [Kern, F. et al. (1998) Nature Medicine 4 : 975-978; Kwok, WW et al. (2001) TRENDSinImmunol. 22 : 583-588 ]. These reagents and methods are used to identify the presence of T-cell clones capable of binding specific MHC-peptide complexes in peripheral blood samples derived from humans or experimental animals, and are suitable for screening multiple potential epitopes from an extremely diverse MHC allotype

Bioassays for T-cell activation provide a practical option for reading the ability of a test peptide/protein sequence to elicit an immune response. Examples of such methods include the work of Petra et al. applying a T-cell proliferation assay to streptokinase followed by stimulation of T-cell lines with synthetic peptides for epitope mapping [Petra, AM et al. (2002) J. Immunol. 168 :155-161]. Similarly, a T-cell proliferation assay utilizing synthetic peptides of the tetanus toxin protein successfully defined the major immune epitopes of the toxin [Reece JC et al. (1993) J. Immunol. 151 :6175-6184]. WO99/53038 discloses a method in which by utilizing isolated subsets of human immune cells, promoting their in vitro differentiation and culturing in the presence of a synthetic peptide of interest, and assaying the expression of any induced cultured T-cells Proliferation, T-cell epitopes in the test protein can be determined. The same technique is also described by Stickler et al. [Stickler, MM et al. (2000) J. Immunotherapy 23 :654-660], in both cases the method was applied to the detection of T- cell epitope. Such techniques require careful cell isolation techniques and cell culture supplemented with multiple cytokines to obtain a satisfactory subset of immune cells (dendritic cells, CD4+ and/or CD8+ T-cells) Fast-throughput screening of donor samples.

Recently, a combined approach combining population-based T-cell proliferation assays and in silico modeling of peptide MHC binding when designing epitope-removing proteins has been developed. WO03/104803]. the

As mentioned above and its consequences, it would be desirable to identify and remove or at least reduce a T-cell epitope on a peptide, polypeptide or protein that is generally therapeutically valuable but initially immunogenic. the

Summary of the invention

The aim of the present invention is to overcome the practical problem that soluble proteins introduced into the human body for therapeutic purposes elicit an immune response leading to the production of host antibodies bound to the soluble proteins. The present invention attempts to solve this problem by providing bouganin proteins with a reduced propensity to elicit an immune response. According to the methods described herein, the inventors have identified regions of the bouganin molecule that contain T-cell epitopes that drive immune responses to the protein. the

The present invention relates to a modified Bouganin protein, the modified Bouganin has a reduced tendency to trigger an immune response. In a preferred embodiment, the modified Bouganin has a reduced tendency to activate T-cells, and the modified Bouganin is modified at one or more amino acid residues in the T-cell epitope. The T-cell epitope is preferably selected from:

a) AKVDRKDLELGVYKL (epitope region R1, SEQ ID NO: 2),

b) LGVYKLEFSIEAIHG (epitope region R2, SEQ ID NO: 3); and

c) NGQEIAKFFLIVIQM (epitope region R3, SEQ ID NO: 4). the

The present invention also relates to a cytotoxin comprising a targeting moiety that binds to the modified Bouganin protein of the present invention. In one embodiment, the targeting moiety is a ligand that binds to cancer cells. In another embodiment, the ligand is an antibody or antibody fragment that binds to cancer cells. In a specific embodiment, the antibody recognizes Ep-CAM or a tumor-node-associated antigen. In a most specific embodiment, the invention provides a cytotoxin comprising VB6-845 or VB6-011. the

In another aspect, the invention provides a method of inhibiting or destroying cancer cells comprising administering a cytotoxin of the invention to the cancer cells. the

The present invention also relates to a method of treating cancer by administering a cytotoxin of the present invention to an animal in need thereof. the

Furthermore, there is also provided a method for preparing a medicament for treating an animal with cancer, comprising the steps of: identifying the T-cell epitope of bouganin, and modifying one or more amino acid residues in the T-cell epitope Thereby preparing modified Bouganin with a reduced propensity to activate T-cells; preparing a cytotoxin with a cancer-binding ligand bound to the modified Bouganin; and suspending the cytotoxin in a pharmaceutically acceptable carrier, diluent or excipient Forming agent. the

In yet another aspect, the present invention provides a pharmaceutical composition for treating cancer, which comprises the cytotoxin of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. the

The cytotoxins, compositions and methods of the invention are useful in the treatment of various forms of cancer such as colorectal, breast, ovarian, pancreatic, head and neck, bladder, gastrointestinal, prostate, small cell and Non-small cell carcinoma, malignancy, glioma, T- and B-cell lymphoma. the

The present invention also provides the T-cell epitope peptide of Bouganin protein and the modified T-cell epitope peptide of the present invention. the

Other features and advantages of the present invention will gradually appear from the following detailed description. It should be understood, however, that the detailed description and specific examples, which illustrate the preferred embodiment of the invention, are provided by way of illustration only since it will become apparent to those skilled in the art that various concepts that are within the spirit and scope of the invention can be derived from the detailed description. Various changes and modifications are evident. the

Brief description of the drawings

The present invention will be described in conjunction with the following drawings:

Figure 1 shows the activity detection results of the modified Bouganin proteins Bou156 (panel A) and Bou157 (panel B) with T-cell epitopes removed. Bou156 includes V123A, D127A, Y133N and I152A substitutions. Bou157 includes V123A, D127A, Y133Q and I152A substitutions. Both assays use wild-type protein and inactivated modified Bouganin (Y70A) as controls. Activity is expressed as % of Bouganin protein concentration in luciferase activity ratio assay. the

Figure 2 shows the T-cell proliferation assay of three synthetic peptides and two different PBMC donor samples. Peptides named DeI-41, DeI-44 and DeI-50 were tested at 1 μM final concentration (panel A) and 5 μM final concentration (panel B). These peptides are derived from the immunogenic region of the bouganin molecule and contain substitutions designed to remove its immunogenicity. the

Figure 3 shows VB6-845, a modified Bouganin cytotoxin with a Fab anti-Ep-CAM, wherein the de-bouganin (Bou156) is linked to the C-terminus of the CH domain via a paired basic amino acid protease linker connected. Accompanying drawing 3A shows the bicistronic unit of encoding original sequence, accompanying drawing 3B shows the nucleic acid coding sequence (SEQ ID NO:15) and aminoacid sequence (SEQ ID NO:16) of this original sequence, and accompanying drawing 3C shows that does not contain pelB Sequence assembly of the VB6-845 protein. the

Figure 4 shows a diagram of the expression vector pING3302. The inserts in the examples were ligated into the 3302 vector using EcoRI and XhoI restriction sites. the

Figure 5 shows a control Fab anti-Ep-CAM construct without the phytotoxin, de-bouganin (VB5-845). Accompanying drawing 5A shows the bicis subunit of encoding original sequence, accompanying drawing 5B shows the nucleic acid coding sequence (SEQ ID NO: 17) and aminoacid sequence (SEQ ID NO: 18) of this original sequence, and accompanying drawing 5C shows that does not contain pelB Sequence assembly of the VB5-845 protein. the

Figure 6 shows the Fab anti-Ep-CAM de-bouganin construct VB6-845- _CL -de-bouganin in which Bou156 is linked to the C-terminus of the _CL domain. Accompanying drawing 6A shows the bicis subunit of encoding original sequence, accompanying drawing 6B shows the nucleic acid coding sequence (SEQ ID NO:19) and aminoacid sequence (SEQ ID NO:20) of this original sequence, and accompanying drawing 6C shows that does not contain pelB Assembly of the sequence VB6-845- _CL -de-Bouganin protein.

Figure 7 shows the Fab anti-Ep-CAM, de-bouganin construct, VB6-845- _NVH -de-bouganin, where Bou156 is linked to the N-terminus of the _VH domain. Accompanying drawing 7A shows the bicis subunit of encoding original sequence, accompanying drawing 7B shows the nucleic acid coding sequence (SEQ ID NO:21) and aminoacid sequence (SEQ ID NO:22) of this original sequence, and accompanying drawing 7C shows not containing pelB Assembly of the sequence VB6-845- _NVH -de-Bouganin protein.

Figure 8 shows the Fab anti-Ep-CAM de-bouganin construct, VB6-845- _NVL -de-bouganin, in which Boul56 is linked to the N-terminus of the _VL domain. Accompanying drawing 8A shows the bicis subunit of encoding original sequence, accompanying drawing 8B shows the nucleic acid coding sequence (SEQ ID NO:23) and aminoacid sequence (SEQ ID NO:24) of this original sequence, and accompanying drawing 8C shows that does not contain pelB Sequence assembly of the VB6-845-NV _L -de-Bouganin protein.

Accompanying drawing 9 shows the expression of VB6-845 (the construct of accompanying drawing 3) and VB6-845-CL-de-bouganin (Bou156) (the construct of accompanying drawing 6) in the supernatant of E104 cells induced at the laboratory level Western blot. the

Figure 10 shows the results of the flow cytometry reactivity study. Accompanying drawing 10A shows that VB6-845 (construct of accompanying drawing 3) and VB6-845- _CL -de-bouganin (construct of accompanying drawing 6) in Ep-CAM-positive cell lines CAL 27 and OVCAR-3 and Ep -CAM-negative cell line A-375 results, while accompanying drawing 10B shows with VB6-845 (construct of accompanying drawing 3) and VB6-845-gelonin (construct of accompanying drawing 14C) and control ( The results of the same tests performed by PBS).

Figure 11 is a graph showing the results of a competition assay between -VB6-845 and Proxinium ^™ in NIH: OVCAR-3 cells as described in Example 7.

Figure 12 is a graph showing the results of the cell-free assay in Example 7. the

Accompanying drawing 13 shows comparison VB6-845 (the construct of accompanying drawing 3), VB6-845-CL-de-bouganin (the construct of accompanying drawing 6) and de-bouganin (Bou156) in CAL 27 (accompanying drawing 3) in embodiment 8 Figure 13A) and MTS cytotoxicity assay results of cytotoxicity in NIH:OVCAR3 (Figure 13B) cells. the

Accompanying drawing 14A and B show the construct of comparison VB6-845 (accompanying drawing 3) of VB6-845 (accompanying drawing 3), VB6-845-gelonin (construct accompanying drawing 14C) and gelonin in CAL 27 (accompanying drawing 14C) in Example 8 14A) and MTS cytotoxicity assay results of cytotoxicity in NIH: OVCAR3 (Fig. 14B) cells. Figure 14C shows the nucleic acid coding sequence (SEQ ID NO: 25) and the amino acid sequence (SEQ ID NO: 26) of the VB6-845-gelonin construct. the

Accompanying drawing 15 shows the nucleic acid coding sequence (SEQ ID NO:27) and aminoacid sequence (SEQ ID NO:28) of VB6-011 original sequence. the

Figure 16 shows the MTS cytotoxicity test results of Example 9, the cytotoxicity of VB6-011 in MB-435S cells. the

Detailed description of the invention

The present inventors have identified T-cell epitopes in Bouganin, and have designed and prepared modified Bouganin proteins that have a reduced propensity to activate human T cells compared to non-modified Bouganin proteins. the

(A) Modified Bouganin protein

The present invention relates to a modified Bouganin protein, wherein the bouganin has been modified for a reduced propensity to elicit an immune response, preferably a T-cell response, compared to a non-modified-Bouganin protein. The mature Bouganin protein is a single-chain polypeptide of 250 amino acids with a molecular weight of approximately 26,200 Da [Den Hartog et al. (2002) EuR.J.Biochem. 269 :1772-1779; US Patent No. 6,680,296]. Bouganin was originally a type 1 ribosome-inactivating protein (RIP) isolated from the plant Bougainvilleaspectabilis Willd [Bolognesi et al. (1997) Planta 203 :422-429]. These plant-derived RIPs are RNA N-glycosidases that depurinate the primary ribosomal RNA of the cell, thereby destroying the ribosome and leading to abort of protein synthesis and cell death.

The amino acid sequence (expressed in single-letter code) of mature Bouganin protein is:

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKR

FVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKV

PTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYK

LEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLY

GSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPW

VVNKVSQISPDMGILKFKSSK [SEQ ID NO: 1]. the

The term "non-modified Bouganin protein" means a Bouganin protein that has not been modified to reduce its propensity to elicit an immune response. The sequence of wild-type or non-modified Bouganin is shown in SEQ ID NO: 1. However, those skilled in the art will understand that the term "non-modified bouganin" also includes modifications to SEQ ID NO: 1, as long as such modifications do not reduce its propensity to elicit an immune response. Examples of modifications that can be made to SEQ ID NO: 1 include peptide fragments and conservative amino acid substitutions that do not reduce the immunogenicity of the protein. the

The term "modified Bouganin protein" means a Bouganin protein that has been modified compared to a non-modified Bouganin protein (as described above), wherein the modification reduces the propensity of the bouganin to elicit an immune response. Modified bouganin protein can also refer to deimmunized bouganin. A "modified Bouganin protein" may be a modified full-length sequence or a modified non-modified Bouganin protein fragment. A "modified bouganin protein" may also contain other changes that do not alter the immunogenicity of the peptide compared to the wild-type bouganin sequence. The modified Bouganin protein preferably has the same biological activity as the non-modified Bouganin. the

The term "reduced propensity to elicit an immune response" as used herein means that the modified Bouganin protein is less immunogenic than non-modified Bouganin. the

The term "immune response" includes both cellular and humoral immune responses. In a preferred embodiment, the modified Bouganin has a reduced tendency to activate T-cells. the

The term "reduced propensity to activate human T-cells" as used herein means that the modified Bouganin protein has a lower propensity to activate human T-cells than the non-modified Bouganin protein. Those skilled in the art can test whether the modified Bouganin has a lower tendency to activate T-cells using methods known in the art, including evaluating the stimulation index of the protein. the

As used herein, the term "stimulation index" refers to a measure of the ability of a modified or non-modified Bouganin protein to activate human T cells. For example, modified or non-modified Bouganin proteins, or peptides thereof, can be tested for their ability to elicit a human T-cell proliferative response in vitro. When performing this type of method using naïve human T-cells from healthy donors, the inventors have determined that a stimulation index equal to or greater than 2.0 is an effective factor for inducing proliferation in the operation of such an assay. measure. Stimulation index is generally obtained by dividing the fraction of proliferation measured with the test peptide (calculated as radioactivity per minute if ³ H-thymidine incorporation is used, for example) by the fraction measured in cells not exposed to the test peptide.

