Compositions and methods for modifying toxic effects of proteinaceous compounds

Abstract

The present invention provides methods to produce immunotoxins (ITs) and cytokines with a reduced ability to promote vascular leak syndrome (VLS). The invention also provides ITs and cytokines which have been mutated to lack amino acid sequences which induce VLS.

Description

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the field of physiology and cancer biology, and particularly concerns toxins and other proteins which induce or cause vascular leak syndrome (VLS). The invention provides immunotoxins (ITs) and cytokines which have been mutated to lack amino acid sequences which induce VLS and other toxic side effects. Disclosed are methods for mutating DNA segments encoding cytokines or immunotoxins so that an immunotoxin is produced that lacks sequences that induce VLS and other toxic side effects.

[0005] 2. Description of Related Art

[0006] VLS is often observed during bacterial sepsis and may involve IL-2 and a variety of other cytokines (Baluna and Vitetta, 1996). The mechanisms underlying VLS are unclear and are likely to involve a cascade of events which are initiated in endothelial cells (ECs) and involve inflammatory cascades and cytokines (Engert et al., 1997). VLS has a complex etiology involving damage to vascular endothelial cells (ECs) and extravasation of fluids and proteins resulting in interstitial edema, weight gain and, in its most severe form, kidney damage, aphasia, and pulmonary edema (Sausville and Vitetta, 1997; Baluna and Vitetta, 1996; Engert et al., 1997). Vascular leak syndrome (VLS) has been a major problem with all ITs thus far tested in humans, as well as cytokines such as interleukin 2 (IL-2), TNF and adenovirus vectors (Rosenberg et al., 1987; Rosensten et al., 1986).

[0007] ITs are hybrid molecules consisting of monoclonal antibodies (MAbs) or other cell-binding ligands, which are biochemically or genetically linked to toxins, toxin subunits, or ribosome inactivating proteins (RIPs) from plants, fungi or bacteria (Vitetta et al., 1993). Over the past two decades, ITs containing deglycosylated (dg) ricin A chain (dgRTA) have been developed, structurally optimized for stability and activity and evaluated for activity both in vitro, and in vivo in rodents, monkeys and humans (Vitetta et al., 1993; Sausville and Vitetta, 1997; Baluna and Vitetta, 1996).

[0008] It has been postulated that dgRTA-ITs induces VLS by damaging vascular endothelial cells (Soler-Rodriguez et al., 1993; Baluna et al, 1996). IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA) and other toxins, damage human ECs in vitro and in vivo (Dutcher et al., 1991; Rosenberg et a., 1987; Vial and Descotes, 1992). Studies using human umbilical vein ECs (HUVECs) demonstrated that dgRTA or ITs prepared with dgRTA can damage these cells within one hour (Soler-Rodriguez et al., 1993) while the inhibition of protein synthesis required 4 hrs or longer. DgRTA-ITs also interfere with fibronectin (Fn)-mediated adhesion (Baluna et al., 1996). Fn inhibits dgRTA-mediated damage to human umbilical vein endothelial cells (HUVECs) (Baluna et al., 1996). Cell adhesion to Fn is mediated by integrins which recognize RGD and LDV sequences in the Fn molecule (Makarem and Humphries, 1991; Wayner and Kovach, 1992).

[0009] Three MAbs linked to dgRTA have been evaluated in Phase I trials in over 200 patients with relapsed chemorefractory lymphoma, myeloma, Hodgkin's disease and graft vs. host disease (GVHD) (Sausville and Vitetta, 1997). These ITs have shown no evidence of myelotoxicity or hepatotoxicity, but all have induced VLS at the maximum tolerated dose (MTD) as defined by hypoalbuminemia, weight gain, and in the most severe cases, pulmonary edema and hypotension (Baluna et al, 1996). In addition, they have induced myalgia and, in 3% of patients, rhabdomyalyosis at the MTD (Sausville and Vitetta, 1997); this side effect may also be related to VLS and result from muscle edema. Further, aphasias have occurred in <5% of patients, these may be due to edema in the cerebral microvasculture.

[0010] Despite this dose limiting toxicity (DLT) clinical responses using dgRTA-ITs have been encouraging with 15-30% of chemorefractory relapsed lymphoma patients experiencing objective partial or complete response in Phase I clinical trials (Sausville and Vitetta, 1997). However, the DLT, VLS, has decreased the enthusiasm for continuing on to Phase II and III trials in patients.

[0011] Clearly, further development of dgRTA-ITs as well as other ITs containing toxins and RIPs, as well as cytokines as clinical agents would be greatly facilitated by the elimination or reduction of VLS. If VLS could be avoided or reduced it would permit the use of much higher doses of a variety of therapeutic agents such as ITs, gene therapy and cytokines without the dose limiting side effects currently encountered.

SUMMARY OF THE INVENTION

[0012] The present invention overcomes the deficiencies in the art by providing methods for modulating the ability of various proteinaceous compounds to induce toxic effects and proteinaceous compositions that have been modified such that they have a modulated ability to induce toxic effects. In some embodiments, the invention allows for the production of ITs with a reduced ability to promote or induce such toxic effects, including, for example, VLS. ITs made in accordance with the invention are for any number of therapeutic applications, for example, the treatment of GVHD, non-Hodgkin's and Hodgkin's lymphoma, myloma, and some solid tumors. The present invention also provides methods for reducing the VLS promoting ability of proteinaceous compositions through a mutation of sequences that induce or promote any of a number of toxic effects. The present invention provides, for example, ITs, IL-2, TNF, and adenovirus, as well as other proteins or viruses with a reduced ability to promote toxic effects, and methods of using such compounds.

[0013] Amino acids flanking (flanking regions) the (x)D(y) tri-peptide sequence may be altered to reduce a proteinaceous composition's ability to induce VLS. As used herein, a “proteinaceous composition” refers to a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene, a polypeptide of greater than about 100 amino acids, and/or a peptide of from about 3 to about 100 amino acids, including peptides of 3, 4, 5, 6, etc., 10, 11, 12, 13, 14, etc., 20, 21, 22, etc, 30, 40, 50, 60, etc. 100, 110, 120, etc. 200, 220, 240, etc, 300, 350, 400, etc, 500, 600, 700, etc., and 1000 amino acids in length. In certain aspects, the method of altering this sequence and/or flanking amino acids is by removal or substitution of the amino acid or amino acid sequence.