In one embodiment, the present invention provides a modified Bouganin protein that is biologically active and has a lower propensity to activate human T cells than a non-modified Bouganin protein. the

In another embodiment, the present invention provides a modified Bouganin protein that has a lower tendency to activate human T cells than non-modified Bouganin protein and has a lower tendency than non-modified Bouganin protein. biological activity. In yet another embodiment, the present invention provides a modified Bouganin protein, which has a reduced tendency to activate human T cells and has no biological activity. Such modified proteins can, for example, be used as controls, for assays or tolerize subjects. the

As used herein, the term "biological activity" refers to the ability of a modified or non-modified Bouganin protein to inhibit protein synthesis on ribosomes, which can be determined by various methods. It should be noted that the modified Bouganin protein is still biologically active, even if such activity is lower than that of the non-modified protein, however it needs to have a certain level of detectable activity. For example, the biological activity of a modified or non-modified Bouganin protein can be determined by identifying its N-glycosidase activity, and in particular has sufficient activity to provide significant inhibition of protein translation. One such suitable assay involves testing the activity of the variant bouganin protein compared to non-modified bouganin in a cell-free protein synthesis assay. A transcription/translation coupling mixture containing methionine, DNA encoding the reporter protein luciferase, and serial dilutions of non-modified and modified Bouganin proteins were incubated. Translated luciferase levels can be readily measured with a luminescence counter after addition of the substrate reagent. The amount of luminescence measured is inversely proportional to the bouganin N-glycosidase activity present in the reaction. Negative controls such as inactivated Bouganin protein, for example comprising a Y70A substitution, are usually provided. the

In a preferred embodiment, one or more T-cell epitopes in the Bouganin protein sequence of the modified Bouganin peptide are modified. the

The term "T-cell epitope" means, capable of binding major histocompatibility complex (MHC) class II, capable of stimulating T-cells and/or capable of binding (not necessarily measurable) in complex with MHC class II activation of) the amino acid sequence of T-cells. the

In one aspect, the overall method for obtaining modified Bouganin proteins containing modified T-cell epitopes that can be used in the present invention comprises the following steps:

(i) determine the amino acid sequence of the protein or portion thereof;

(ii) Determination of peptide and MHC by, for example, using in vitro or in silico techniques or bioassays

Molecular combinations that identify one or more potential amino acid sequences in the protein

T-cell epitopes;

(iii) Designing new sequence variants with one or more modified amino acids located in the identified T-cell epitopes, based on determination of peptide binding to MHC molecules using in vitro or in silico techniques or bioassays As determined, in this way the activity of the T-cell epitope is substantially reduced or eliminated. Such sequence variant epitopes are constructed in such a way as to avoid the formation of new potential T-cell epitopes due to mutations in the sequence, unless such new potential T-cell epitopes are subsequently modified in such a manner as to essentially or Removal of T-cell epitope activity;

(iv) constructing such sequence variants by recombinant DNA techniques, and testing said variants according to well-known recombinant techniques to identify one or more variants with the desired properties; and

(v) optionally repeating steps (ii) to (iv). the

In one embodiment, step (iii) is performed by substituting, adding or deleting amino acid residues in any T-cell epitope in the non-modified Bouganin protein. In another embodiment, the method for preparing the modified Bouganin protein is carried out with reference to homologous protein sequences and/or computer models. the

Identification of potential T-cell epitopes according to step (ii) can be accomplished as previously described in the art. Suitable methods are disclosed in WO98/59244, WO98/52976, WO00/34317, WO02/069232 and can be used to identify the binding propensity of bouganin-derived peptides to MHC class II molecules. To identify biologically relevant peptides, the inventors have developed a method for exploiting ex vivo human T-cell proliferation assays. This approach has proven to be a very efficient one and involves testing overlapping bouganin-derived peptide sequences in a protocol to scan and test the entire bouganin sequence. Synthetic peptides were tested for their ability to elicit a proliferative response in human T-cells cultured in vitro. The present inventors have performed this type of method using naive human T-cells taken from healthy donors and have established that a stimulation index equal to or greater than 2.0 is a valid measure of induction of proliferation in the operation of such an assay. The stimulation index is generally obtained by dividing the fraction of proliferation measured with the test peptide (calculated as radioactivity per minute if 3H-thymidine incorporation is used, for example) by the fraction measured in cells not exposed to the test peptide. the

Accordingly, in the present study, in a T-cell proliferation assay using PBMC (peripheral blood mononuclear cells) taken from natural donors (ie, in the absence of known bouganin sensitization), the 89 synthetic 15-mer peptides (listed in Table 1). Twenty donor PBMC samples were selected for optimal MHC class II allotype coverage. Triplicate cultures were stimulated with each peptide for 7 days before proliferation was assessed ^by3H -thymidine incorporation. All peptides were diluted to two concentrations: 1 μM and 5 μM. The stimulation index (SI) was calculated as the amount of ³ H incorporated into the cells divided by the amount of ³ H incorporated in the sham-stimulated control.

This method has identified the most immunogenic regions of the bouganin molecule in humans. Therefore, in a specific embodiment, one or more amino acid residues selected from the following T-cell epitopes in the modified Bouganin protein are modified:

a) AKVDRKDLELGVYKL, referred to herein as epitope region R1 (SEQ ID NO: 2);

b) LGVYKLEFSIEAIHG, referred to herein as epitope region R2 (SEQ ID NO: 3); and

c) NGQEIAKFFLIVIQM, referred to herein as epitope region R3 (SEQ ID NO: 4). the

These T-cell epitopes have been identified based on SI>2 given in two or more donor PBMC samples. The peptide sequences disclosed above give the key information needed to construct modified Bouganin proteins in which one or more of these peptides are weakened. the

In one embodiment of the invention, the modified Bouganin protein of the invention 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 present invention also contemplates a modified Bouganin protein wherein preferably 1 to 9 amino acid residues in the T-cell epitope are modified. In another embodiment, 1 to 5 amino acid residues are modified. The term "modified" as used herein means that amino acid residues are modified by substitution, addition or deletion, preferably by substitution, but Bouganin protein has a reduced tendency to stimulate human T cells. In another embodiment, the modified protein has biological activity. More preferably, the modified Bouganin protein of the present invention is modified by substitution at a position corresponding to any specific amino acid in the above sequence (a), (b) or (c). the

One embodiment of the invention includes bouganin proteins for which the MHC class II ligands identified in any of the epitopes R1-R3 have been modified to abolish binding or reduce the MHC isotypes to which the peptide is able to bind The number of shapes. Amino acids in the R1 to R3 region that abolish binding or reduce the number of MHC allotypes that the peptide is capable of binding can be modified by substitution, addition or deletion. the

To remove a T-cell epitope, amino acid substitutions are made at appropriate positions in the peptide sequence that are expected to achieve substantial attenuation or elimination of the activity of the T-cell epitope. In practice, suitable positions are in one embodiment amino acid residues that bind within the pocket of the MHC class II binding groove. the

In one embodiment, the binding within the first pocket of the cleft at the so-called P1 or P1 anchor position of the peptide is modified. The quality of the binding interaction between the peptide P1 anchor residue and the first pocket of the MHC class II binding groove is considered to be the main determinant of the overall binding affinity of the entire peptide molecule. Appropriate substitutions at this position of the peptide will be made with a residue that is less easily accommodated in the pocket, for example, by a more hydrophilic residue. Binding amino acid residues in the peptide that are located in the equivalent of other pocket regions in the MHC binding cleft were also considered. the

It will be appreciated that single amino acid substitutions, deletions or additions in a given potential T-cell epitope are a preferred route of removal of the epitope. Combinatorial modifications (i.e. substitutions, deletions and additions) in a single epitope can be considered, for example, are particularly suitable when defined epitopes overlap each other, as in the present invention the epitope regions R1 and R2 overlap by 5 residues. In addition, single amino acid modifications in a given epitope, or combined modifications in a single epitope, can be made at positions analogous to the "pocket residues" of the MHC class II binding groove, but not anywhere in the peptide sequence. Modifications can be made by reference to homologous structures or structural methods using chip-based techniques known in the art. All such modifications are within the scope of this invention. the

The peptide regions R1-R3 of Bouganin were analyzed to indicate MHC class II ligands in their respective sequences. For this analysis the software tools for developing such protocols given in WO98/59244 and WO02/069232 were used. The software simulates the antigen presentation process at the level of peptide MHC class II binding interactions to provide a binding score for any given peptide sequence. Such scores were determined for many of the major MHC class II allotypes present in the population. Since this protocol is able to test any peptide sequence, it is possible to predict the outcome of amino acid substitutions, additions or deletions for the interaction of the peptide with the MHC class II binding groove. From this it is possible to design new sequence compositions comprising a reduced number of peptides capable of interacting with MHC class II and thus acting as immunogenic T-cell epitopes. the

Under this approach, in one embodiment of the invention, substitutions within epitope region R1 include changes at positions V123, D127 and/or E129. Similarly, for epitope region R2, in one embodiment the substitution is at Y133. This residue falls within the overlapping region of R1 and R2, but substitution at position Y133 is sufficient to remove R2-associated MHC class II ligand and is not sufficient by itself to remove R1-associated MHC class II ligand. For epitope region R3, in one embodiment of the invention, residues E151 and/or I152 are substituted. the

In all cases, substitutions were made to one or more alternative amino acid residues. Analysis of R1 with MHCII simulation software showed that amino acid residues 123, 127, 129 and 131 are the key residues in this epitope for binding MHC II molecules. Residue 123 is a preferred mutation site for the R1 region because it is located on the surface of the molecule, remote from the active site and variable in RIP sequence alignments. However, not all substitutions result in active molecules, thus mutations need to be validated in bioactivity assays. Thus, for example in R1, the substitutions V123T, V123A and V123Q are preferred alternative substitution examples. Residue 131 was found to be absolutely conserved in RIP, so it may not be amenable to mutation. Residues 127 and 129 are not highly conserved, but only a limited number of residues were found to have an effect on MHC II binding. Substitution groups: D127G, D127A, E129Q and E129G are also preferred substitutions. For R2, residue 133 was shown as a possible candidate for removal of MHC II binding, and its apparent surface location (determined from the model) coupled with the fact that it is not highly conserved in RIP makes it a candidate for mutation Good candidate residues. Preferred alternative substitutions were found to be Y133N, Y133T, Y133A, Y133R, Y133D, Y133E, Y133Q, Y133G, Y133K, Y133H and Y133S. For R3, amino acid residues 152, 155 and 158 were identified as critical residues for MHC II binding. However, residues 155 and 158 are part of a highly conserved hydrophobic sequence, thus suggesting that their mutation would fail to produce a biologically active molecule. Weakly conserved residues were found to be more likely candidates. For R3, the substituent groups: I152Q and I152A are also preferred substitutions. the

Correspondingly, the present invention provides a modified Bouganin protein, wherein the bouganin is modified at one or more positions of the following X ¹ , X ² , X ³ , X ⁴ or X ⁵ :

a) AKX ¹ DRKX ² LX ³ LGVX ⁴ KL (epitope region R1, SEQ ID NO: 5);

b) LGVX ⁴ KLEFSIEAIHG (epitope region R2, SEQ ID NO: 6); and

c) NGQEX ⁵ AKFFLIVIQM (epitope region R3, SEQ ID NO: 7)

Wherein X ¹ to X ⁵ can be any amino acid.

In a particular embodiment, X is ^{T or} A or Q; ^X is G or A; X is Q or G; X is N or D or T or A or R or Q or E or ^G or H or K or S; and ^X5 is Q or A (epitope region R1, SEQ ID NO:8; epitope region R2, SEQ ID NO:9; epitope region R3, SEQ ID NO:10).

Based on a combination of peptide epitope mapping studies using ex vivo T-cell assays, chip-based MHC peptide binding simulations, and structural considerations from sequence homology analyses, an optimal set of substitutions can be compiled. Finally, if a certain active peptide is preferred, the modified protein containing one or more substitutions can be tested for in vitro activity. the

Accordingly, in another embodiment, the present invention provides a modified Bouganin peptide, which comprises the amino acid sequence:

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK

RFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLD

KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKX ¹ DRKX ² LX ³ L

GVX ⁴ KLEFSIEAIHGKTINGQEX ⁵ AKFFLIVIQMVSEAARFKYIETE

VVDRGLYGSFKPNFKVLNLENNWGDISDAIHKS SPQCTTINPALQ

LISPSNDPWVVNKVSQISPDMGILKFKSSK

X ¹ to X ⁵ can be any amino acid (SEQ ID NO: 11).

In a preferred embodiment, X ^is T or A or Q; ^X is G or A; X is Q or G; ^X is ^N or D or T or A or R or Q or E or G or H or K or S; and ^X5 is Q or A (SEQ ID NO: 12).

In a specific embodiment, the modified Bouganin protein comprises the amino acid sequence:

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK

RFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLD

KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAK A DRK A LELG

V N KLEFSIEAIHGKTINGQE A AKFFLIVIQMVSEAARFKYIETEVV

DRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLIS 

PSNDPWVVNKVS QISPDMGILKFKS SK (SEQ ID NO: 13). the

In yet another embodiment, the modified Bouganin protein comprises the amino acid sequence:

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK

RFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLD

KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAK A DRK A LELG

V Q KLEFSIEAIHGKTINGQE A AKFFLIVIQMVSEAARFKYIETEVV

DRGLYGSFKPNFKVLNLENNWGDISDAIHKS SPQCTTINPALQLIS

PSNDPWVVNKVSQISPDMGILKFKSSK (SEQ ID NO: 14). the

Underlined residues were replaced by residues from non-modified Bouganin proteins. the

As will be apparent to those skilled in the art, several alternative sets of modifications can be achieved to remove unwanted epitopes. However the resulting sequences will remain largely homologous to the proteins disclosed herein and they thus fall within the scope of the present invention. Obvious chemical equivalents of the sequences disclosed herein are also contemplated to fall within the scope of the present invention. Such equivalents include proteins that perform substantially the same function in substantially the same manner. the

In another embodiment the modified Bouganin protein of the invention has 1, 2, 3, 4, 5 or more amino acid modifications in the T-cell epitope of the protein. the

In yet another embodiment, a modified Bouganin protein of the invention elicits a reduced stimulation index when tested in a T-cell assay compared to a non-modified Bouganin protein. the

In another embodiment of the present invention, the T-cell epitopes of the Bouganin protein are mapped using a T-cell assay, and then these epitopes are modified so that when tested again in a T-cell assay, they are modified Bouganin The protein stimulates a lower stimulation index than the non-modified Bouganin protein, preferably the stimulation index is less than 2.0. the

It is clear to those skilled in the art that if the modified Bouganin protein has substantially reduced or no biological activity, it needs further amino acid residue substitution, addition or deletion to restore the biological activity of the modified Bouganin protein. However, such modified Bouganin proteins with substantially reduced or no biological activity are still included within the scope of the present invention and can be used as controls in assays, or for tolerization. the

In one embodiment, the modified bouganin is mutated at the tyrosine residue at position 70 to produce an inactive bouganin. In a specific embodiment, tyrosine position 70 is substituted with alanine. In a preferred embodiment, the modified Bouganin has the sequence:

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK

RFVLVDITTTSKKTVKVAIDVTDVAVVGYQDKWDGKDRAVFLD

KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELG

VYKLEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVD

RGLYGSFKPNFKVLNLENNWGDISDAIHKS SPQCTTINPALQLISP

SNDPWVVNKVSQISPDMGILKFKSSK [SEQ ID NO: 129]. the

Under the concept of the present invention, the epitopes are weakened by mutations so that sequences are no longer able to function as T-cell epitopes. It is possible to achieve target sequence directed mutagenesis using recombinant DNA methods, and many such techniques are available and known in the art. In practice, many modified bouganin proteins can be prepared and tested to obtain the desired immune and functional characteristics. It is especially important that when modifications are made to the protein sequence, the contemplated changes do not introduce new immunogenic epitopes. In practice this event can be avoided by retesting the contemplated sequences for the presence or absence of epitopes and/or MHC class II ligands by any suitable method. the

The modified Bouganin proteins of the invention may also comprise or be used to obtain or design "peptidomimetics". A "peptidomimetic" is a structure that acts as a peptide surrogate in an intermolecular interaction (for review, see Morgan et al. (1989), Ann. Reports Med. Chem. 24 :243-252). Peptidomimetics include synthetic structures that may or may not contain amino acids and/or peptide bonds but retain the structural and functional characteristics of the peptides of the invention, including biological activity and reduced propensity to activate human T cells. Peptide mimetics also include peptides, oligopeptides (Simon et al. (1972) Proc. Natl. Acad, Sci USA 89 :9367).