[0014] In certain embodiments, a modified proteinaceous composition may comprise a protein having a (x)D(y) sequence and at least one amino acid mutation that alters the ability of a (x)D(y) sequence to induce VLS. Proteins of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations. Mutation(s) may be in the flanking regions, in the active site of the protein, and/or in a (x)D(y) sequence in the protein. Amino acid(s) defined as being located within the flanking region may include an amino acid located within approximately 10 angstroms of the aspartic acid in the (x)D(y) sequence of the native protein conformation, located greater than approximately 6 angstroms from an enzymatic site residue (active site) of the native protein conformation, located at least partially exposed to the surface of the native protein conformation, or a combination of two or more of these parameters. A mutation in an amino acid in the flanking regions may be in combination with any other mutation described herein. A protein comprising an (x)D(y) may be a toxin, cytokine, or a viral protein. In certain embodiments the protein is a toxin. A toxin includes, but is not limited to a ricin A chain toxin (RTA), Abrin A chain, Diptheria Toxin (DT) A chain, Pseudomonas exotoxin (PE), Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, or Barley Toxin. In particular embodiments, the toxin is a ricin A chain toxin.

[0015] In various embodiments, a modified proteinaceous composition comprises a protein having a (x)D(y) sequence and at least one amino acid mutation in a flanking sequence that alters the toxicity of the protein. The protein(s) of the compositions described herein may further comprise other mutations as described herein. In certain embodiments, such as vaccine compositions and methods of vaccination, an inactivated protein may be preferred (e.g., a protein comprising an inactivating alteration in the active site). Mutant amino acids of the protein(s) may comprise amino acid(s) flanking the (x)D(y) sequence as described above. A protein may be a toxin, cytokine, or a viral protein, as described herein. In various embodiments the toxin is a ricin A chain toxin.

[0016] In various embodiments a ricin A chain toxin comprises at least one mutation in a flanking region and may be in combination with a mutation or alteration in the active site of the protein, which may be used in vaccination against ricin A chain toxin; and/or in a (x)D(y) sequence in the protein, as described herein. In particular embodiments, a mutation in the ricin A chain toxin comprises at least one mutation in an amino acid in the flanking region sequence, which includes, but is not limited to R48, N97 or R48 and N97. Mutation of a protein includes substitution, modification, or deletion of a native amino acid. In particular, the native amino acid may be substituted with an alanine residue. Compositions of the present invention may be provided as a pharmaceutical or pharmaceutically acceptable composition.

[0017] The invention also provides a modified proteinaceous composition that has altered, relative to the sequence of a native proteinaceous composition, at least one amino acid of a sequence comprising (x)D(y), prepared according to the methods described above and elsewhere in this specification in combination with an alteration in a flanking region sequence. In certain embodiments, the proteinaceous composition comprises a toxin, a cytokine, a viral sequence or a combination thereof. In certain aspects, the toxin is, for example, a plant toxin, a fungal toxin, a bacterial toxin, a RIP or a combination thereof. In additional aspects, the toxin comprises Abrin A chain, Diphtheria Toxin (DT) A-Chain, Pseudomonas exotoxin, RTA, Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, Barley toxin or a combination thereof. In other embodiments, the proteinaceous composition comprises a cytokine, such as for example, Interleukin-2. In other aspects, the proteinaceous composition comprises a viral sequence, such as, for example, an adenoviral sequence.

[0018] In certain facets, the composition further comprises an antibody. In particular aspects, the composition further comprises an IT. In additional facets, the IT further comprises at least a second agent, such as, for example, at least one effector molecule. In particular aspects, the effector molecule is a toxin, an anti-tumor agent, a therapeutic enzyme, an antiviral agent, a virus, a cytokine, a growth factor, or a combination thereof. In other facets, the agent is at least one reporter molecule.

[0019] The invention additionally provides an IT, comprising at least one proteinaceous molecule with a reduced ability to induce VLS, apoptosis, disintegrin-like activity or EC damage, wherein the proteinaceous molecule has at least one (x)D(y) and/or flanking region sequence altered.

[0020] In certain embodiments, the composition comprises a therapeutic agent, such as, for example, at least one IT, antibody, cytokine, virus or a combination thereof. In specific embodiments, the composition and the therapeutic agent are covalently conjugated. In particular facets, the composition is a therapeutic agent.

[0021] The invention also provides a RTA with a reduced ability to promote toxicity in a patient, wherein the (x)D(y) sequence comprising positions 74 to 76 is altered. In certain embodiments, the leucine at position 74 is altered, the aspartate at position 75 is altered, and/or the valine at position 76 is altered. In specific facets, the (x)D(y) sequence further comprises positions of from about 1 to about 6 residues of an (x) or an (y) of the (x)D(y) tripeptide sequence. In various embodiments the alterations in the (x)D(y) sequence is in combination with an alteration(s) in a flanking region sequence.

[0022] The invention provides a proteinaceous composition that has a reduced ability to induce VLS. In an aspect, the proteinaceous composition that has been altered to remove at least one amino acid sequence contiguous with the composition comprising the sequence (x)D(y). In certain aspects of the present invention, the proteinaceous composition may be a ribosome-inactivating protein (RIP), including but not limited to gelonin, momordin, pokeweed antiviral protein (PAP), saporin, or trichosanthin; a toxin or toxin subunit, including but not limited to abrin A chain, diphtheria toxin (DT) A-chain, Pseudomonas exotoxin-A (PE38-lys), RTA, Shiga toxin A chain, or barley toxin; a cytokine including but not limited to IL-2. Proteins, polypeptides and/or peptides may be derived from RIPs, toxins or cytokines to be used in the methods and compositions of the present invention. In certain aspects, the proteinaceous composition may be used to make an IT with a reduced ability to promote or enhance VLS. In other aspects, the proteinaceous composition for use in an IT is a RIP and/or toxin sequence.

[0023] Certain embodiments of the invention include a method of reducing the ability of a proteinaceous composition to induce VLS, comprising the steps of: identifying a protein comprising at least one amino acid sequence of (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine; and altering at least one amino acid residue in the flanking regions, as described above, of the (x)D(y) sequence, wherein the ability to induce VLS is reduced.

[0024] Various embodiments include methods of preparing an immunotoxin with a reduced ability to induce VLS comprising the steps of: identifying a toxin comprising at least one amino acid sequence of (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine; altering at least one amino acid residue in the flanking region of the (x)D(y) sequence, as described above, wherein the ability to induce VLS is reduced; and conjugating said toxin to a composition comprising at least one antibody to produce an immunotoxin, wherein the immunotoxin produced possesses a reduced ability to promote VLS when compared to a like immunotoxin, wherein the flanking amino acid sequence was not altered from the toxin.

[0025] In some embodiments, the invention provides a method of modifying the ability of a proteinaceous composition to induce a toxic effect, comprising the steps of: identifying at least one amino acid sequence comprising the sequence (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine; and altering the amino acid sequence comprising the sequence (x)D(y). In certain embodiments, the altering comprises at least one mutation of the amino acid sequence. In other embodiments, the amino acid sequence is removed. In particular aspects, the amino acid sequence comprises the sequence (x)D(y), wherein the (x)D(y) sequence is GDL, GDS, GDV, IDL, IDS, IDV, LDL, LDS, LDV, LDS, VDL or VDV. In certain more specific embodiments, the invention provides a modified proteinaceous composition that has altered, relative to the sequence of a native proteinaceous composition, at least one amino acid of a sequence comprising (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine, for use as a medicament.

[0026] The proteinaceous composition can be any presently known or discovered in the future that has the (x)D(y) tripeptide sequence. In some embodiments, the proteinaceous composition comprises a toxin, a cytokine, a viral sequence or a combination thereof. In particular aspects, the toxin comprises a plant toxin, a fungal toxin, a bacterial toxin, a RIP or a combination thereof. In certain facets, the toxin comprises Abrin A chain, Diphtheria Toxin (DT) A-Chain, Pseudomonas exotoxin, RTA, Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, Barley toxin or a combination thereof. In other embodiments, the proteinaceous composition comprises a cytokine, such as, for example, Interleukin-2. In further embodiments, the proteinaceous composition comprises a viral sequence, such as, for example, an adenoviral sequence. In certain aspects, the proteinaceous composition further comprises, or is comprised in an IT.

[0027] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.

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

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] FIGS. 1A and 1B. The in vivo effect of RFB4-RTA-peptides. (FIG. 1A) SCID mice with vascularized human skin xenografts were injected with 200 μg of RFB4-dgRTA (solid), RFB4-LDV+ (open), RFB4-GQT (cross-hatched) or saline (hatched), and the wet/dry weight ratios of biopsies of the human skin were determined. (FIG. 1B) SCID mice were injected as described in FIG. 1A and the wet/dry weight ratios of lungs were determined. The values represent the mean of three experiments ±SD. The asterisks indicate a statistically significant difference from saline (−) treated mice (*,p<0.02, **p<0.01).

[0031] FIGS. 2A and 2B. Inhibition of the binding of dgRTA and RFB4-LDV+to HUVECs. (FIG. 2A) 105 HuvECs were incubated on ice for 30 min with FITC-dgRTA, in the presence or absence of 100-fold excess of dgRTA (solid), RFB4-LDV+ (crosshatched), RFB4 (shaded), Fn (hatched) or PE38-lys (open) in 100 μl PBS/BSA/Azide. The percent inhibition of binding to HUVECs is presented. The values represent the means ±SD of three studies. (FIG. 2B) The same as FIG. 2A, except the 105 HUVECs were incubated on ice for 30 min with FITC-RFB4-LDV+.

[0032] FIG. 3. Profile of Acid-Treated Sepharose 4B Column-Purification of Ricin

[0033] FIG. 4. Profile of Sephacryl S-200 Column-Separation of RCA-1 and RCA-2.

[0034] FIG. 5. Profile of DEAE Sepharose Column and Acid-Treated Sepharose 4B Column-Separation of dgRTA and dgRTB Chains.

[0035] FIG. 6. Profile of Blue-Sepharose CL-4B Column—Purification of dgRTA.

[0036] FIG. 7. Profile of Asialofetuin-Sepharose Column—Purification of dgRTA.

[0037] FIG. 8. Ribbon diagram of RTA. Ribbon representation of the X-ray crystallographic structure of ricin A chain, with active site residue side chains, the LDV motif, and R48 and N97.

[0038] FIG. 9. In vivo VLS effect of RFB4-RTA ITs. The 125I-albumin retention in the lungs of SCID mice treated with various RTA-ITs was normalized by weight to retention by PBS-treated mice for comparison. (n=number of mice. P values compare each group with the PBS group.)

[0039] FIG. 10. SCID/Daudi survival curves. SCID mice (n=10) treated as described in Methods were monitored for survival or paralysis, either of which is an endpoint in this model. (open diamond) PBS, (closed diamond) RFB4, (open triangle) RFB4-R48A, (closed triangle) RFB4-N97A, (open square) RFB4-wt rRTA. P values (by log-rank or Wilcoxon tests): RFB4-R48A vs RFB4, P=0.0093 or P=0.0037, RFB4-N97A vs RFB4, P=0.0018 or P=0.00012, RFB4-wt rRTA vs RFB4, P=0.0022 or P=0.00012.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0040] Cell damage, particularly endothelial cell damage, whether produced by toxins, such as from snake bites or molecules causing septic shock, or therapeutic agents, such as ITs or interleukins, remains a problem for patients. Types of cell damage include VLS, disintegrin-like activities and apoptosis.

[0041] To devise methods and compositions to alleviate VLS, candidate sequences of molecules which cause VLS were evaluated to determine whether RTA, toxins, RIPS and IL-2 might share structural motifs responsible for interfering with cell-cell and cell-matrix interactions, and thereby damage human ECs. In comparing the sequences of VLS-inducing toxins, RIPS and IL-2, a (x)D(y) consensus motif was identified where (x) could be L, I, G or V and (y) could be V, L or S. In the case of RTA and IL-2, molecular modeling indicated that these motifs were completely or substantially exposed on the surface of their respective molecules. A similar motif is shared by viral disintegrins, which disrupts the function of integrins, indicating that RTA, IL-2 and perhaps other toxins may damage ECs by virtue of their (x)D(y) motifs and hence, may be disintegrins.

[0042] This vascular leak promoting activity of this motif was surprising and unexpected, since LDV homologue sequences also play a role in the vascular functions of a variety of non-toxic molecules including vascular cell adhesion molecule 1 (VCAM-1), which contains the IDS sequence, and the γ chain of fibrinogen, which contains the GDV sequence (Clements et al., 1994). LDV constitutes the minimal active site in the CS1 domain of fibronectin responsible for its binding to the α4β1 integrin receptor (Makarem and Humphries, 1991; Wayner and Kovach, 1992; Nowlin et al., 1993). Though fibronectin possesses this sequence, it does not damage HUVECs. Instead, FN protects HUVECs from RTA-mediated damage (Baluna et al., 1996), in direct contrast to the VLS activity of toxic agents that possess this motif.