Peptide mimetics can be designed based on information obtained by systematic substitution of D-amino acids for L-amino acids, substitution of side chains with groups with different electrical characteristics, and by systematic substitution of peptide bonds with amino linkages. Positional conformational constraints can also be introduced to determine active conformational requirements for candidate peptidomimetics. These mimetics include isosteric amino bonds, or D-amino acids to stabilize or facilitate the inverted configuration and aid in stabilizing the molecule. Cyclic amino acid analogs can be used to constrain amino acid residues to a particular conformational state. These mimetics may also include mimetics of the secondary structure of the proteins of the invention. These structures can model the three-dimensional orientation of amino acid residues into the known secondary configuration of the protein. N-substituted amino acid oligopeptide peptidomimetics can also be used and can also be used as structural motifs to generate chemically diverse libraries of new molecules. the

The molecules of the invention can be prepared by any of several methods, but are most preferably accomplished using conventional recombinant techniques. Using the protein sequences and information provided herein to deduce the polynucleotide (DNA) encoding any preferred protein sequence is a relatively straightforward approach. This can be done, for example, using computer software tools, such as the DNSstar software suite [DNAstar Inc, Madison, WI, USA] or similar software. Any such DNA sequence capable of encoding a preferred polypeptide of the present invention or a significant homologue thereof shall be understood as an embodiment of the present invention. the

As a general plan, the gene encoding any modified Bouganin protein sequence can be prepared by gene synthesis and cloned into a suitable expression vector. Expression is introduced sequentially into the host cell and the selected and cultured cells. The protein of the invention is purified from the culture medium and formulated for therapeutic administration. As an alternative, the wild-type bouganin gene sequence is obtained by, for example, following a cDNA cloning strategy using RNA prepared from root tissue of Bougainvillea spectabilis Willd plants. The wild-type gene can be used as a template for mutation and for the construction of preferred variant sequences. In this regard, it is convenient to utilize the "overlap extension PCR" strategy described by Higuchi et al. [Higuchi et al. (1988) Nucleic Acids Res. 16 :7351], although other methods and systems are also readily applicable.

Likewise, the biological activity of proteins of the invention can be assessed in a number of ways. In one embodiment, modified Bouganin molecules are identified by N-glycosidase activity, particularly by an activity sufficient to cause significant inhibition of protein translation. One such suitable assay involves testing the activity of the modified Bouganin protein compared to non-modified Bouganin in a cell-free protein synthesis system. A transcription/translation coupling mixture containing methionine, DNA encoding the reporter protein luciferase, and serial dilutions of non-modified and modified Bouganin proteins was co-cultured. The level of translated luciferase can be easily detected with a luminometer by adding the substrate reagent. The measured luminescence is inversely proportional to the bouganin N-glycosidase activity present in the reaction. It is routine to provide an inactivated Bouganin protein, for example comprising a Y70A substitution, as a negative control. the

The construction of preferred and active bouganin molecules, including bouganin molecules fused to antibodies or other targeting moieties of interest, can be accomplished by recombinant DNA techniques. Methods of purifying and manipulating recombinant proteins, including fusion proteins, are well known in the art. The required techniques are well described in the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" ( R.I.Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M.Weir & C.C. Blackwell, eds.); "Gene TransferVectors for Mammalian Cell" (J.M.Miller&M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991). the

The proteins and peptides of the invention can be prepared using recombinant DNA methods. It is also possible to utilize techniques well known in protein chemistry such as solid-phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85 : 2149-2154) or synthesis in a homogeneous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15I and II, Thieme, Stuttgart) prepared the proteins of the invention by chemical synthesis.

The present invention also provides an isolated and purified nucleic acid molecule comprising a sequence encoding the modified Bouganin protein or peptide of the present invention, preferably encoding the protein described herein as SEQ ID NO: 13 or SEQ ID NO: 14. the

As used herein, the term "isolated and purified" refers to nucleic acids from substantially cell-free material or media produced by recombinant DNA techniques, chemical precursors, or other compounds produced by chemical synthesis. An "isolated and purified" nucleic acid is also substantially free of sequences flanking its native sequence (ie, sequences located at the 5' and 3' ends of the nucleic acid). the

The term "nucleic acid" as used herein refers to a sequence of nucleotides or nucleoside monomers consisting of natural bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences or portions thereof comprising non-natural monomers that serve a similar function. The nucleic acid sequence of the present invention may be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and may contain natural bases including adenine, guanine, cytosine, thymine and uracil. Sequences may also contain, for example, adenine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyladenines, 5-halouracil, 5-halocytosine, 6-aza Uracil, 6-azacytosine, and 6-azathymine, pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thioadenine, 8-thio-alkyl Adenine, 8-hydroxyadenine and other 8-substituted adenine, 8-haloguanine, 8-aminoguanine, 8-thioguanine, 8-sulfanylguanine, 8-hydroxyguanine and other 8 -substitution of guanine, other nitrogen and deaza uracil, thymine, cytosine, adenine, or modified bases such as guanine, 5-trifluoromethyluracil and 5-trifluorocytosine. the

In one embodiment, the purified and isolated nucleic acid molecules comprise the sequences encoding proteins and peptides of the present invention, preferably SEQ ID NO: 13 or SEQ ID NO: 14, comprising:

(a) Nucleic acid sequence, wherein T can also be U;

(b) a nucleic acid sequence complementary to (a);

(c) Nucleic acid sequence homologous to (a) or (b);

(d) at least 15% of (a) to (c) that hybridize to (a) to (c) under stringent conditions

bases, preferably fragments of 20 to 30 bases, or

(e) is codon different from any of (a) to (c) due to degeneracy of the genetic code

nucleic acid sequence molecule. the

Furthermore, it is understood that the present invention includes nucleic acid sequence molecules and fragments thereof comprising nucleic acid sequences having substantial sequence homology to nucleic acid sequences encoding the proteins and peptides of the present invention. The term "sequences having substantial sequence homology" means those nucleic acid sequence sequences having slight or insignificant variations from these sequences, i.e., sequences that produce functionally equivalent proteins in substantially the same manner. These changes can contribute to positional mutations or structural modifications. the

Nucleic acid sequences having substantial sequence homology include nucleic acid sequences that are at least 80%, preferably 90% identical to nucleic acid sequences encoding proteins and peptides of the present invention. the

Another aspect of the present invention provides nucleic acid molecules and fragments thereof having at least 15 bases that are capable of hybridizing to the nucleic acid sequence molecules of the present invention under hybridization conditions, preferably stringent conditions. Conditions of appropriate stringency to promote hybridization of DNA are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following conditions can be used: 6.0x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a 2.0x SSC wash at 50°C. Stringency can be chosen according to the conditions used in the washing steps. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the washing steps can be at high stringency conditions of about 65°C. the

Correspondingly, molecules having nucleic acid sequences encoding the proteins and peptides of the present invention can be integrated into appropriate expression vectors to ensure good expression of the proteins and peptides according to methods known in the art. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression "vector suitable for transforming host cells" means that the expression vector comprises the nucleic acid sequence molecule of the present invention and the regulatory sequence operably linked to the nucleic acid sequence molecule selected according to the host cell used for expression . "Operably linked"means that the nucleic acid sequence is linked to regulatory sequences in a manner that permits expression of the nucleic acid sequence. the

The present invention thus contemplates recombinant expression vectors of the invention comprising the nucleic acid sequences of the invention or fragments thereof and the regulatory sequences necessary for the transcription and translation of the inserted protein sequences. Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, or viral genes (see, e.g., the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Selection of appropriate regulatory sequences is dependent on the chosen host cell and can be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences including translation initiation signals. In addition, depending on the host chosen and the vector used, other sequences such as origins of replication, additional DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility to the sequence may be incorporated into the expression vector. It will be appreciated that the required regulatory sequences may be provided by the native protein and/or its flanking regions. the

The recombinant expression vectors of the invention may also contain selectable markers to facilitate selection of host cells transformed or transfected with the recombinant molecules of the invention. Examples of selectable marker genes include genes encoding specific drug resistance-conferring proteins such as G418 and hygromycin, β-galactosidase, chloramphenicol acylase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of selectable marker proteins such as β-galactosidase, chloramphenicol acylase, or firefly luciferase. G418 can be selected if the selectable marker gene encodes a protein that confers antibiotic resistance, such as hygromycin resistance, on transformed cells. Cells incorporating the selectable marker gene will survive, while other cells will die. This allows visualization and detection of the expression of the recombinant expression vectors of the invention, especially the determination of the effect of mutations on expression and phenotype. It is understood that the selectable marker can be introduced on a vector independent of the nucleic acid sequence of interest. the

Recombinant expression vectors may also contain genes encoding fusion moieties that enhance expression of the recombinant protein, increase the solubility of the recombinant protein, and serve as affinity purification ligands to aid in the purification of the target recombinant protein. For example, a protease cleavage site can be added to the target recombinant protein to allow separation of the recombinant protein from the fusion portion following purification of the fusion protein. the

A recombinant expression vector can be introduced into a host cell to produce a transformed host cell. The term "transformed host cell" is intended to include prokaryotic and eukaryotic cells that have been transformed or transfected with an expression vector of the invention. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to include nucleic acid sequences (e.g., vectors) via a number of possibilities known in the art. One of the techniques is the introduction into cells. Prokaryotic cells can be transformed by, for example, click or calcium chloride-mediated transformation. Nucleic acid sequences can be introduced into mammalian cells by conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, click or microinjection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)) and other such laboratory textbooks. the

Suitable host cells include various prokaryotic and eukaryotic host cells. For example, proteins of the invention may be expressed in cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991). the

Nucleic acid sequences are "operably linked" if they are placed in such a way that they are functionally related to another nucleic acid sequence. For example, the DNA of a presequence or secretory leader sequence is operably linked to a polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. operably linked; or the position of the ribosome binding site favors translation, it is operably linked to the coding sequence. In general, "operably linked" means that the DNA sequences being linked are contiguous, and, for a secretory leader sequence, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at conventional restriction sites. If such sites are not available, synthetic oligonucleotide linkers can be used according to conventional practice. the

In some embodiments, the expression vector comprises a nucleic acid sequence encoding a modified Bouganin having a reduced number of potential T cell epitopes operably linked to an expression control sequence. In various embodiments, an expression vector comprising a nucleic acid sequence encoding a protein or peptide of the present invention, or a degenerate variant thereof, will comprise at least the RIP coding domain of said nucleic acid sequence operably linked to appropriate expression control and selection sequences . Degeneracy with respect to polynucleotides refers to the generally recognized fact that many amino acids in the genetic code are specified by more than one codon. Codon degeneracy allows 20 different amino acids to be encoded by 64 possible triplet sequences of the four different bases that make up DNA. the

The term "RIP coding domain" or "ribosome inactivating protein coding domain" as used herein means the functional domain that confers the biological activity of bouganin. the

The nucleic acid sequence molecules of the invention can also be chemically synthesized by standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis that has been fully automated in commercially available DNA synthesizers, such as peptide synthesis (see, e.g., Itakura et al., U.S. Patent 4,598,049; Caruthers et al., US Patent 4,458,066; and Itakura, US Patents 4,401,796 and 4,373,071). the

The present invention also provides a nucleic acid sequence encoding a fusion protein comprising the novel protein of the present invention and a selected protein, or a selectable marker protein. the

Another aspect of the invention is a cultured cell comprising at least one vector as described above. the

Another aspect of the present invention is a method for modified Bouganin, comprising culturing the above-mentioned cells under conditions allowing the expression of the modified Bouganin from the expression vector and purifying bouganin from the cells. the

(B) Modified Bouganin cytotoxin:

As previously mentioned, bouganin is a type I ribosome-inactivating protein (RIP) that depurates the primary ribosomal RNA of the cell leading to the termination of protein synthesis and cell death. Therefore, the modified Bouganin of the present invention can be used to prepare cytotoxins. Cytotoxins comprising modified Bouganin proteins are preferred over cytotoxins comprising non-modified Bouganin proteins because the former are less immunogenic and less likely to be destroyed by the immune system before reaching the target. the

Accordingly, the present invention also provides a cytotoxin comprising (a) a modified Bouganin protein of the present invention and (b) a target moiety attached to (a). the

The term "modified Bouganin protein of the present invention" is used for convenience of introduction, which includes any or all modified Bouganin proteins described herein, such as the modified Bouganin protein described in the above-mentioned part (A) and the accompanying drawings and examples . the

As used herein, the term "target moiety" refers to a substance, method, or technique that delivers a modified Bouganin protein to a target cell. In one embodiment the targeting moiety is an antibody. In one embodiment the targeting moiety may be a liposome. In one embodiment the liposome can be attached to an antibody. In another embodiment the targeting moiety is a protein that can be directed to a specific binding interaction with a specific target cell. Such protein moieties include various polypeptide ligands for specific cell surface receptors, thereby including many cytokines, peptide hormones and other biological response modifiers. Primary examples include eg vascular endothelial growth factor, epidermal growth factor, heregulin, interleukins, interferons, tumor necrosis factor and other protein and glycoprotein molecules. Fusion proteins of the bouganin of the present invention with these or other molecules are contemplated, depending on the protein ligand domain may comprise a modified bouganin moiety in either N-terminal or C-terminal orientation. The targeting moiety can be linked directly to the protein of the invention or via a linker. In one embodiment, the linker is a ether linker or a chemical linker. Likewise, chemical cross-linking of purified ligands to the modified Bouganin protein is also contemplated and is within the scope of the present invention. the

In a preferred embodiment, the present invention provides a cytotoxin, which comprises (a) the modified Bouganin protein of the present invention, (b) attached to (a) a ligand capable of binding to cancer cells. the

A ligand can be any molecule capable of binding to a cancer cell, including but not limited to a protein. In one embodiment, the ligand is an antibody or antibody fragment that recognizes the surface of cancer cells. the

Accordingly, the cytotoxins of the present invention are useful in the treatment of various forms of cancer such as colorectal, breast, ovarian, pancreatic, head and neck, bladder, gastrointestinal, prostate, small cell and non-small cell Lung cancer, sarcoma, glioma, T- and B-cell lymphoma. the

In one embodiment, the cancer cell binding ligand comprises an intact immunoglobulin molecule that binds to the cancer cell. If the cancer cell binding ligand is an antibody or a fragment thereof, the cytotoxin is preferably an immunotoxin. In another embodiment, the cancer cell binding ligand is a Fab, Fab', scFv, single domain antibody fragment, or dimer of Fv fragments stabilized by disulfide bonds. In another embodiment, the cancer antibody comprises a variable heavy chain, variable light chain, Fab, Fab', scFv, single domain antibody fragment, or Fv fragment stabilized by disulfide bonds. The portion of the cancer cell binding ligand may be derived from one or more species, preferably comprises a human-derived portion, most preferably fully human or humanized. Regions designed to facilitate purification and toxin coupling may also be included or added to the cancer cell binding moiety. the

In a specific embodiment, the cancer cell binding ligand recognizes Ep-CAM. Ep-CAM (Epithelial Cell Adhesion Molecule, also known as 17-1A, KSA, EGP-2, and GA733-2) is a protein found in many solid tumors, including lung, breast, ovarian, colorectal, and head and neck squamous Transmembrane proteins that are highly expressed but weakly expressed in most normal epithelial tissues. the

Accordingly, in one embodiment, the present invention provides an Ep-CAM-targeted-modified Bouganin cytotoxin comprising: (a) having a reduced propensity to activate T-cells compared to non-modified Bouganin proteins The modified Bouganin protein, (b) attached to (a) ligands (such as antibodies or antibody fragments) that can bind to Ep-CAM on cancer cells. 