[0043] To determine whether this motif was responsible for EC damage, short LDV or LDL containing peptides from RTA or IL-2, respectively, were generated, attached to a mouse MAb and studied for the ability to bind to and damage HUVECs in vitro and to damage mouse lung vasculature and human vasculature in skin xenografts in vivo. One active site mutant of RTA and several LDV mutants were generated. These LDV mutants contained conservative changes which, when modeled, would not be expected to affect the active site of the RTA. Antibody-conjugated peptides from RTA containing the sequence (L74, D75, V76), but not peptides with deleted or altered sequence, induced EC damage in vitro and vascular damage in vivo in the two animal models (Baluna et al., 1999). These results demonstrated that the VLS-inducing site does not require the active site. It is contemplated that the noncontiguous active site of the RTA, which does not encompass LDV, is either not required to damage ECs, or only partly contributes to vascular damage.

[0044] These results demonstrate that an active site may not be required to induce vascular damage, and that one or more active peptides or polypeptides may be made with reduced VLS promoting activity. With this discovery, it is now possible that one or more amino acid deletion(s) or mutation(s) of the (x)D(y) sequence(s), and/or of a flanking region sequence, may reduce or prevent VLS and improve the therapeutic index or the tolerated dose of VLS-inducing molecules. It is expected that one or more peptides and small molecule drug inhibitors comprising at least one mutated motif and/or one or more flanking region sequence can be created that reduce or eliminate the VLS induced by VLS promoting agents.

[0045] In various embodiments, other residues that are positioned in the physical region, space, or vicinity of the active site and/or the (x)D(y) motif may be mutated or altered to abrogate, reduce, or eliminate VLS. The amino acids targeted for mutation in the flanking regions include amino acids on or near the surface of a native protein, in particular RTA and derivatives thereof. The alteration may remove or substitute a charged residue in the region of a (x)D(y) motif, which may negate or reverse the charge in a particular area on the surface of the protein. The alteration may also change size and/or hydrophilicity of an amino acid in the physical region, space or vicinity of the (x)D(y) sequence or active site of a protein. Two exemplary flanking residues are arginine 48 (R48) and asparagine 97, which are found physically adjacent to the (x)D(y) motif in the three dimensional structure of RTA.

[0046] In certain embodiments, it is contemplated that disintegrin or disintegrin-like activity of proteinaceous compositions may be reduced or enhanced. Disintegrins possess various activity(ies) including an ability to damage ECs, an ability to interfere with cell adherence and/or an ability to interfere with platelet aggregation. It is contemplated that one or more amino acid deletion(s) or mutation(s) of the (x)D(y) sequence(s), and/or one or more flanking region residues, may reduce or prevent the disintegrin-like activity of one or more molecules comprising these sequences. It is expected that one or more peptides and small molecule drug inhibitors comprising at least one mutated motif and/or one or more flanking sequence can be created that reduce or eliminate the disintegrin-like activity of such agents.

[0047] Additionally, the LDV site of RTA induced apoptosis in ECs. It has been reported that many toxins and ITs induce apoptosis as well as inhibit protein synthesis. It is also contemplated that one or more amino acid deletion(s) or mutation(s) of the (x)D(y) sequence(s), and/or at least one flanking region residue, may reduce or prevent the apoptotic activity of one or more molecules comprising these sequences. It is expected that one or more peptides and small molecule drug inhibitors comprising at least one mutated motif and/or at least one flanking sequence can be created that reduce or eliminate the apoptotic activity of such agents. Thus apoptotic activity may cause or contribute to toxin or cytokine induced VLS.

[0048] It is also contemplated that one or more amino acid deletion(s) or mutation(s) of the (x)D(y) sequence(s), and/or one or more flanking region residues, may reduce or prevent the ability of molecules comprising these sequences to induce EC damage. It is expected that one or more peptides and small molecule drug inhibitors comprising at least one mutated motif and/or one ore more flanking residues can be created that reduce or eliminate the EC damaging activity of such agents.

[0049] Described herein are methods for generating and compositions comprising agents with reduced or enhanced VLS promoting abilities based upon mutations in the (x)D(y), (x)D(y)T, or flanking regions of (x)D(y) motifs within proteins, polypeptides, peptides or other proteinaceous materials which remove or add such sequences, respectively. It is contemplated that the same mutations described for reducing or enhancing VLS promoting ability will also reduce or enhance, respectively, the apoptotic activity, EC damaging and/or one or more disintegrin-like activities of polypeptides, peptides or proteins. Thus, it will be understood that all methods described herein for producing proteins, polypeptides and peptides with enhanced or reduced VLS promoting ability will be applied to produce proteins, polypeptides and peptides with reduced apoptotic activity, EC damaging and/or one or more disintegrin-like activities. All such methods, and compositions identified or produced by such methods, are encompassed by the present invention.

[0050] A. Identification of an (X)D(Y) Motif in VLS-Inducing Agents

[0051] Homologous structural motifs in RTA, other toxins, RIPs and IL-2, which may affect cell-cell and cell-matrix interactions and thereby damage human ECs, have been identified and tested for their ability to promote VLS in model systems. The (x)D(y) motif where x=L, I, G or V and y=V, L or S (Table 1) is common in the sequences of RTA, other toxins, RIPs and cytokines which induce VLS. This motif is also shared by viral disintegrins which disrupt the function of integrins (Coulson et al., 1997).

TABLE 1
Non-Limiting Examples of (x)D(y) Motifs in Molecules Which Induce VLS
				GenBank or GenPept
Category	Agent inducing VLS	(X)D(Y) Motif	Location	Accession #
Toxins1	Abrin A chain	IDV	68-70	X76721
		GDL	114-116
		VDS	229-231
	Barley toxin	LDV	171-173	U77463
	Diphtheria Toxin (DT) A-Chain	VDS	6-8	576189
		VDS	28-30
		IDS	289-291
		LDV	441-443
	Pseudomonas exotoxin-(PE38-lys)2	GDL	348-350	K01397
		GDV	430-432
		GDL	605-607
	Ricin Toxin A-Chain (RTA)	LDV	74-76	A23903
	Shiga toxin A chain	VDS	36-38	M19437
		IDS	63-65
		VDV	74-76
		GDS	132-134
		LDL	162-164
		VDL	219-221
RIPs3	Gelonin	IDV	114-116	L12243
	Momordin	LDV	64-66	576194
		LDS	132-134
	Momordin	LDS	165-167	P16094
	Pokeweed Antiviral Protein (PAP)	VDS	179-181	X98079
		GDL	308-310
	Saporin	LDL	6-8	X69132
		IDL	143-145
	Trichosanthin	GDV	23-25	U25675
		IDV	87-89
		LDS	155-157
Cytokines	Interleukin-2 (IL-2)	LDL	19-21	1311005
1The enzymatically active chain of the holotoxin
2PE38 refers to enzymatically active Domain III (residues 405 to 613) plus residues 253-354 and 381-404 in PE.
3Ribosome-inactivating proteins (RIPs) which are homologues of the enzymatically active A chains of plant toxins

[0052] 1. Localization of (x)D(y) Motifs in RTA, Disintegrins, PE38-lys and IL-2

[0053] With the discovery of the importance of the (x)D(y) sequence in promoting VLS, it is now possible to create RTA mutants which will retain their enzymatic activity, which is important for making effective ITs, but which also have their VLS-inducing properties reduced.