In a specific embodiment, the cytotoxin comprises: (a) a modified Bouganin protein having a reduced propensity to activate T-cells compared to a non-modified Bouganin protein, (b) attached to (a) a humanized An antibody or antibody fragment capable of binding to the extracellular domain of human Ep-CAM and comprising complementarity determining region (CDR) sequences from the Moc-31 antibody. the

Suitable Ep-CAM-targeted-modified Bouganins according to the invention include, without limitation, VB6-845 and variants thereof, other cytotoxins including other single or double chain immunoglobulins that selectively bind Ep-CAM, and variants thereof. body. The term "VB6-845" as used herein means a cytotoxin comprising a Fab of an anti-Ep-CAM scFv antibody linked to a modified form of bouganin, Bou 156 (SEQ ID NO: 13). The amino acid sequence and nucleic acid of VB6-845 are connected and shown in Figure 3B (respectively SEQ ID NO: 16 and SEQ ID NO: 15). the

In another embodiment, the cancer cell binding ligand recognizes a tumor-associated antigen that is specifically found on neoplastic cells but not on normal cells. In a preferred embodiment, the ligand is an antibody to a tumor-associated antigen. Anti-tumor-associated antigen antibodies specifically recognize cancer cells derived from various cancers but not normal non-cancerous cells. the

Thus in another embodiment, the present invention provides a cytotoxin comprising: (a) a modified Bouganin protein having a reduced propensity to activate T-cells compared to a non-modified Bouganin protein; (b) attached to Ligands (such as antibodies or antibody fragments) capable of binding to tumor-associated antigens on cancer cells in (a). the

Suitable tumor-associated antigen-targeting modified Bouganins according to the invention include, without limitation, VB6-011 and variants thereof, other cytotoxins or variants thereof that selectively bind to tumor-associated antigens, other single- or double-chain immunoglobulins body. The term "VB6-011" as used herein means a cytotoxin comprising the Fab of the H11 human monoclonal antibody linked to a modified form of bouganin, Bou 156 (SEQ ID NO: 13). The H11 antibody was obtained by fusing peripheral blood lymphocytes of a 64-year-old male cancer patient with a human myeloma cell line to make a hybridoma. The hybridoma NBGM1/H11 produces IgMk, which is reengineered into a Fab antibody form to form VB6-011 (see US 6207153 or the preparation of H11 antibody-secreting hybridoma in WO 97/44461 for details). The amino acid sequence and nucleic acid sequence of VB6-011 are shown in accompanying drawing 15 (respectively being SEQ ID NO: 28 and SEQ ID NO: 27). the

In a specific non-limiting embodiment, the cytotoxin comprises VB6-845 (Fig. 3B, SEQ ID No. 16) or VB6-011 (Fig. 15, SEQ ID NO: 28). In other non-limiting embodiments, the cytotoxin comprises a variant of VB6-845 or VB6-011. the

The VB6-845 variant also binds to the Ep-CAM epitope or binds to an Ep-CAM epitope that is substantially similar to that of VB6-845, and this variant can competitively inhibit the binding of VB6-845 to Ep-CAM under physiological conditions At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. VB6-845 variants comprise the same modified Bouganin as VB6-845, or may comprise a different modified Bouganin according to the invention. In another non-limiting embodiment, the cytotoxin comprises an Ep-CAM-binding moiety comprising a MOC31 variable region or a variant thereof. In yet another embodiment, the cytotoxin comprises an Ep-CAM-binding moiety comprising 4D5MOCB[C2] or a portion thereof. The binding of any of these cytotoxins to Ep-CAM can be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40% by competing with reference MOC31 or 4D5MOCB antibodies under physiological conditions %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. the

The VB6-011 variant binds to the same tumor-associated epitope as VB6-011 or binds to a tumor-associated epitope that is substantially similar to that bound by VB6-011. Under physiological conditions, the variant can competitively inhibit the binding of VB6-011 and Tumor-associated antigen binding of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, or 95%. VB6-011 variants may comprise the same modified Bouganin as VB6-011, or may comprise a different modified Bouganin of the invention. In another non-limiting embodiment, the cytotoxin comprises a tumor-associated antigen-binding moiety comprising a H11 monoclonal antibody, an H11 antigen-binding fragment, or a variant thereof. The binding of any of these cytotoxins to VB6-011 can be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% by competition with the reference H11 antibody under physiological conditions %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. the

In a preferred embodiment, the binding affinity of the Ep-CAM-binding moiety or the tumor-associated antigen-binding moiety is at least four orders of magnitude lower than the binding affinity of VB6-845 or VB6-011, preferably at least three orders of magnitude, as determined according to standard experimental techniques. order of magnitude, more preferably two orders of magnitude lower. In a non-limiting embodiment, under physiological conditions, the Ep-CAM-binding moiety can competitively block known anti-Ep-CAM antibodies, such as but not limited to or MT201, binding to Ep-CAM at least 0.1%, 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% %, 70%, 75%, 80%, 85%, 90%, or 95%. In a non-limiting embodiment, under physiological conditions, the tumor-associated antigen binding portion can competitively block the binding of known anti-tumor-associated antigen antibodies, such as but not limited to H11, to the tumor-associated antigen by at least 0.1%, 1 %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Those skilled in the art will appreciate that residues that determine specificity can be identified. The term "specificity determining residues", also referred to as "SDR", refers to the residues forming part of the antibody determinants of an antibody, especially the CDR residues, a single substitution with alanine independently of any other mutation can The binding affinity of the antibody for the epitope is reduced by at least 10-fold, preferably by at least 100-fold, more preferably by at least 1000-fold. The loss of affinity emphasizes the importance of the residue for the ability of the antibody to bind the epitope. See, eg, 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): 441 . the

The effect of single or multiple mutations on binding activity, especially on binding affinity, can be evaluated simultaneously to assess the importance of a particular series of amino acids for the binding interaction (eg, contribution of light or heavy chain CDR2 to binding). The effect of an amino acid mutation can also be assessed to assess the contribution of individual amino acids when evaluated individually. Such assessment can be done, 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 mutation (see, e.g., Cunningham and Wells, 1989, "High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis," Science 244:1081-1085; Bass et al., 1991, "A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor," ProcNatl Acad Sci. USA 88: 4498-4502) to complete. In the alanine scanning mutagenesis technique, single alanine mutations are introduced at multiple residues in the molecule, and the resulting molecules are tested for biological activity to identify key amino acid residues for the molecule's activity. the

Physical analysis of structures determined from, for example, nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined 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-4at 2.25 A resolution", FEBS Lett.309:59-64) to identify ligand-receptor or other biological interaction sites point. In addition, the importance of a particular single amino acid, or amino acid sequence, can be assessed by comparing the amino acid sequences of related polypeptides or similar binding sites. the

Furthermore, those skilled in the art will appreciate that an increase in avidity can compensate for a decrease in affinity. The affinity of the cytotoxin for the cancer cell receptor is a measure of the binding strength of the Ep-CAM-binding moiety to Ep-CAM. The functional binding strength between Ep-CAM and the Ep-CAM-binding moiety represents the total strength of all affinity bonds, thus individual components can bind with relatively low affinity, but multimers of such components can prove effective. biological effects. Indeed, multiple binding of Ep-CAM-binding sites and Ep-CAM epitopes may be more demonstrative than additional biological effects, ie multivalency has the advantage that the equilibrium constant may be many orders of magnitude. the

Similarly, the affinity of a cytotoxin for a cancer cell receptor is a measure of the strength of the binding of the tumor-associated antigen-binding moiety to the tumor-associated antigen, which may have multiple binding sites. The functional binding strength of tumor-associated antigens and tumor-associated antigen-binding moieties represents the total strength of all affinity bonds, thus individual components may bind with low affinity, but multimers of such components may demonstrate potent biological effects. In fact, multiple interactions of tumor-associated antigen-binding sites with tumor-associated antigen epitopes are more telling than additive biological activities, ie, multivalency has the advantage that the equilibrium constant may be many orders of magnitude. the

In one non-limiting embodiment, the Ep-CAM-binding moiety has a substantially similar structure to 4D5MocB. This substantially similar structure can be characterized with reference to an epitope map reflecting the binding site of the Ep-CAM-binding portion of the cytotoxin to the Ep-CAM molecule. In another non-limiting embodiment, an epitope map of the tumor-associated antigen-binding portion can be generated, and this substantially similar structure can be referenced to an epitope reflecting the binding site of the cytotoxic tumor-associated antigen-binding portion to the tumor-associated antigen molecule. Figure to describe the features. the

The cytotoxins of the present invention can be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid phase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964)) or synthesis in homogeneous liquid (Houbenweyl, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15I and II, Thieme, Stuttgart (1987)). the

In one embodiment, both the cancer binding ligand and the modified Bouganin are proteins and can be coupled using techniques well known in the art. There are hundreds of crosslinkers that can couple these two proteins. (See e.g. "Chemistry of Protein Conjugation and Crosslinking". 1991, Shans Wong, CRC Press, Ann Arbor). Crosslinkers can generally be selected based on available or inserted reactive functional groups on the ligand or toxin. Furthermore, if there are no reactive groups, photoactivatable crosslinkers can be used. In certain cases it may be desirable to include a spacer between the ligand and the toxin. Cross-linking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyl adipimidate, and Bis (diazobenzidine) and heterobifunctional agents: mMaleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-m Maleimidobenzoyl-N-Hydroxysuccinimide. the

Ligand-bouganin toxin fusion proteins can also be prepared using recombinant DNA techniques. In this case, the DNA sequence encoding the cancer-binding ligand is fused to the DNA sequence encoding the modified Bouganin protein, resulting in a chimeric DNA molecule. The chimeric DNA sequence is transfected into a host cell capable of expressing the ligand-bouganin fusion protein. Fusion proteins can be recovered and purified from cell culture using techniques known in the art. the

Antibodies specific for cell surface proteins such as Ep-CAM and tumor-associated antigens can be prepared using conventional techniques. Mammals, such as mice, hamsters or rabbits, can be immunized with an immunogenic form of the peptide that elicits an antibody response in the mammal. Techniques for rendering the peptide immunogenic include conjugation to carriers or other techniques well known in the art. For example, peptides can be administered in the presence of an adjuvant. The progress of immunization can be monitored by detecting antibody titers in plasma or serum. Antibody levels are assessed by standard ELISA or other immunoassay procedures and the immunogen as antigen. After immunization, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. the

To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion methods, thereby immortalizing these cells and giving rise to hybridoma cells. Such techniques are known in the art, (e.g. hybridoma technology originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) and such as human B-cell hybridoma technology (Kozbor et al., Immunol. Today 4 : 72 (1983)), the EBV-hybridoma technique for producing human monoclonal antibodies (Cole et al., Monoclonal Antibodies in Cancer Therapy Allen R., Bliss, Inc., pages 77-96 (1985)), and screening combination antibodies Other techniques such as libraries (Huse et al., Science 246:1275 (1989)). To generate antibodies that specifically react with peptides, hybridoma cells can be screened immunochemically and monoclonal antibodies isolated.

As used herein, the term "antibody" is intended to include monoclonal and polyclonal antibodies, antibody fragments (such as Fab and F(ab')2, and single-chain antibodies (scFv) that are also capable of specifically reacting with a cell surface component. )), and chimeric antibodies. Antibodies can be fragmented using conventional techniques, and available fragments can be screened in the same manner as described above. For example, F(ab')2 fragments can be produced by treating the antibody with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bonds to generate Fab' fragments. Single-chain antibodies bind the antigen-binding region of the antibody on a stably folded polypeptide chain. Single chain antibodies can be produced by recombinant techniques. the

Chimeric antibody derivatives, ie, antibody molecules combined with non-human animal variable regions and human constant regions are also contemplated as being within the scope of the present invention. A chimeric antibody molecule can include, for example, an antigen binding domain from an antibody of mouse, rat or other species and a human constant region. Chimeric antibodies comprising immunoglobulin variable regions that recognize cell surface antigens can be prepared conventionally (see, e.g., Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81:6851 (1985); Takeda et al., Nature 314:452 (1985), Cabilly et al., US Patent 4816567; Boss et al., US Patent 4816397; Tanaguchi et al., European Patent 171496; European Patent 173,494, Hong Kong Patent 2177096B). Chimeric antibodies are expected to be less immunogenic in humans than corresponding non-chimeric antibodies. Chimeric antibodies can be stabilized by the method described in Pluckthun et al., WO 00/61635. the

Monoclonal or chimeric antibodies specifically reactive with cell surface components can be further humanized by generating human constant region chimeras in which portions of the variable region, especially the conserved frame regions of the antigen-binding region, are of human origin, And only the hypervariable region is non-human. Such immunoglobulin molecules can be obtained by methods known in the art (for example, Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312 (1983); ); Olsson et al., Meth. Enzymol., 92:3-16 (1982) and PCT publication WO92/06193 or EP 239,400) preparation. Humanized antibodies can also be prepared commercially (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.). In addition, monoclonal or chimeric antibodies may be less immunogenic by reducing the number of potential T-cell epitopes on them that specifically react with cell surface components. the

Specific antibodies, or antibody fragments or portions thereof, that specifically react with cell surface components can also be produced by screening expression libraries of genes encoding immunoglobulins and expressed in bacteria bearing cell surface components. For example, complete Fab fragments, VH regions and Fv regions can be expressed in bacteria using phage expression libraries (see e.g. 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, SCID-hu mice, such as the model developed by Genpharm, can be used to generate antibodies and fragments thereof. the

In all cases where the modified Bouganin protein will be fused to an antibody sequence, it is best to use one in which T cell epitopes or sequences capable of binding MHC class II molecules or stimulating T cells or co-binding T cells with class II MHC molecules have been removed antibody sequence. the

In yet another embodiment of the present invention, the modified Bouganin protein can be linked to a protein that is not an antibody protein but can still be directed to specifically bind to a specific target cell. Such protein moieties include various polypeptide ligands presenting specific cell surface receptors, and thus include many cytokines, peptide and polypeptide hormones and other biological response modifiers. Prime examples include proteins such as vascular endothelial growth factor, epidermal growth factor, heregulin, interleukins, interferons, tumor necrosis factor and other protein and glycoprotein molecules. Fusion proteins of these and other molecules with the bouganin of the invention are also contemplated, which may contain a modified bouganin moiety in the N-terminal or C-terminal direction of the ligand domain of the protein. Likewise, chemical cross-linking of purified ligands to modified Bouganin proteins is also contemplated and falls within the scope of the present invention. the

In another embodiment, the modified Bouganin protein of the present invention can be used as a complex comprising a water-soluble polymer such as hydroxypropylmethacrylamide or other polymers, wherein the modified Bouganin protein is co-polymerized with the polymer. interact with polymers through valent bonding or non-covalent bonding. Such an embodiment may also include an antigen binding domain, such as an antibody or antibody fragment, associated with the polymer bouganin complex. the

(C) Use of Cytotoxins

The modified Bouganin protein of the present invention can be used to specifically inhibit or destroy mammalian cells infected with cancer. Allowing RIP to enter cells and effectively killing cancer cells is an advantage of the cytotoxins of the present invention with lower immunogenicity. Therefore, this cytotoxin can be used to specifically target cancer cells. Once inside cancer cells, bouganin depurinates the primary ribosomal RNA, thereby destroying the ribosome and leading to a halt in protein synthesis and cell death. the

Accordingly, in one embodiment, the invention provides a method of inhibiting or destroying cancer cells comprising administering a cytotoxin of the invention to an animal in need thereof. The invention also includes the use of the cytotoxins of the invention for inhibiting or destroying cancer cells. The present invention also includes the use of the cytotoxin of the present invention in the preparation of drugs for inhibiting or destroying cancer cells. The type of cancer cells inhibited or destroyed by the cytotoxin can be determined by the antigen specificity of the antibody portion thereof. the

In another embodiment, the present invention provides a method of inhibiting or destroying cancer cells, comprising the steps of preparing the cytotoxin of the present invention and administering the cytotoxin to the cells. The cancer may be any type of cancer including, but not limited to, colorectal, breast, ovarian, pancreatic, head and neck, bladder, liver, kidney, melanoma, gastrointestinal, prostate, small cell, and Non-small cell lung cancer, sarcoma, glioma, and T- and B-cell lymphoma. the

The ability of the cytotoxins of the invention to selectively inhibit or destroy cancer cells can be readily tested in vitro using animal cancer cell lines. The selective inhibitory effect of the cytotoxins of the invention can be determined, for example, by demonstrating selective inhibition of proliferation of cancer cells. the

Toxicity can be measured in terms of cell viability, eg, the viability of normal and cancerous cell cultures exposed to a cytotoxin can be compared. Cell viability can be assessed using known techniques, such as orcin blue exclusion assays. the

In another example, many models are available for testing the cytotoxicity of cytotoxins. Thompson, E.W et al. (Breast Cancer Res. Treatment 31: 357-370 (1994)) describe a method for reconstituting the basement membrane (collagen, layer fibronectin, Matrigel or gelatin) to determine in vitro models of human breast cancer cell invasion. Other applicable cancer cell models include cultured ovarian adenoma cells (Young, T.N. et al., Gyneco.L.Oncol. 62:89-99 (1996); Moore, D.H. et al., Gyneco.L.Oncol.65 : 78-82 (1997)), human follicular thyroid carcinoma cells (Demeure, M.J. et al., World J.Surg. 16: 770-776 (1992)), human melanoma (A-2058) and fibrosarcoma ( HT-1080) cell line (Mackay, A.R. et al. Lab.Invest. 70:781-783 (1994)), and lung squamous (HS-24) and adenocarcinoma (SB-3) cell lines (Spiess, E. et al., J. Histochem. Cytochem. 42:917-929 (1994)). In vivo test system including tumor implantation and determination of tumor growth and metastasis in athymic mice (Thompson, E.W et al., Breast Cancer Res. Treatment 31:357-370 (1994); Shi, Y.E et al., Cancer Res.53 : 1409-1415 (1993)) have also been described. the

The invention also relates to methods of treating cancer comprising administering to an animal in need thereof an effective amount of one or more cytotoxins of the invention. The invention includes the use of the cytotoxins of the invention to treat cancer. The present invention also includes the use of the cytotoxin of the present invention in the preparation of medicines for treating cancer. the

The term "animal" includes all members of the animal kingdom, including humans. the

The term "treating cancer" refers to inhibition of cancer cell replication, cancer spread (metastasis), tumor growth, reduction of cancer cell number and tumor growth, reduction of cancer malignancy, or amelioration of cancer-related symptoms. the

In a preferred embodiment, the animal is a human. In another embodiment, the cancer is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer, liver cancer, kidney cancer, melanoma, gastrointestinal cancer, prostate cancer, small cell and non- Small cell lung cancer, sarcoma, glioma, and T- and B-cell lymphoma. the

The clinical outcome of cancer treatment with the cytotoxins of the invention is readily ascertainable by one skilled in the art, eg, a physician. For example, standard clinical assays for measuring clinical markers of cancer can be strong indicators of treatment efficacy. Such experiments may include, without limitation, physical examination, performance scales, disease markers, 12-lead ECG, tumor measurements, biopsy, cytology, cytology, calculation of tumor maximum diameter, radiography, digital imaging of tumors, Vital signs, weight, adverse event records, infectious period assessment, concomitant medication assessment, pain assessment, blood or serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis. In addition, the synergistic effect of combination therapy comprising a cytotoxin and another cancer therapeutic agent can be determined by comparative studies with untreated patients. the

Removal of malignancy can be assessed using criteria accepted by those skilled 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 Institution of Canada," Fe J Natl ;92(3):205-16. the

The effective dosage of a particular cytotoxin construct depends on various factors, including cancer type, tumor size, cancer stage, toxicity of the cytotoxin to the patient, target specificity for cancer cells, and age, weight and health of the patient. the

Cytotoxins comprising modified Bouganin can be administered by i.v. perfusion at minute to time intervals depending on the dose and concentration of the cytotoxin in the perfusate. the

In one embodiment, the cytotoxin is infused at 3 hour intervals. the

In one embodiment, the effective dosage range of the cytotoxin administered i.v. by infusion is about 1 to 100 mg/kg/dose. In other embodiments, the dosage range is about 2 to 5 mg/kg/dose. In particular embodiments, the dosage may be at least about 2, 4, 8, 13, 20, 28, 40, 50 mg/kg/dose. the

In one embodiment, a single dose is administered about weekly for about 1, 2, 3, 4, 5, or 6 weeks. Single administrations may be administered on consecutive weeks, or alternatively, one or more weeks may be skipped. After this cycle, the next cycle can begin about 1, 2, 4, 6 or 12 weeks later. The treatment regimen may comprise 1, 2, 3, 4, 5, 6 or more cycles with about 1, 2, 4, 6 or 12 weeks between cycles. the

In another embodiment, the administration is monthly for about 1, 2, 3, 4, 5 or 6 consecutive months. After this cycle, the next cycle can begin about 1, 2, 4, 6, or 12 months later. The treatment regimen includes 1, 2, 3, 4, 5, 6 or more cycles with about 1, 2, 4, 6 or 12 months between cycles. the

In certain specific non-limiting embodiments, the effective dose of the cytotoxin is between about 1 and 50 mg/kg/tumor/day, wherein the patient is administered once daily. About a single dose per day (optionally skipping one or more days), for about 1, 2, 3, 4, 5, 6 or 7 consecutive days. After this cycle, the next cycle begins about 1, 2, 3, 4, 5 or 6 weeks later. The treatment regimen may comprise 1, 2, 3, 4, 5, 6 or more cycles with about 1, 2, 3, 4, 5 or 6 weeks between cycles. the

The injection volume is preferably at least an effective amount, which is an appropriate amount for the type and/or location of the tumor. The maximum volume for a single administration is between about 25% and 75% of the tumor volume, eg, about one quarter, one third, or three quarters of the estimated target tumor volume. In a specific non-limiting embodiment, the maximum injection volume for a single administration is about 30% of the tumor volume. the

In another embodiment, a solution comprising 1 to 10 mg cytotoxin/mL is perfused at a rate of 100 cc per hour for three hours. Cytotoxins can be diluted in suitable physiologically compatible solutions. the

The effective dose of another cancer therapeutic agent administered with the cytotoxin in the circulation also varies depending on the mode of administration. One or more cancer therapeutic agents can be delivered into the tumor, or administered by other means. Typically, chemotherapeutic agents are administered systemically. Standard dosages and treatment regimens are known in the art (see, eg, the latest editions of the Merck Index and the Physician's Desk Reference; NCCN Practice Guidelines in Oncology)). the

Cytotoxic combination therapy can sensitize a cancer or tumor to the administration of other cancer therapeutic agents. Accordingly, the present invention contemplates combination therapy for the prophylaxis, treatment and/or prevention of cancer recurrence comprising administering an effective amount of a cytotoxin before, after, or simultaneously with administration of a reduced dose of a cancer therapeutic agent. For example, initial treatment with cytotoxins can increase the sensitivity of a cancer or tumor to subsequent administration of cancer chemotherapeutic agents. This dose is close to, or below, the lower range of standard doses used when the cancer therapeutic is administered alone, or in the absence of cytotoxins. When administered concurrently, the cytotoxin can be administered separately from the cancer therapeutic agent, alternatively, by a different mode of administration. the

In another embodiment, the cytotoxin is administered in combination with at least one other immunotherapeutic agent. the

In another embodiment, the cytotoxin is administered in combination with a radiation therapy regimen. The therapy may also include surgery and/or chemotherapy. For example, cytotoxins can be administered in combination with radiation therapy and cisplatin (Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), and/or paclitaxel (Taxol). Treatment with cytotoxicity also allows the use of lower radiation doses and/or less frequent radiation treatments, which can, for example, reduce the incidence of severe sore throat that impedes swallowing function and thus potentially leads to weight loss or dehydration. the

In another embodiment, the cytotoxin is administered in combination with one or more cytokines including, without limitation, lymphokines, tumor necrosis factor, tumor necrosis factor-like cytokines, lymphotoxins, interferons, macrophages Cellular inflammatory proteins, granulocyte-monocyte colony-stimulating factor, interleukins (including but not limited to interleukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin-15, interleukin-18), and variants thereof, including Its pharmaceutically acceptable salt. the

In yet another embodiment, the cytotoxin is administered in combination with a cancer vaccine, including but not limited to autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin , BCG live vaccine, melanoma cell lineage proteins, and mutated tumor tumor-specific antigens. the

In yet another embodiment, the cytotoxin is administered in combination with hormone therapy. Hormone therapy agents include, without limitation, hormone agonists, hormone antagonists (e.g., flunitramide, tamoxifen, leuprolide acetate (LUPRON)) and steroids (e.g., dexamethasone, retinoids, betamethasone, hydrocortisone cortisone, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, testosterone, progesterone). the

In yet another embodiment, the cytotoxin is administered in combination with a gene therapy regimen to treat or prevent cancer. the

In yet another embodiment, the Ep-CAM-targeted cytotoxin is administered in combination with one or more agents that increase Ep-CAM expression in the tumor cells of interest. Preferably, Ep-CAM expression is increased such that a greater number of Ep-CAM molecules are expressed on the tumor cell surface. For example, the agent can inhibit endocytosis of normally circulating Ep-CAM antigen. Such combination therapy may improve the clinical effect of Ep-CAM-targeted cytotoxins alone, or with other cancer therapeutics or radiation therapy. In a specific non-limiting embodiment, the agent that increases the expression of Ep-CAM in tumor cells is vinorelbine tartrate (Navelbine) and/or paclitax (Taxol). See, eg, Thurmond et al., 2003, "Adenocarcinoma cell exposed in vitro to Navelbine or Taxol increase Ep-CAM expression through novel mechanism." Cancer Immunol Immunother. Jul;52(7):429-37. the

Combination therapy can thus increase the sensitivity of the cancer or tumor to the administered cytotoxin and/or other cancer therapeutic agent. In this way, it is possible that shorter treatment cycles would be required thereby reducing toxic events. Accordingly, the present invention provides a method of treatment or prevention comprising administering to a patient in need thereof an effective amount of a cytotoxin and at least one other cancer therapeutic agent for a short therapeutic cycle. Cycle duration can vary depending on the particular cancer therapeutic being used. The invention also contemplates continuous or discontinuous dosing, or daily dosing in divided doses. Those skilled in the art will understand the appropriate duration of circulation for a particular cancer therapeutic, and the present invention contemplates ongoing assessment of the optimal duration of treatment for each cancer therapeutic. Specific guidelines are known to those skilled 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 Fe 2 Natlst Cancer ;92(3):205-16. the

Alternatively, longer treatment cycles may be required. Accordingly, cycle durations may vary between about 10 to 56, 12 to 48, 14 to 28, 16 to 24, or 18 to 20 days. Cycle duration can vary depending on the cancer therapeutic being used. the

The present invention contemplates at least one cycle, preferably more than one cycle, during which a cancer therapeutic agent or series of therapeutic agents are administered. Appropriate total number of cycles and cycle intervals will be understood by those skilled in the art. The number of cycles can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 cycles. The cycle interval may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. The present invention contemplates continuous assessment of the optimal treatment schedule for each cytotoxin and other cancer therapeutic agent. the

In another embodiment, there is provided a method of preparing a medicament for treating cancerous mammals, comprising the steps of: identifying bouganin T-cell epitopes with a reduced propensity to activate T-cells; preparing T-cell epitopes with one or more T-cells; - A cytotoxin of the invention for a cellular epitope and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient. the

The present invention also provides a pharmaceutical composition for treating a cancer-affected mammal comprising the cytotoxin of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. the

The cytotoxins of the invention can be formulated into pharmaceutical compositions for administration to a subject in biocompatible form suitable for in vivo administration. "Biocompatible form suitable for in vivo administration" means the form of the administered substance in which the therapeutic effects outweigh any toxic effects. These substances can be administered to living organisms including humans and animals. Administration of a therapeutically active amount of a pharmaceutical composition of the invention is defined as an effective amount at dosages and for durations necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, body weight of the individual, and the ability of the antibody to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses per day may be administered or the dose may be appropriately reduced as indicated by the exigencies of the therapeutic situation. the

The active substance may be administered by convenient means such as injection (subcutaneous, intravenous, intramuscular, etc.), oral administration, inhalation, transdermal administration (eg, topical cream or ointment, etc.), or suppositories. Depending on the route of administration, the active substance may be coated with a substance to protect the compound from enzymes, acids, and other natural conditions that would otherwise inactivate the compound. the

The compositions described herein can be prepared by known methods of preparing pharmaceutically acceptable compositions that can be administered to a subject, whereby an effective amount of the active material is combined in admixture with a pharmaceutically acceptable carrier. Suitable vectors are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). In this regard, the composition includes, although not specifically limited to, substances contained together with one or more pharmaceutically acceptable carriers, diluents, in a buffer having an appropriate pH and isotonicity with physiological fluids. the

The pharmaceutical composition is useful for treating cancer in animals including mammals, preferably humans. These compositions are expected to be especially suitable for the treatment of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer, lung cancer, renal cancer, melanoma, gastrointestinal cancer, prostate cancer, small cell and non-small cell lung cancer , sarcomas, gliomas, and T- and B-cell lymphomas. The dose and type of cytotoxin administered depends on various factors that are readily monitored in the human recipient. Such factors include the pathology and severity (grade and stage) of the neoplasia. the

Pharmaceutical compositions suitable for direct administration include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injection solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats, and The blood of the body is a substantially isotonic solute. Other ingredients that may be present in such compositions include, for example, water, ethanol, polyols, glycerol, and vegetable oils. Extemporaneous injectable solutions and suspensions can be prepared from sterile powders, granules or tablets. Cytotoxins may be applied, but not limited to, in the form of lyophilized powders for reconstitution with sterile water or saline solution prior to administration to the patient. the

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include substantially chemically inert 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 solution, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride ( DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain the therapeutically effective compound, together with an appropriate amount of carrier, so that the form is administered directly to the patient. the

In another embodiment, a pharmaceutical composition comprises a cytotoxin and one or more additional cancer therapeutic agents, optionally contained in a pharmaceutically acceptable carrier. the

The compositions may be in the form of pharmaceutically acceptable salts including, without limitation, those formed with free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, sorrel, tartaric acid, and the like, those formed with free amino groups such as those derived from sodium, potassium, amine , Calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylarninoethanol, histidine, procaine and other free carboxyl salts. the