[0054] The LDV motif in RTA (residues 74-76, SEQ ID NO:1) is at the C-terminus of a β-strand of the first domain near the Tyr-80 residue which is involved in the active site (Mlsna et al., 1993). The active site (residues 80, 123, 177, 180, 209 and 211) of the enzyme does not include the LDV sequence so that the enzymatic activity of RTA should not be affected by mutations or deletions in this sequence (Munishkin and Wool, 1995).

[0055] To examine the crystal structure of RTA and IL-2, space filling models of the three dimensional structures of RTA (PDB accession number lbr5.pdb) and IL-2 (PDB accession number 1irl.pdb) were compared. Specifically, the atoms of the LDV residues of RTA, atoms of the LDL residues of IL-2, and the atoms of the active site residues of RTA (Y80, Y123, E177, R180, N209 and W211) were analyzed regarding their relative positioning in the structure. The models were generated with the Insight II program (MSI). Examinations of the crystal structure of RTA indicate that this motif is only partially exposed, but structural fluctuations in the molecule may increase its accessibility. From this and other data described herein, it is contemplated that either alterations in the (x)D(y) motif, the C-terminal flanking amino acid(s), the N-terminal flanking amino acid(s), the flanking region sequence or a combination thereof, may result in the loss of VLS-inducing activity by a variety of agents.

[0056] Another family of proteins called disintegrins usually contain an RGD sequence. In the case of one disintegrin, which is present in rotavirus, an LDV sequence is present (Coulson et al, 1997). Disintegrins damage ECs or interfere with cell adherence and/or platelet aggregation (McLane et al., 1998; Huang, 1998; Tselepis et al., 1997). In the snake venom disintegrin kistrin, LDV can be substituted for RGD without compromising disintegrin function (Tselepis et al., 1997). Thus, RTA and a variety of other molecules may be disintegrins which share properties with kisrtin (Blobel and White, 1992; Lazarus and McDowell, 1993) in damaging human ECs. In certain embodiments, it is contemplated that disintegrins or molecules that possess disintegrin-like activity may be altered or produced to possess a reduced ability to damage ECs, a reduced ability to interfere with cell adherence and/or a reduced ability to interfere with platelet aggregation. Such molecules may be produced by mutating at least one residue in the (x)D(y) sequence or at least one flanking residue. It is also contemplated that peptides or peptide mimics of the (x)D(y) and/or flanking sequences may be made that block the activity of disintegrins.

[0057] In PE38-lys the GDL sequence is distal from the active site (Li et al, 1995). Thus, it is contemplated that PE38-lys may be similarly mutated to reduce or eliminate its VLS promoting activity without completely eliminating its activity.

[0058] In IL-2, the LDL sequence at residues 19-21 (SEQ ID NO:2) is located in an α-helix and is also partially exposed. A mutation in Asp-20, in the LDL motif (Table 1) eliminates binding of IL-2 to the β chain of the IL-2 receptor and subsequent cell proliferation (Collins et al., 1988). It has been reported that IL-2 directly increases the permeability of the vascular endothelium to albumin in vitro and that this effect can be inhibited by anti-IL-2 receptor MAbs (Downie et al., 1992). The results of Example 1 demonstrate that the LDL sequence in IL-2 damages HUVECs. However, in contrast to RTA, the Asp-20 in the LDL of IL-2 is involved in receptor binding and functional activity (Collins et al., 1988). Thus, it is contemplated that in certain embodiments, mutations in IL-2's (x)D(y) sequence and/or flanking sequence(s) to eliminate or reduce VLS must preserve the Asp-20 or the biological activity of IL-2 may be reduced.

[0059] 2. Mutations in Flanking Sequences

[0060] The (x)D(y) sequence may not be solely responsible for the promotion of VLS. In certain embodiments, it is contemplated that additional sequences that flank the (x)D(y) sequence may be mutated to enhance or reduce a peptide, polypeptide or protein's ability to promote VLS. These flanking sequences may include amino acids that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from an (x)D(y) sequence. As well as amino acids located within the flanking region approximately 10 angstroms from the aspartic acid in the (x)D(y) sequence in the native protein conformation, located greater than approximately 6 angstroms from an active site residue of the native protein conformation, located at least partially exposed to the surface of the native protein conformation, or a combination of two or more of these parameters.

[0061] For example, LDV constitutes the minimal active site in the CS1 domain of fibronectin responsible for its binding to the α4β1 integrin receptor (Makarem and Humphries, 1991; Wayner and Kovach, 1992; Nowlin et al, 1993). However, fibronectin (FN) does not damage HUVECs. Instead, FN protects HUVECs from RTA-mediated damage (Baluna et al, 1996). Unlike RTA, FN has a C-terminal LDV-flanking proline instead of a threonine.

[0062] In disintegrins, residues flanking RGD, play a role in ligand binding (Lu et al., 1996). The difference between the ability of an LDV or homologue-containing molecule to promote vascular integrity (e.g., FN) or disrupt it (e.g., RTA) may depend on the orientation, or availability for interaction (i.e., binding), of the LDV motif and hence, on flanking sequences. Therefore, a change in one or more amino acids of this sequence or one or more amino acids of the N- or C-terminal flanking sequences may convert a molecule from one that damages endothelial cells (distintegrin-like) to one that enhances their growth. It is contemplated that changes in one or more flanking residues of the (x)D(y) sequence may enhance or reduce the ability of a molecule to promote VLS. It is further contemplated that changes that expose the (x)D(y) sequence to the external surface of the protein so as to interact with other proteins, such as receptors, would enhance VLS promoting activity, while conformations that are less exposed may reduce VLS promoting activity.

[0063] a. Ricin A Chain—Structural Analysis to Identify Candidate Residues for VLS Mutations

[0064] Identification of an amino acid as a flanking region residue may be determined by at least one of the ricin A chain crystal structures, which are available from NCBI (GenBank), http://www.ncbi.nlm.nih.gov/Structure/. These two exemplary structures are two structures of RTA alone, without mutation or co-crystallization with other entities in a complex. These structures may be analyzed and manipulated by using the software available from the NCBI, Cn3D v.4.0. Exemplary sequences or structures that may be used include, but are not limited to RTA (GenBank accession number 1RTC) D. Mlsna, A. F. Monzingo, B. J. Katzin, S. Ernst & J. D. Robertus, 29-Oct-92; and rRTA (GenBank accession number: 11FT) S. A. Weston, A. D. Tucker, D. R. Thatcher, D. J. Derbyshire & R. A. Pauptit, 5-Jul-96, as well as other sequences identified as containing an (x)D(y) sequences that are readily available in these and other databases.