The present invention relates to modified Bouganin, compositions comprising such modified Bouganin protein or fragments of modified Bouganin protein and related compositions shall be deemed to fall within the scope of the present invention. A relevant example of this is the development of a peptide-mediated tolerance-inducing strategy in which one or more of the disclosed peptides is administered to a patient for immunotherapeutic purposes. Correspondingly, a synthetic peptide molecule, such as one of various synthetic peptides comprising all or part of any of the above-mentioned epitope regions R1-R3. Such peptides are considered as embodiments of the present invention. the

Yet another aspect of the invention relates to methods of treating humans with modified Bouganin compositions. For administration to an individual, any modified composition should be made preferably at least 80% pure and free of pyrogens and other contaminants. the

The invention also provides kits comprising an effective amount of a cytotoxin and, optionally, one or more other cancer therapeutic agents, and instructions for treating cancer therewith. the

(D) T-cell epitope peptide

Another embodiment of the invention is a T-cell epitope peptide. In this embodiment, the T-cell epitope peptide is capable of eliciting a stimulation index greater than 1.8, more preferably 2.0, in a T-cell assay. The T-cell epitope peptides of the present invention are capable of binding to MHC class II. the

In one embodiment of the invention, the T-cell epitope peptide comprises at least 9 contiguous amino acid residues of any of the sequences of R1, R2 or R3 (above). In another embodiment, the T-cell epitope peptide sequence has greater than 90% amino acid identity to any of the peptide sequences R1, R2 or R3; more preferably the T-cell epitope peptide and the peptide sequences R1, R2 or any of R3 have greater than 80% amino acid identity. the

The term "peptide" as used herein is a compound comprising two or more amino acids. These amino acids are linked by peptide bonds (as defined below). Twenty different natural amino acids are involved in the biosynthesis of peptides, and any number of these amino acids can be linked in any order to form a peptide chain or peptide ring. The naturally occurring amino acids involved in the biosynthesis of peptides are all in the L-configuration. Synthetic peptides can be prepared by conventional methods using L-amino acids, D-amino acids, or various combinations of amino acids in two different configurations. Short peptides, eg, having less than ten amino acid units, are often referred to as "oligopeptides". Other peptides comprise a large number of amino acid residues, eg, as many as 100 or more, and are referred to as "polypeptides". A "polypeptide" is conventionally considered to be any peptide comprising three or more amino acids, while an "oligopeptide" is generally considered to be a special type of "short". Thus, as used herein, it is understood that any reference to "polypeptide" includes oligopeptides. Furthermore, any reference to "peptide" includes polypeptides, oligopeptides, and proteins. Various arrangements of amino acids form different polypeptides and proteins. The number of polypeptides that can be formed—and thus the number of different proteins that can be formed—is practically unlimited. the

Another embodiment of the present invention is the use of the T-cell epitope peptide of the present invention to prepare the modified Bouganin protein and the modified T-cell epitope peptide of the present invention. the

Yet another embodiment of the present invention is a modified T-cell epitope peptide that has been modified such that the modified T-cell epitope peptide has a reduced ability to activate human T cells compared to a non-modified T-cell epitope peptide Propensity. In one embodiment, the improved T-cell epitope peptide of the invention comprises a peptide such that the epitope peptide elicits a lower stimulation rate when tested in a T-cell assay compared to a non-modified T-cell epitope peptide. index modification. the

In one embodiment the improved T-cell epitope peptide of the present invention has the following sequence:

AKX1DKX2LX3LGVX4KL

Wherein at least one of X1, X2, X3, and X4 is modified from a non-modified sequence, as follows:

X1 is T or A or Q;

X2 is G or A;

X3 is Q or G; and

X4 is N or D or T or A or R or Q or E or G or H or K or S (SEQ ID NO: 8). the

In another embodiment the improved T-cell epitope peptide of the present invention has the following sequence:

LGVX4KLEFSIEAIHG

Wherein X4 is N or D or T or A or R or Q or E or G or H or K or S (SEQID NO:9). the

In another embodiment the improved T-cell epitope peptide of the present invention has the following sequence:

NGQEX5AKFFLIVIQM

Wherein X5 is Q or A (SEQ ID NO: 10). the

The present invention also provides nucleic acid sequences encoding T-cell epitope peptides or improved T-cell epitope peptides of the present invention. the

The following Figures, Sequence Listing and Examples are provided to aid in the understanding of the present invention. It will be appreciated that modifications may be made to the methods presented without departing from the spirit of the invention. the

The following non-limiting examples are illustrative of the invention:

Example 1: Method for mapping epitopes in bouganin using a naive human T-cell proliferation assay

Peptides covering the mature Bouganin protein sequence were synthesized as described by Den Hartog et al. [ibid]. Each peptide was 15 amino acids in length, and successive peptides overlapped by 12 residues. The sequences of these peptides and their numbers are shown in Table 1. the

These peptides were used in T-cell proliferation experiments with PBMCs (peripheral blood mononuclear cells) from naive donors (ie no known bouganin sensitization). Twenty donor PBMCs were pooled for optimal MHC class II allotype coverage. Allotype coverage was over 85%. HLA-DR allotypes are shown in Table 2. the

PBMCs were stimulated with each individual peptide in triplicate cultures for 7 days before proliferation was measured by 3H-Thymidine (3H-Thy) incorporation. All peptides were tested at two different concentrations (1 μM and 5 μM). Stimulation index (S.I.) was calculated as 3H incorporation divided by 3H incorporation in sham-stimulated control cells. the

Human blood buffy coats kept for less than 12 hours were obtained from the National Blood Service (Addenbrooks Hospital, Cambridge, UK). Ficoll-paque was obtained from Amersham Pharmacia Biotech (Amersham, UK). Serum-free AIM V medium Gibco-BRL containing L-glutamic acid, 50 μg/ml streptomycin, 10 μg/ml gentamycin and 0.1% human serum albumin for culturing primary human lymphocytes (Paisley, UK ). Synthetic peptides were obtained from Eurosequence (Groningen, The Netherlands) and Babraham Technix (Cambridge, UK). the

Red blood cells and white blood cells are separated from plasma and platelets by gentle centrifugation of the buffy coat. The top phase (containing plasma and platelets) was removed and discarded. Red and white blood cells were diluted 1:1 in phosphate-buffered saline (PBS) before being plated onto 15 ml ficoll-paque (Amersham Pharmacia, Amersham UK). PBMCs were harvested from the surface of serum+PBS/ficoll paque by centrifugation according to the manufacturer's recommended conditions. PBMC were mixed with PBS (1:1) and collected by centrifugation. Remove and discard the supernatant and resuspend the PBMC pellets in 50 ml PBS. Cells were centrifuged again and the PBS supernatant was discarded. Cells were resuspended in 50 ml of AIM V medium at which point they were counted and viability assessed by orcin blue dye exclusion. Collect the cells by centrifugation again and discard the supernatant. Resuspend cells and store at 3×10 ⁷ per milliliter. The preservation medium was 90% (v/v) heat-inactivated AB human serum (Sigma, Poole, UK) and 10% (v/v) DMSO (Sigma, Poole, UK). Cells were transferred to a regulated freezer (Sigma) and placed at -70°C overnight. When required for use, thaw cells quickly in a 37°C water bath before transferring them to 10 ml of pre-warmed AIM V medium.

Stimulate PBMCs at a density of 2 × 105 per well in 96-well flat-bottomed plates with protein and peptide antigens. PBMCs were cultured at 37°C for 7 days before being pulsed with 3H-Thy (Amersham-Pharmacia, Amersham, UK). In each donor experiment, two control peptides designated C-32 and C-49, which had previously been shown to be immunogenic, and a potential whole protein non-recall antigen keyhole limpet hemocyanin (KLH) were used. C-32 = sequence PKYVKQNTLKLAT from residues 307-319 of hemagglutinin (SEQ ID NO: 127). C-49=Sequence KVVDQIKKISKPVQH (SEQ ID NO: 128) from Chlamydia HSP60. the

Peptides were dissolved in DMSO to a final concentration of 10 mM, after which these stocks were diluted 500-fold in AIM V medium (final concentration 20 μM). Peptides were added to flat bottom 96-well plates to final concentrations of 1 and 5 μM in a volume of 100 μl. Viability of molten PBMCs was assessed by tosmol blue stain exclusion, after which cells were resuspended to a density of 2 x 106 cells/ml and 100 μl (2 x 105 PBMCs/well) were transferred to each well containing the peptide. Triplicate well cultures were assayed for each peptide concentration. Plates were incubated for 7 days at 37°C in a humidified atmosphere with 5% CO2. Cells were pulsed with 1 μCi 3H-Thy/well for 18-21 hours before harvesting onto filter sheets. CPM values were determined with a Wallac microplate beta topplate counter (Perkin Elmer). Results are expressed as stimulation index, which is obtained by dividing the proliferation fraction (eg radioactivity counts per minute) determined for the test peptide by the fraction determined for cells not exposed to the test peptide. the

Analysis of the above experimental results revealed the presence of four T cell epitopes corresponding to peptides 41, 44 and 50 in the mature processed region of the protein and peptide 88 in the unprocessed form. Since peptide 88 is not part of the mature peptide, it was ignored in the scheme of the present invention. the

For peptide 41 (designated epitope region R1), there are four donors corresponding to this peptide; donors 4, 5, 10 and 11. Their S.I. at 5 μM were 3.6, 4.9, 2.1 and 2.0, respectively. the

For peptide 44 (designated epitope region R2), there are two donors corresponding to this peptide; donor 4 (S.I. = 3.5) and 11 (S.I. = 2.3). Neighboring peptides 43 and 45, due to their 12 amino acid overlap with peptide 44, induced lower levels of T cell proliferation. the

For peptide 50, there are two donors corresponding to this peptide; donor 4 (S.I. = 2.9) and 14 (S.I. = 2.0). Peptide 51 induced lower levels of T cell proliferation in Donor 14 (S.I. > 1.9). the

Histology of all PBMC samples was determined using a commercially available reagent system (Dynal, Wirral, UK). The assay was performed according to the supplier's recommended procedures, standard auxiliary reagents, and agarose electrophoresis system. The allotype specificity for each corresponding donor sample is shown in Table 2. the

Example 2: Cloning of bouganin from Bougainvillea spectabilis

Total RNA was isolated from Bougainvillea spectabilis leaves using the 'SV Total RNA Isolation System and the protocol provided by the supplier (Promega, Southampton, UK). Fresh leaf tissue was ground into a fine powder under liquid nitrogen, and about 50 mg of the ground tissue was taken for RNA isolation. RNA quality and quantity were checked by visualization in 1% agarose gel, and the bouganin gene was amplified from total RNA using “Access RT-PCR System” (Promega) and specific primers OL1032 and OL1033, using approximately 1 μg RNA for each reaction. Primer sequences are shown in Table 3 below. This reaction yielded a 1242bp fragment including the native leader sequence and the full-length bouganin sequence. The fragment was cloned into the pGEM-T Easy vector (Promega) according to the kit instructions, and named pBou1. Sequences were verified by DNA sequencing. the

Using the pBou1 plasmid as a template, the bouganin gene was transformed into pET21a (Novagen, Nottingham, UK) by PCR. A pelB (pectate lyase pectinase?) leader sequence was added to the 5' end and a sequence encoding a 6×histidine tag was added to the 3' end of the bouganin coding sequence. The pelB leader sequence was amplified from the vector pPMI-his using primers OL1322 (incorporating an Nde1 site) and primer OL1067 [Molloy, P. et al. (1995) J. Applied Bacteriology, 78 :359-365] . The bouganin-his fragment was amplified from pBou1 using OL1068 and OL1323 (incorporating a Not1 site). The pelB leader sequence was fused to the bouganin-his fragment cassette using overlap PCR, and the resulting fragment was amplified into pGEM-TEasy (Promega). After sequence verification, the pelB-bouganin-his fragment was cloned as Nde1-Not1 fragment into Nde1-Not1 digested pET21a. This clone was named pBou32.

Embodiment 3: Construction mutant bouganin protein

A number of modified (mutated) bouganin proteins were designed using data provided by T-cell epitope mapping methods and using software capable of modeling the binding of peptides to the human MHC class II binding groove. The latter approach is described in detail elsewhere [WO 02/069232]. Variant genes were constructed and the functional activity of the mutant proteins was tested. Typically, "single mutant" proteins each containing only one amino acid substitution are first constructed and tested, and genes for active modified proteins are then combined to produce multiple substitution modified proteins. the

Mutant genes were constructed by the overlap PCR method, which utilizes mutations in "overlap primers" to introduce mutated amino acid codons into the gene. This procedure is well known in the art and is described in detail elsewhere [Higuchi, et al. (1900) Nucl. Acids Res. 16:7351]. A total of 37 single mutant modified proteins that retain functional activity were constructed and tested. In addition, a negative control modified protein containing the Y70A substitution was constructed and tested in all experiments. In fact, one of the 37 "single mutation" modified proteins contained two adjacent substitutions (E151T and I152E), which were also counted as single mutations herein. The substitutions tested and the corresponding activity values are shown in Table 4. the

A total of 11 modified proteins with multiple substitutions that retained activity were constructed and tested. The mutations tested and the corresponding activity values are shown in Table 5. the

Table 6 describes the sequence of the modified protein by substitution. Table 7 lists some specific sequences. the

In all examples, protein purification and testing were performed according to the procedures given in Examples 4 and 5 below. the

Embodiment 4: Expression and purification of Bouganin protein

Plasmid pBou32 was transformed into BL21(DE3) (Novagen) competitor cells following the manufacturer's instructions and selected on LB (Invitrogen, Paisley, UK) plates containing 50 μg/ml carbenicillin. Inoculate 5 ml of 2×YT (Invitrogen) liquid medium with a fresh clone obtained from the transformation, without antibiotics, and cultivate to OD600=1.5-2.0 with shaking at 250 rpm at 37°C. The culture was then centrifuged at 2500 rpm for 15 minutes at room temperature and the cells were resuspended in 5 ml of fresh 2×YT plus 1 mM IPTG. The culture was shaken at 30° C. for 1.5 hours at 300 rpm, the cells were collected by centrifugation, and the supernatant was discarded. the

The cell pellet was resuspended in 1ml PEB2 (50mM Tris-HCl pH8, 20% sucrose, 1mg/ml lysozyme, 1×Complete Protease Inhibitor Tablet (Roche, Lewes, UK)), and incubated on ice for 1 hour with gentle agitation. The cell debris was centrifuged at 14000 rpm at 4°C, and the centrifuged pellet was discarded. The resulting supernatant is the "periplasmic fraction". Bouganin protein was isolated from the periplasmic fraction by nickel affinity column chromatography using a commercially available "spincolumn" following the manufacturer's (Qiagen, Crawley, UK) instructions. The resulting material was dialyzed against 4 liters of phosphate-buffered saline (0.138M NaCl, 0.0027M KCl, pH 7.4) overnight at 4°C using a 10,000 molecular weight cut-off 'Slide-A-Lyzer' (Pierce, Chester, UK). After dialysis, protein concentration was estimated using the Micro BCA Assay Kit (Pierce), and samples were stored at -20°C. the

Bouganin protein concentration was further determined with an ELISA-based detection system. Briefly, antisera against bouganin were generated by genetic immunization of two rats with a bouganin expressing plasmid (Genovac, Freiburg, Germany). For ELISA, recombinant bouganin was captured via its His-tag onto a Ni-agarose-coated plate, followed by rat antiserum and secondary HRP-conjugated anti-rat Fc antibody (Sigma, Poole, UK) to test. As a standard, wild-type bouganin expressed in E. coli and quantified by total protein assay was used in each assay in large preparations. the

Example 5: Determination of bouganin activity

The activity of wild-type and modified (mutant) bouganin proteins was tested by measuring their ability to synthesize protein in a cell-free protein synthesis assay. the

A mixture of 10 μl TNT transcription/translation association mix (Promega), 20 μM methionine, 120 ng pT7 luciferase DNA (Promega) and serial dilutions of WT and mutant Bouganin proteins was incubated for one hour at 30°C in a final volume of 12.5 μl , after which the reaction was terminated by adding 100 [mu]l "SteadyGlow" luciferase detection reagent (Promega). Luciferase activity was measured with a Wallac luminometer. The detection of active Bouganin protein is the decrease of the measured luciferase activity. At least 5 concentrations were tested for each modified Bouganin protein, and each data point was set as a replicate. Positive and negative controls were included in each experiment. the

The results for the single mutant proteins are shown in Table 4. Table 5 shows the results of the modified Bouganin protein by multiple mutations. In each case, results are expressed relative to wild-type protein activity. All assays included an inactivating mutant Bouganin protein with a Y70A substitution. the

In addition, luciferase assay results can also be plotted, showing % luciferase activity relative to control versus protein concentration of added bouganin. An example of such a graph is shown in Figure 1, which depicts the results of assays for two different multiple mutant bouganin proteins. the

Example 6: Determination of T-cell epitope deletion of variant bouganin sequences.