[0065] Methods for identifying residues that, when altered, may eliminate, reduce or abrogate VLS include, but are not limited to a) identifying all residues within 10 A of a (x)D(y) sequence and in particular an LDV sequence—e.g., determining the distance of a residue from the aspartic acid (D), RTA D75 in particular; b) determining, for each residue, the distance from the ‘active site’—e.g., the six major active site residues of RTA (Y80, Y123, E177, R180, N209, W211) were selected and the distance of each candidate was ‘measured’ to the nearest point of the active site; c) visually inspecting, for each residue, surface exposure—rated ‘Y’—yes, ‘P’—partial, or ‘N’—no; and/or d) visually inspecting each residue for where on the surface of the protein they appear, rated as ‘F’ for front face, or ‘B’ for backside, or both (either on the ‘edge’ or extending through the molecule) relative to (x)D(y), LDV and/or the active site.

[0066] In the case of RTA, tables comparing residues from separate RTA structures may be compiled and respective distances between amino acid analyzed and confirmed. Typically, surface/buried front/backside differences are evaluated and, for each residue, a consensus is reached (due to the subjective nature of these evaluations, the differences, for example, between partial and exposed, may be hard to define—any exposure to the surface is typically classified as at least ‘partially’ exposed. An exemplary list for RTA is provided in Table 2 (80 residues, including LDVT and four residues of the active site (AS) within 10 Å of LDV are provided).

[0067] In addition, the methods may also include e) removing from the list all ‘turn’ residues: such as P (proline) G (glycine) (8 residues in the case of RTA), generally, these cannot be changed without completely disrupting the integrity of the molecule; f) removing from the list all buried residues (those labeled ‘N’ from above; in the case of RTA 17 residues, L74 is for comparison purposes) and all residues exposed exclusively on the backside of the molecule (as determined above, in the case of RTA 27 residues, V76 is for comparison purposes); g) removing from the list, all residues within 6 A of the active site; this distance was arbitrarily chosen but based on the loss of activity of the RTA D75 mutant, which is 3.0 A away, and the retention of RTA activity of two V76 mutants, which is 6.0 A away (6 residues). For example, this method reduces the exemplary list in Table 2 to the exemplary list is Table 3 (22 residues, which still includes LDVT and active site residues for comparison).

[0068] The remaining 14 residues (minus LDVT and active site) are listed in Table 4 roughly in order of distance from the LDV region of RTA. This conservative list of residues, when altered may eliminate, reduce, or abrogate the VLS activity of RTA, although alteration of other residues from Table 2 and 3 may also have the appropriate result. This selection was typically based on 1) proximity to the (x)D(y), in the case of RTA the LDV region; 2) distance from the active site; and 3) surface exposure on the same face of the molecule as the (x)D(y) sequence. For example, both N97 and R48 of RTA, which meet these structural criteria, have a reduced, eliminated, or abrogated VLS while retaining RTA activity. One or more of these alterations, as described herein, may be used alone or in combination with other alterations to produce a protein with a reduced toxicity, a reduced VLS character, a reduced enzymatic activity, or a combination thereof.

TABLE 2
An exemplary compiled list of amino acid distances for RTA
Residue#	Å from D	Å from LDV	Å from AS	Surface	Face
Y6	9.0-9.5	7.5-8.0	12.0-12.5	Y	B
P7	10.0	7.5	12.0	Y	B
I8	12.0-12.5	9.5	14.0	Y	B
I9	10.0-11.0	6.5-7.0	12.0	Y	B
F24	12.5	7.0-7.5	9.5	P	B
I25	13.0	8.5	6.5	P	B
A27	12.0	9.0	11.0	Y	B
V28	9.0	4.5-5.0	7.5	N
R29	10.5	7.5-8.0	5.0	Y	B
G30	11.5	8.0-8.5	10.0	Y	B
R31	8.5	4.5	11.5	Y	B
L32	4.5-5.0	4.0	6.5	N
T33	9.0	6.0-6.5	9.0-10.5	P	B
T34	11.0-11.5	7.0	13.0	Y	B
P43	12.0-12.5	9.5	7.0-7.5	Y	B
V44	11.0	7.5	8.0	P	B
L45	6.0	3.5	4.0	P	Both
P46	7.5	3.5-4.0	9.0-9.5	Y	Both
N47	8.0-8.5	7.0	11.0	Y	F
R48	5.5-7.0	5.5-7.0	11.0-11.5	Y	F
V49	9.5	9.5	14.0	Y	F
G50	11.5	10.0	16.7-17.0	Y	F
L51	6.0	4.0-4.5	11.5	Y	Both
P52	9.0-9.5	7.5-8.0	14.5	Y	Both
I53	9.0	8.5	13.0	Y	F
N54	10.5	8.5	14.5	Y	B
Q55	7.5	4.0	11.5-12.0	Y	B
R56	3.0-3.5	3.0-3.5	8.0	Y	F
F57	4.0-4.5	3.5	6.5-7.0	P	B
I58	5.5	3.0-3.5	7.5	P	B
L59	8.0	5.5	9.5	Y	B
V60	9.0	4.5	9.5	N
E61	12.5	8.0-8.5	12.5-13.0	Y	B
L62	13.0-13.5	8.0-8.5	12.0	P	B
V70	14.0	10.0	13.0	P	B
T71	11.0	7.5-8.0	10.5-11.0	Y	B
L72	7.5-8.0	4.0-4.5	7.5	N
A73	4.5	1.5	4.0	N
L74	1.5	—	3.5	N
D75	—	—	3.0-3.5	Y	F
V76	1.5	—	6.0	Y	B
T77	3.0	1.5	5.0-5.5	Y	F
N78	3.5-4.0	3.5	3.0	Y	F
A79	3.0	3.0	1.5	N
Y80	3.0-3.5	3.0-3.5	-	Y	F
V81	5.5	4.0-4.5	1.5	Y	F
V82	4.0	4.0	3.5	Y	F
G83	7.5	6.0	6.5	N
Y84	10.5	8.0	9.5	N
R85	12.5-13.0	9.5-10.0	12.5	Y	B
Y91	11.5-12.0	10.0-10.5	11.0	Y	B
F92	10.0	9.0	10.0	Y	F
F93	9.5-10.0	9.5-10.0	5.5	P	F
P95	9.5	9.5	10.5-11.0	Y	F
D96	8.5-9.5	8.5-9.5	9.0	Y	F
N97	8.5	8.5	12.0	Y	F
E99	8.5	8.5	13.5	Y	F
D100	5.0	5.0	10.0	P	F
A101	9.0	9.0	13.0	Y	F
E102	10.0-10.5	10.0-10.5	14.0-14.5	Y	F
A103	6.5-7.0	6.5-7.0	11.5	Y	B
I104	5.5-7.0	5.5-7.0	8.5-9.0	Y	B
L107	7.5	6.5-7.0	9.0-9.5	Y	B
F108	10.0	7.0-7.5	10.5	Y	B
F168	12.0-12.5	10.0	6.0	N
C171	11.5-12.0	7.5	6.0-6.5	N
I172	10.0-10.5	8.0	3.0-3.5	N
Q173	13.0	9.5-10.0	3.5	N
M174	13.5	9.0	5.5	N
I175	9.5	4.5	3.5-4.0	N
S176	7.0-7.5	4.5-5.0	1.5	N
E177	10.5	8.0	-	Y	F
A178	12.5	9.5	1.5	N
A179	9.5	6.5-7.0	1.5	N
R180	7.5-8.0	7.5-8.0	-	Y	F
W211	8.0	7.5	-	Y	F
L254	8.5	7.0-7.5	3.0	Y	B
M255	10.0	7.0-7.5	4.0	N
V256	9.0-9.5	7.5-8.0	3.5-4.0	Y	F
Y257	10.5	8.0-8.5	6.5	Y	F

[0069]

TABLE 3
An exemplary compiled list of amino acid distances
for RTA using additional criteria.
Residue#	A from D	A from LDV	A from AS	Surface	Face
N47	8.0-8.5	7.0	11.0	Y	F
R48	5.5-7.0	5.5-7.0	11.0-11.5	Y	F
V49	9.5	9.5	14.0	Y	F
L51	6.0	4.0-4.5	11.5	Y	Both
I53	9.0	8.5	13.0	Y	F
R56	3.0-3.5	3.0-3.5	8.0	Y	F
L74	1.5	—	3.5	N
D75	—	—	3.0-3.5	Y	F
V76	1.5	—	6.0	Y	B
T77	3.0	1.5	5.0-5.5	Y	F
Y80	3.0-3.5	3.0-3.5	—	Y	F
F92	10.0	9.0	10.0	Y	F
D96	8.5-9.5	8.5-9.5	9.0	Y	F
N97	8.5	8.5	12.0	Y	F
E99	8.5	8.5	13.5	Y	F
D100	5.0	5.0	10.0	P	F
A101	9.0	9.0	13.0	Y	F
E102	10.0-10.5	10.0-10.5	14.0-14.5	Y	F
E177	10.5	8.0	—	Y	F
R180	7.5-8.0	7.5-8.0	—	Y	F
W211	8.0	7.5	—	Y	F
Y257	10.5	8.0-8.5	6.5	Y	F

[0070]

TABLE 4
An exemplary compiled list of particular
flanking residues for RTA
Residue#	A fromD	A from LDV	A from AS	Surface	Face
R56	3.0-3.5	3.0-3.5	8.0	Y	F
D100	5.0	5.0	10.0	P	F
R48	5.5-7.0	5.5-7.0	11.0-11.5	Y	F
L51	6.0	4.0-4.5	11.5	Y	Both
N47	8.0-8.5	7.0	11.0	Y	F
N97	8.5	8.5	12.0	Y	F
E99	8.5	8.5	13.5	Y	F
D96	8.5-9.5	8.5-9.5	9.0	Y	F
I53	9.0	8.5	13.0	Y	F
A101	9.0	9.0	13.0	Y	F
V49	9.5	9.5	14.0	Y	F
F92	10.0	9.0	10.0	Y	F
Y257	10.5	8.0-8.5	6.5	Y	F
E102	10.0-10.5	10.0-10.5	14.0-14.5	Y	F

[0071] B. Production of Compositions with Altered VLS Activity

[0072] With the identification of the (x)D(y) and the (x)D(y)T motifs as inducing VLS, inducing apoptosis, and other effects, it is possible that the creation of a new family of molecules of VLS inhibitors will allow these molecules to exert maximal beneficial effects. For example, a reduced toxicity of anti-cancer therapeutic agents using the compositions and methods disclosed herein may allow larger tumors or more advanced disease to be treated. It is now possible to identify or synthesize small drug molecule(s) which block the interaction between cells and VLS promoting, apoptosis promoting, EC damaging, and/or distintegrin-like molecules. In certain embodiments, peptides or drug-mimetics based on the (x)D(y) and/or (x)D(y)T motif or its flanking sequences may be used to inhibit VLS or other activities in vivo. It is possible to create peptides or peptide-carrier conjugates which compete with the LDV motif binding site on endothelial cells and prevent VLS or other actions in a variety of other situations including sepsis, IL-2 therapy, etc.

[0073] In certain embodiments, it is also possible that one or more (x)D(y), (x)D(y)T motifs and/or particular flanking sequences added to larger molecules will increase extravasation into tissues. In light of the present disclosure, peptides containing (x)D(y) and/or (x)D(y)T sequences being tested as anti-inflammatory or anti-metastatic agents (Jackson et al., 1997; Maeda et al., 1997; Greenspoon et al., 1994) should be monitored for both increased extravasation and for toxic effects on vasculature. However, in certain embodiments, it may be desirable to produce proteinaceous compositions that enhance extravasation into tissues. Improvement in extravasation of a therapeutic composition, or promoting extravasation for a therapeutic composition with a protein, polypeptide or peptide of the present invention may allow greater access of the therapeutic agent to tissues. Thus, methods of enhancing or decreasing extravasation of one or more proteins, polypeptides, peptides or therapeutic agents are provided. Preferred therapeutic agents include, but are not limited to one or more ITs, antibodies, cytokines, virus, drugs and the like.

[0074] To produce peptides, polypeptides or proteins that lack the (x)D(y) and/or (x)D(y)T sequence, one could delete or mutate the conserved aspartic acid (D), substitute another amino acid for the aspartic acid, or insert one or more amino acids at or adjacent to its position. Any amino acid that may replace the (D) residue in the sequence as a consequence of a deletion or mutation event.