A multiple modified protein named Bou156 was selected for further testing by immunogenicity assay. This variant contains substitutions V123A, D127A, Y133N and I152A. Immunogenicity tests involved the use of living cells that could be disrupted by tests using the whole Bouganin protein, so these assays were done using synthetic peptides containing the substitutions incorporated in variant Bou156. The peptides tested are listed in Table 8. These assays were performed according to the procedure described in Example 1 (above) using a pool of 20 individual PBMC donors. These peptides were replicated three times for each donor sample at two different final peptide concentrations (1 μM and 5 μM). the

Results are expressed as SI per peptide per donor sample and are shown in Figure 2. DeI-41 is the peptide sequence AKADRKALELGVNKL (SEQ ID NO: 29). Del-44 is the peptide sequence LGVNKLEFSIEAIHG (SEQ ID NO: 30). DeI-50 is the peptide sequence NGQEAAKFFLIVIQM (SEQ ID NO: 31). All modified peptides did not induce a T cell response in either donor (S.I.<2). In contrast, the immunogenic control peptide stimulated T cells of 6 donors (S.I. > 2). the

Example 7: VB6-845: Recombinant Engineering of Ep-CAM-specific Fab Antibody for Optimal Delivery of De-bouganin.

For this example and Example 8, the deimmunized bouganin used was Bou156. the

Tumor-targeting cytotoxins consist of antibody variable regions linked to bacterial, fungal, or plant toxins. This study exemplifies that deimmunized bouganin constructs of the invention, including deimmunized bouganins linked to a targeting moiety, have reduced immunogenicity while still retaining their biological activity. Table 12 demonstrates the binding of the Ep-CAM antibody to several types of tumors and thus shows that it can be used to treat these types of cancers. the

De-immunized Bouganin construct: Ep-CAM-directed target moiety linked to de-bouganin

VB5-845, a Fab version of an anti-Ep-CAM scFv antibody, was genetically linked to a de-immunized form of bouganin (de-bouganin), Bou 156, a potent, plant-derived, type I ribosome-inactivating protein (RIP), forming the antibody-toxin construct VB6-845. Figure 3 illustrates construct VB6-845. Figure 3A is a schematic representation of the bicis-trans unit of pro-VB6-845 with the pelB leader sequence. Accompanying drawing 3B provides amino acid sequence (SEQ ID NO: 16) and nucleic acid coding sequence (SEQ ID NO: 15). Figure 3C schematically illustrates the assembly of the VB6-845 protein, which is described in detail below. Testing of this construct demonstrated that the construct retained its biological activity (cytotoxicity) and specificity of the target moiety (Ep-CAM antibody). the

Orientation orientation of deimmunized bouganin construct

To determine the most ideal antibody-de-bouganin orientation, several forms of bicistronic expression units were generated, expressed, and tested for potency. the

In each case, bicistronic units were cloned into the pING3302 vector (Fig. 4) under the control of the arabinose-inducible araBAD promoter and transformed into E104 E. coli. Upon induction, the presence of the pelB leader sequence directs the secretion of the Fab-de-bouganin fusion protein into the culture supernatant. The cleavable linker enables de-bouganin to be cleaved from the target moiety and exert its biological activity. In one embodiment, the linker is a paired basic amino acid protease linker, although those skilled in the art will appreciate that other cleavable linkers may also be suitable. Preferred linkers can be selected according to target specificity, and circumstances. Construct samples were prepared and tested as follows:

Figure 3: VB6-845 in which de-bouganin (Bou156) is linked to the C-terminus of the CH domain via a paired basic amino acid protease linker. Accompanying drawing 3A shows the dicistronic unit of original sequence, accompanying drawing 3B shows the nucleic acid coding sequence (SEQ ID NO:15) of original sequence and aminoacid sequence (SEQ ID NO:16) and accompanying drawing 3C shows not containing pelB sequence Assembly of VB6-845 protein. the

Figure 5 shows a control Fab anti-Ep-CAM construct without the phytotoxin, de-bouganin (VB5-845). Accompanying drawing 5A shows the double cis subunit of original sequence, accompanying drawing 5B shows the nucleic acid coding sequence (SEQ ID NO:17) of original sequence and aminoacid sequence (SEQ IDNO:18), and accompanying drawing 5C shows that assembled does not contain VB5-845 protein of pelB sequence. the

Figure 6 shows the Fab anti-Ep-CAM de-bouganin construct, VB6-845-CL-de-bouganin, in which Bou156 is linked to the C-terminus of the CL domain. Accompanying drawing 6A shows the bicis norm subunit of original sequence, accompanying drawing 6B shows the nucleic acid coding sequence (SEQ IDNO:19) and aminoacid sequence (SEQ ID NO:20) of original sequence, and accompanying drawing 6C shows not containing pelB sequence Assembly of VB6-845-CL-de-Bouganin protein. the

Figure 7 shows the Fab anti-Ep-CAM, de-bouganin construct, VB6-845-NVH-de-bouganin, where Bou156 is linked to the N-terminus of the VH domain. Accompanying drawing 7A shows the bicis subunit of coding original sequence, accompanying drawing 7B shows the nucleic acid coding sequence (SEQ IDNO:21) and aminoacid sequence (SEQ ID NO:22) of original sequence, and accompanying drawing 7C shows not containing pelB sequence Assembly of VB6-845-NVH-de-Bouganin protein. the

Figure 8 shows the Fab anti-Ep-CAM de-bouganin construct, VB6-845-NVL-de-bouganin, in which Bou156 is linked to the N-terminus of the VL domain. Accompanying drawing 8A shows the bicis subunit of encoding original sequence, accompanying drawing 8B shows the nucleic acid coding sequence (SEQ ID NO:23) and aminoacid sequence (SEQ ID NO:24) of original sequence, and accompanying drawing 8C shows not containing pelB sequence Assembly of VB6-845-NVL-de-Bouganin protein. the

In one embodiment, the de-bouganin molecule is attached to the C-terminus of the heavy or light chain. The most ideal configuration includes a pelB leader sequence adjacent to the VH-CH domain and an N-terminal histidine affinity tag as the first unit. This is followed by _{a second unit comprising the pelB-VL-CL domain} linked to de-bouganin by a protease sensitive linker. (Fig. 6) For constructs in which de-bouganin was relocalized at the N-terminus, Western blot analysis showed no detectable product, only C-terminally linked de-bouganin (constructs of Figs. 3 and 6) produced An intact soluble protein was obtained with good binding properties to Ep-CAM-positive cell lines (Fig. 9), as shown in reactivity experiments by flow cytometry. Figure 9 shows the expression of VB6-845 and VB6-845CL-de-bouganin in supernatants of induced E104 cells at laboratory levels in Western blot analysis. Under non-inducing conditions, 16 µl aliquots of the supernatant were loaded onto an SDS-PAGE acrylamide gel and analyzed using rabbit polyclonal anti-4D5 antibody followed by goat anti-rabbit (1/2000), Or use goat anti-human κ-light chain-HRP antibody (1/1000) for Western blot analysis to verify the identity and size of the recombinant protein. Arrows indicate full-length VB6-845 (construct of Figure 3) and VB6-845-CL-de-bouganin (construct of Figure 6). Western blotting of non-induced E104 culture supernatants showed no corresponding bands, demonstrating the specificity of the antibody (not shown).

VB6-845 (accompanying drawing 3) and VB6-845-CL-de-bouganin (accompanying drawing 6) compared with control (Ep-CAM-negative cell line, A-375) to Ep-CAM positive cell line CAL 27 and NIH: The reactivity test results of OVCAR-3 are shown in Figure 10A. These results are comparable to the same reactivity experiments performed with another anti-Ep-CAM construct, VB6-845-gelonin, in which de-bouganin was replaced by another phytotoxin gelonin (See accompanying drawing 14C showing its amino acid sequence (SEQ ID NO: 26) and nucleic acid sequence (SEQ ID NO: 25)). The results of reactivity experiments with gelonin constructs are shown in Figure 10B. Addition of a second de-bouganin domain to a molecule with the most ideal orientation produced no product. the

Flow cytometry experiments were completed by incubating the construct or control with 0.45 x ¹⁰⁶ cells for one hour on ice. After washing, the surface of cells bound to the construct was probed with rabbit anti-bouganin (Figure 10A) or mouse anti-His tag (Figure 10B) on ice. Cells were washed and incubated with FITC-conjugated sheep anti-rabbit IgG (Figure 10A) and FITC-conjugated sheep anti-mouse (IgG) (Figure 10B) for 30 minutes on ice. Cells were then washed and resuspended in PBS 5% FCS containing propidium iodide for antibody binding assessment by flow cytometry. No change in median fluorescence was detected after incubation of VB6-845 and VB6-845-CL-de-bouganin with A-375. In contrast, significant changes in median fluorescence were observed with the Ep-CAM positive cell lines, CAL 27 and NIH:OVCAR-3 (Fig. 10A). As noted above, the results for VB6-845 were similar to those for the gelonin construct (Fig. 10B).

Ep-CAM specificity

Competition experiments of VB6-845 (the construct of FIG. 3 ) with Proxinium ^TM , an scFv form of VB6-845 but containing Pseudomonas exotoxin A, demonstrated that ^when VB6-845 was engineered The Ep-CAM specificity was unchanged in Fab format (Fig. 11).

Accompanying drawing 11 illustrates the result of the flow cytometry of competition experiment, 1 and 10 μ g/mL VB6-845 and the Proxinium ^TM and NIH: OVCAR-3 cell of increasing concentration (concentration range 0 to 100 μ g/mL) (Ep-CAM positive tumor cell line) co-culture. After incubation for one hour at 4°C, cells were washed and bound VB6-845 was detected with biotinylated rabbit anti-bouganin followed by streptavidin. The same experiment was performed with 4B5-PE as a negative control. Reaction conditions are shown in accompanying drawing 11.

potency (biological activity)

Furthermore, cell-free (Fig. 12) and MTS (Fig. 13A and B) assays demonstrated that de-bouganin retained its potency when coupled to the Fab fragment. The MTS assay for determining potency is performed using standard techniques known in the art and is described in more detail in Example 8 below. With Ep-CAM-positive cell lines, CAL 27 and NIH:OVCAR-3, the _IC50 of VB6-845 was 3 to 4 nM and 2 to 3 nM, respectively. For VB6-845- _CL -de-bouganin, its potency against 1 to 2 nM of CAL 27 and against NIH:OVCAR-3 was determined. The development of Fab anti-Ep-CAM constructs that include human tumor-targeting antibody fragments linked to deimmunized bouganin should allow repeated systemic administration of this drug and thus be able to yield greater clinical benefit.

Harvest of constructs

Constructs can be isolated from cell culture using techniques known in the art. For example, if the His-tag is located at the N-terminus of the peptide construct, the Fab-Bouganin protein can be purified using Ni ²⁺ -chelate capture. For example, the following scheme can be adopted.

Fed-batch fermentation of the VB6-845 variant was accomplished in a 15L CHEMAP fermenter with TB medium. At an _OD600 of 20 (mid-log), the cultures were induced with feed and an inducer comprising 50% glycerol and 200 g/l L-arabinose. 30 hours after induction, the culture was harvested, centrifuged at 8000 rpm for 30 min, and the VB6-845 variants were purified using CM Sepharose and Metal-Charged chelating Sepharose columns, followed by size exclusion columns. Briefly, supernatants were concentrated and diafiltered against 20 mM sodium phosphate pH 6.9 ± 0.1. The diafiltration concentrated supernatant was then applied to a CM sepharose column equilibrated with 20 mM sodium phosphate, 25 mM NaCl pH 6.9±0.1. The column was washed with 20 mM sodium phosphate, 25 mM NaCl pH 6.9±0.1, after which bound VB6-845 was eluted with 20 mM sodium phosphate, 150 mM NaCl pH 7.5±0.1. The CM Sepharose eluate was adjusted to contain Triton-X100 at a final concentration of 0.25%, and applied to a charge-chelating Sepharose column. Then use 20mM sodium phosphate, 150mM NaCl, 0.25% triton-X100 pH 7.5±0.1, then use 20mM sodium phosphate, 150mM NaCl pH 7.5±0.1, then use 20mM sodium phosphate, 150mM NaCl, 10mM imidazole pH 7.5±0.1 in sequence with 3 different concentration of wash buffer to wash the chelating sepharose column. VB6-845 was then eluted with 20 mM sodium phosphate, 150 mM NaCl, 250 mM imidazole pH 7.5±0.1 and collected in 2 mL fractions. In order to obtain a purity of >80%, the absorption of each fraction on A ₂₈₀ was determined, and each fraction and the collected material were applied to a size exclusion column S200. In one embodiment, to increase protein purity and remove endotoxin, the collected SEC fractions were diluted five-fold with 20 mM NaPO ₄ , pH 7.5, and passed through 20 mM NaPO ₄ , 25 mM NaCl at a rate of about 5 ml/min. pH 7.5 equilibrated Q-Sepharose 15ml fast flow column. After the sample was passed through the column, the column was washed with 10 CV of equilibration buffer, and the washing solution was mixed with the initial Q-Sepharose flow-through and collected. The effluent was concentrated tenfold to a final concentration of 7.5 mg/ml using a 30 kDa MWCO membrane (Sartorius hydrosart membrane). Then add Tween-80 at a final concentration of 0.1%. The final product was filter sterilized and stored at -80°C. Samples used in each step of the method were analyzed by Western blot after immunoblotting with anti-4D5 antibody. Colloidal blue staining verified purity. Expression levels of VB6-845 variants were determined by Western blot analysis and ELISA.

Example 8: Functional and biological characteristics of a recombinant Ep-CAM-specific Fab antibody genetically linked to de-immune Bouganin (de-bouganin), VB6-845.

Chemotherapeutic agents are highly cytotoxic agents that often represent the standard of care in the treatment of many solid tumor cancers. The cytotoxic effects of these drugs rapidly target dividing cells, both normal and neoplastic, thus causing various clinical side effects. VB6-845 is a Fab antibody to a deimmunized form of bouganin linked to a plant-derived toxin. Unlike chemotherapeutic agents that lack precise tumor target specificity, VB6-845 restricts its cytotoxic effects only to Ep-CAM-positive tumor targets. In this study, flow cytometry and cytotoxicity were measured to assess the potency and selectivity of VB6-845. the

Flow Cytometry

The tumor cell lines used in this study were purchased from ATCC and propagated according to ATCC's recommendations, except cell lines C-4I and TOV-112D which were grown in RPMI 1640 or DMEM supplemented with 10% FCS, respectively. Tumor cells were collected when the confluence rate reached 60-70% and the viability exceeded 90%. Human normal mammary epithelial cells (HMEC) were purchased from CAMBREX and maintained in specific media according to the procedures provided by CAMBREX. Cells were collected when the confluence rate reached 70% and the viability exceeded 90%. the

Gynecological cell lines from endometrial ovarian and cervical cancer indications were tested for binding to VB6-845 on a flow cytometer (Table 9). 10 μg/mL VB6-845 was added to each cell line (3×10 ⁵ cells), and incubated at 4° C. for 2 hours. A-375 and CAL 27 were used as negative and positive cell line controls, respectively. After washing away unbound substances, a mouse monoclonal anti-histidine antibody (Amersham Pharmacia, Cat#27471001) diluted 800-fold in PBS containing 10% FCS was added, and incubation was continued at 4°C for 1 hour. Then, FITC-labeled anti-mouse IgG (The Binding Site, Cat#AF271) diluted 100 times in PBS-10% FCS was added and incubated for 30 minutes. Finally, after removal of dead cells by propidium iodide staining, cells were analyzed on a FACS Calibur.

Cytotoxicity

The killing levels of VB6-845 in cells listed in flow cytometry studies are shown in Table 10, indicating that this construct retained its de-bouganin cytotoxic activity against Ep-CAM-positive cell lines. The cytotoxicity was comparable to the activity of another Fab VB6-845 variant containing a different toxin of plant origin, gelonin (Fig. 14). Figure 14A compares gelonin, the Fab anti-Ep-CAM-gelonin construct (VB6-845-gelonin) and the Fab anti-Ep-CAM-de-bouganin (Bou156) construct ( VB6-845) toxicity in CAL 27 (FIG. 14A) and NIH:OVCAR-3 cells (FIG. 14B). The nucleic acid and amino acid sequences of the VB6-845-gelonin construct are shown in Figure 14C. the

To investigate the specificity and selectivity of VB6-845 (construct of accompanying drawing 3), tested VB6-845 (90% pure) and 17 kinds of chemotherapeutic drugs (LKB Laboratories Inc.) to Ep-CAM-positive (NIH: Cytotoxicity of OVCAR-3) and Ep-CAM-negative (HMEC, DAUDI, A-375) cell lines (Table 11). the

MTS detection is accomplished using standard techniques known in the art. Specifically, 50 microliters of cells (2×104 cells/ml) were seeded per well, and the plate was incubated at 37° C. for 2 hours in 5% CO 2 . Then add 50 microliters of spiked drug (i.e. construct to be tested or control) with gradually increasing concentration to the medium. Media with or without cells were used as positive and negative controls, respectively. Plates were kept at 37°C, 5% CO2 for 5 days. On day 5, inhibition of cell proliferation was assessed by adding 20 microliters of MTS reagent (Promega, Cat#G5430). Continue to incubate the plate at 37°C and 5% CO2 for 2 hours, and read the OD value at 490nm with a plate reader spectrophotometer. The background values were subtracted from the sample values obtained for each concentration and the results expressed as a percentage of viable cells. Calculate the IC50 of each drug on the cell line. the

When measuring cytotoxicity against NIH:OVCAR-3, an Ep-CAM-positive ovarian sarcoma, with a panel of standard chemotherapeutic agents, VB6-845 was more effective than 12 of the 17 tested (Table 11 ). Although five chemotherapeutic agents were more cytotoxic, they showed greater toxicity due to their lack of any cell-specific killing. Of the five chemotherapeutic agents (paclitaxel, carboplatin, cisplatin, doxorubicin, and topotecan) recommended for ovarian cancer, only two (paclitaxel and topotecan) are more cytotoxic. Although VB6-845 demonstrated more potent cytolytic activity at 1 to 2 nM, this potent killing was limited to the Ep-CAM-positive tumor cell line NIH:OVCAR-3. Although VB6-845 showed some killing on Ep-CAM-negative cell lines, its cytotoxic effect was at least 220-fold lower and at most more than 1000-fold lower. VB6-845 thus represents an effective antibody-directed therapeutic alternative to chemotherapeutic agents used in combination with other less toxic profiles, with great promise in the treatment of many different types of solid tumors. the

Example 9: VB6-011: Recombinant Engineering of Tumor-Associated Antigen-Specific Fab Antibody for Optimal Delivery of De-bouganin.

Tumor-targeting cytotoxins consist of variable regions linked to bacterial, fungal or plant toxins. This study demonstrates that deimmunized bouganin constructs of the invention, including deimmunized bouganin linked to a targeting moiety, have reduced immunogenicity while still retaining their biological activity. Table 13 demonstrates the binding of tumor associated antigen antibody to several types of tumors and thus shows that it can be used to treat these types of cancers. the

De-immunized bouganin constructs: tumor-associated antigen-directed targeting moieties linked to de-bouganin

The H11 antibody, a monoclonal antibody that recognizes a tumor-associated antigen, is genetically linked to a de-bouganin form (de-bouganin), Bou 156, a potent, plant-derived, type I ribosome-inactivating protein (RIP), resulting in the antibody-toxin construct VB6-011. Figure 15 shows the nucleic acid coding sequence and amino acid sequence. Testing of this construct demonstrated that the construct retained its biological activity (cytotoxicity). the

potency (biological activity)

MTS assay confirmed that de-bouganin still maintains its potency after coupling with Fab fragment (Fig. 16). The MTS assay used to determine potency was accomplished using standard techniques in the art, as described in detail in Example 8. the

Cytotoxicity

To study the specificity and selectivity of VB6-011, its cytotoxic activity on MB-435S cells was tested. MTS detection is accomplished using standard techniques known in the art. Specifically, 50 microliters of cells (2×10 ⁴ cells/ml) were seeded per well, and the plate was incubated at 37° C. in 5% CO ₂ for 2 hours. Then add 50 microliters of spiked drug (that is, construct to be tested or control) with increasing concentration to the culture medium. Media with or without cells were used as positive and negative controls, respectively. Plates were kept at 37 °C, 5% _CO2 for 5 days. On day 5, inhibition of cell proliferation was assessed by adding 20 microliters of MTS reagent (Promega, Cat#G5430). Continue to incubate the plate at 37°C, 5% CO ₂ for 2 hours, and read the OD value at 490nm with a plate readerspectrophotometer. The background values were subtracted from the sample values obtained for each concentration and the results expressed as a percentage of viable cells. The results showed that the IC ₅₀ value of VB6-011 was 350nM.

While this invention has been described with reference to what are presently considered to be the preferred embodiments of the invention, it is to be understood that the invention is not limited to the disclosed examples. On the contrary, the invention is intended to cover various modifications and equivalent arrangements falling within the spirit and scope of the claimed invention. the

All publications, patents, and patent applications are herein incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically or individually indicated to be incorporated by reference in its entirety. the

Table 1

Bouganin sequence peptide. Underlined residues are absent in the mature peptide. the

Table 2

Donor number Donor storage code Allotype 1 BC63 DRB1*04, DRB1*07, DRB4*01 2 BC86 DRB1*04, DRB1*15, DRB5 3 BC90 DRB1*07, DRB1*15, DRB4*01, DRB5 4 BC134 DRB1*01, DRB1*03, DRB3 5 BC167 DRB1*01, DRB1*07 and DRB4*01 6 BC216 DRB1*14, DRB1*15, DRB3, DRB5 7 BC217 DRB1*04, DRB1*12, DRB3, DRB4*01 8 BC233 DRB1*04, DRB1*11 and DRB3, DRB4*01 9 BC241 DRB1*07, DRB1*11, DRB3, DRB4*01 10 BC246 DRB1*01, DRB1*13 and DRB3 11 BC262 DRB1*03, DRB1*07, DRB3, DRB4*01 12 BC292 DRB1*07, DRB1*13, DRB3, DRB4*01 13 BC293 DRB1*04, DRB1*10, DRB4*01 14 BC231 DRB1*03 or DRB1*03, DRB1*13 and DRB3 15 BC301 DRB1*07, DRB1*14, DRB3 16 BC326 DRB1*03, DRB1*15, DRB3, DRB5 17 BC316 DRB1*13, DRB1*15, DRB3, DRB5 18 BC321 DRB1*01, DRB1*15, DRB5 19 BC382 DRB1*04, DRB1*08, DRB4*01 20 BC336 DRB1*01, DRB1*11, DRB3

MHC allotypes of PBMC donors

table 3

Primer SEQ ID NO sequence

OL1032 121 CATTACAAACGTCTACCAAGTTT

OL1033 122 TTACAAAAGTAGATAAGTAATGTG

OL1322 123 GATATACATATGAAATACCTATTGCCTACG

OL1067 124 TGACACAGTGTTGTACGCTGGTTGGGCAGCGAGTAA

OL1068 125 GCTGCCCAACCAGCGTACAACACTGTGTCATTTAAC

OL1323 126 CGAGTGCGGCCGCTCAATGGTGATGGTGATGGTGT

Construction of primer sequences used in WT bouganin gene

Table 4

Single substitution bouganin variants constructed and tested. the

*Activity in luciferase assay:

++ = equal to or higher than WT protein. + = within 2 times of WT activity. +/- = within 3-fold of WT activity. -- = less than one-third of WT activity. WT = wild type protein. the

**Clone ID. Only functionally active variants are named. the

table 5

Multiple substitution bouganin variants constructed and tested. the

*Activity in luciferase assay:

++ = equal to or higher than WT protein. + = within 2 times of WT activity. +/- = within 3-fold of WT activity. -- = less than one-third of WT activity. WT = wild type protein. the

Table 6

Clone ID replace* protein Bou32 WT SEQ ID No 1 Bou156 V123A, D127A, Y133N, I152A SEQ ID No 13 Bou157 V123A, D127A, Y133Q, I152A SEQ ID No 14 Bou143 V123Q, Y133Q, I152Q the Bou144 V123A, Y133N, I152A the Bou145 V123A, Y133Q, I152A the Bou146 V123A, D127G the Bou147 V123A, D127A the Bou148 V123Q, D127G the Bou149 V123Q, D127A the Bou150 V123Q, E129G the Bou151 V123A, E129G the Bou2 V123T the Bou3 V123A the Bou4 V123Q the Bou5 D127G the Bou6 D127A the Bou7 E129Q the Bou8 E129G the Bou9 Y133N the Bou10 Y133T the Bou11 Y133A the Bou12 Y133R the Bou13 Y133D the Bou14 Y133E the Bou15 Y133Q the Bou16 Y133G the Bou17 Y133H the Bou18 Y133K the Bou19 Y133S the Bou20 I152Q the Bou21 I152A the

*Numbering from residue 1 of the bouganin reading frame, thus excluding the PeIB leader sequence included in most constructs. the

Table 7

SEQ ID No 1

protein

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYKLEFSIAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK 

SEQ ID No 13

protein

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKADRKALELGVNKLEFSIAIHGKTINGQEAAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK 

Table 8

Modified and WT Bouganin further tested in T cell assays. the

*Substituted (mutated) residues are underlined. the

Table 9

Combination of VB6-845 with gynecological cell lines by flow cytometry

The results are represented by MF±SEM. The multiple of proliferation

Indications cell line VB6-845 (multiplication factor MF±SEM) endometrium HEC-1-A 42.3±0.9 the RL95-2 4.9±0.7 the SK-UT-1 1.1±0.1 ovaries NIH:OVCAR-3 33.6±6.0 the SK-OV-3 4.3±1.0 the TOV-112G 1.1±0.1 cervix HT-3 29.1±1.2 the C-4I 6.8±0.6 the C-33A 1.1±0.0 melanoma A-375 1.1±0.1

Table 10

VB6-845-mediated cytotoxicity determined by MTS assay

Table 11

Specificity and selectivity of VB6-845 compared to chemotherapeutic agents

Table 12: Indications for VB6-845 tumor cells

¹ N represents the number of cell lines tested in each indication.

² means the fold proliferation of the median fluorescence from all cell lines versus the control antibody in each indication.

Table 13: Indications for VB6-011 tumor cells

¹ N represents the number of cell lines tested in each indication.

^{The 2} values represent the mean calculated from the sum of the median fluorescence from all cell lines versus the mean fold proliferation of the control antibody for each indication. A value of zero means no measurable activity relative to control activity.

Claims (16)

1. A modified Bouganin protein, wherein said Bouganin protein has a weakened tendency to activate T-cells, and the amino acid sequence of the modified Bouganin protein consists of the following sequences: YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFV LVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVAT SKLFPGVTNRVTLTFDGSYQKLVNAAKX ¹ DRKX ² LX ³ LGVX ⁴ KLEFSIEAIHGKTINGQEX ⁵ AKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK1 (SEQ ID NO: X ¹ is T or A or Q; ^X2 is G or A; X ³ is Q or G; ^X4 is N or D or T or A or R or Q or E or G or H or K or S; and X ⁵ is Q or A. 2. according to the modified Bouganin protein of claim 1, wherein the modified Bouganin protein is made up of following sequence: YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFV LVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVAT SKLFPGVTNRVTLTFDGSYQLVNAAKADRKALELGVNKLEFSIEAIH GKTINGQEAAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVL NLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPD MGILKFKSSK (SEQ ID NO: 13). 3. A cytotoxin comprising (a) according to the modified Bouganin protein of claim 1 or 2; (b) Attached to the target moiety of (a). 4. A cytotoxin comprising (a) according to the modified Bouganin protein of claim 1 or 2; (b) Ligand capable of binding cancer cells attached to (a). 5. The cytotoxin of claim 4, wherein the ligand is an antibody or antibody fragment that binds to cancer cells. 6. The cytotoxin of claim 5, wherein the antibody or antibody fragment binds to Ep-CAM on the surface of cancer cells. 7. The cytotoxin of claim 6, comprising the amino acid sequence shown in SEQ ID NO:16. 8. The cytotoxin of claim 5, wherein the antibody or antibody fragment binds a tumor-associated antigen on the surface of a cancer cell. 9. The cytotoxin of claim 8, comprising the amino acid sequence shown in SEQ ID NO:28. 10. Use of the cytotoxin according to any one of claims 3-9 for the preparation of a medicament for inhibiting or destroying cancer cells. 11. The purposes of claim 10, wherein the cancer cells are selected from the cells of the following cancers: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer, liver cancer, kidney cancer, melanoma, gastrointestinal cancer, Prostate cancer, small cell and non-small cell lung cancer, sarcomas, gliomas, and T- and B-cell lymphomas. 12. Use of the cytotoxin according to any one of claims 3-9 for the preparation of a medicament for the treatment of cancer. 13. The use of claim 12, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer, liver cancer, kidney cancer, melanoma, gastrointestinal cancer, prostate cancer, small cell and Non-small cell lung cancer, sarcoma, glioma, and T- and B-cell lymphoma. 14. A pharmaceutical composition comprising a cytotoxin according to any one of claims 3-9 and a pharmaceutically acceptable carrier, diluent or excipient. 15. A nucleic acid molecule encoding a modified Bouganin protein according to claim 1 or 2. 16. A nucleic acid molecule encoding a cytotoxin according to any one of claims 3-9.

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