[0075] Alternatively the (x) residue could be deleted, substituted, or moved by the insertion of one or more amino acids, to remove the (x)D(y) and/or (x)D(y)T sequence. Any amino acid that may replace the (x) residue in the sequence as a consequence of the deletion or mutation event is preferably not leucine (L), isoleucine (I), glycine (G) or valine (V).

[0076] Or the (y) residue could be deleted, substituted, or moved by the insertion of one or more amino acids, to remove the (x)D(y) and/or (x)D(y)T sequence. Any amino acid that may replace the (y) residue in the sequence as a consequence of the deletion or mutation event is preferably not valine (V), leucine (L) or serine (S).

[0077] Additionally, the (x)D(y) and/or (x)D(y)T sequences can be removed by any mutation that alters or changes this sequence. Such mutations include but are not limited to truncations, insertions, substitutions and deletions of amino acids. It is contemplated that chemical modification may also alter a (x)D(y) and/or (x)D(y)T sequence to reduce its ability to induce or promote VLS.

[0078] Thus, it is contemplated that such mutations that affect the (x)D(y) sequence or flanking sequence may alter the ability of a polypeptide to promote VLS or other abilities associated with these sequences. For example, one preferred agent that produced VLS is abrin A chain (GenBank Accession number X76721; SEQ ID NO:3), which contains an IDV sequence at positions 68-70 of its amino acid sequence. A glycine (G) is at position 67. Therefore, a deletion of the isoleucine at position 68 would result in the glycine at position 67 to be directly adjacent to the aspartic acid residue (D) at original position 69. The new sequence created would then be GDV at positions 67-69 of the mutated abrin A chain. This new tripeptide sequence still matches the VLS-inducing sequence (x)D(y) and/or (x)D(y)T. However, it is contemplated that since such a deletion would shift the position of the tri-amino acid sequence in the structure of the mutated abrin A chain protein, polypeptide or peptide being produced. A shift in the position of the tri-amino acid sequence may move it into a less favorable position to contact any cell, receptor or molecule to promote or induce VLS. The resulting mutated abrin A chain protein, polypeptide or peptide may have a reduced ability to promote or induce VLS, and thus would be encompassed by the present invention.

[0079] Similarly, other toxins or compounds that induce VLS, including but not limited to those listed in Table 1, can be mutated so that one or more (x)D(y) and/or one or more flanking residues are removed (i.e., mutated). However, it is contemplated that to produce toxins or compounds that have a reduced ability to induce VLS, it is preferable that any remaining (x)D(y) and/or (x)D(y)T sequences to have a reduced exposure to the surface of the protein, polypeptide or peptide.

[0080] For example, it is contemplated that (x)D(y) and/or (x)D(y)T sequences that are at least partly located in the non-exposed portions of a protein, polypeptide or peptide, or otherwise masked from full or partial exposure to the surface of the molecule, would interact less with cells, receptors or other molecules to promote or induce VLS. Thus, it is contemplated that the complete elimination of (x)D(y) and/or (x)D(y)T sequences from the primary structure of the protein, polypeptide or peptide is not necessary to produce toxins or molecules with a reduced ability to induce or promote VLS. However, removal of all (x)D(y) and/or (x)D(y)T sequences is preferred to insure the composition has the least ability to induce or promote VLS.

[0081] To determine whether a mutation would likely produce a protein, polypeptide or peptide with a less exposed (x)D(y) and/or (x)D(y)T motif, the putative location of the moved or added (x)D(y) and/or (x)D(y)T sequence could be determined by comparison of the mutated sequence to that of the unmutated protein, polypeptide or peptide's secondary and tertiary structure, as determined by such methods known to those of ordinary skill in the art including, but not limited to, X-ray crystallography, NMR or computer modeling. Computer models of various polypeptide and peptide structures are also available in the literature or computer databases. In a non-limiting example, the Entrez database (http://www.ncbi.nlm.nih.gov/Entrez/) may be used by one of ordinary skill in the art to identify target sequences and regions for mutagenesis. The Entrez database is crosslinked to a database of 3-D structures for the identified amino acid sequence, if known. Such molecular models may be used to identify (x)D(y), (x)D(y)T and/or flanking sequences in peptides and polypeptides that are more exposed to contact with external molecules, (e.g. receptors) than similar sequences embedded in the interior of the polypeptide or polypeptide. It is contemplated that (x)D(y), (x)D(y)T and/or flanking sequences that are more exposed to contact with external molecules are more likely to contribute to promoting or reducing VLS and other toxic effects associated with these sequences, and thus should be primary targets for mutagenesis. In certain embodiments, when adding at least one (x)D(y), (x)D(y)T and/or flanking sequence is desirable, regions of the protein that are more exposed to contact with external molecules are preferred as sites to add such a sequence. The mutated or wild-type protein, polypeptide or peptide's structure could be determined by X-ray crystallography or NMR directly before use in in vitro or in vivo assays, as would be known to one of ordinary skill in the art.

[0082] Once an amino acid sequence comprising a (x)D(y) and/or (x)D(y)T sequence is altered in a peptide, polypeptide or protein, or added to a peptide, polypeptide or protein, changes in its ability to promote at least one toxic effect may be assayed by any of the techniques described herein or as would be known to one of ordinary skill in the art.

[0083] As used herein, “alter”, “altered”, “altering”, “alteration” of an amino acid sequence comprising a (x)D(y) sequence, a (x)D(y)T, or a flanking region sequence may include chemical modification of an amino acid sequence comprising a (x)D(y), a (x)D(y)T, and/or a flanking region sequence in a protein, polypeptide or peptide as would be known to those of ordinary skill in the art, as well as any mutation of such an amino acid sequence including but not limited to insertions, deletions, truncations, or substitutions. It is preferred that such changes alters at least one toxic effect (i.e., the ability to promote VLS, EC damage, apoptosis, disintegrin-like activity) of one or more amino acid sequence(s) comprising a (x)D(y) and/or (x)D(y)T sequences. As used herein an amino acid sequence comprising a (x)D(y) sequence or a (x)D(y)T sequence may comprise at least one flanking sequence C- and/or N-terminal to a (x)D(y) and/or a (x)D(y)T tri- or quatra-peptide sequence. Such an “alteration” may be made in synthesized peptides, or in nucleic acid sequences that are expressed to produce mutated proteins, polypeptides or peptides.

[0084] In an aspect of the invention, the alteration of an amino acid sequence comprising a (x)D(y), a (x)D(y)T, and/or a flanking region sequence comprises removal of the amino acid sequence. As used herein “remove”, “removed”, “removing” or “removal” of an amino acid sequence comprising a (x)D(y), a (x)D(y)T, and/or a flanking region sequence refers to a mutation in the primary amino acid sequence that eliminates the presence or reduces the activity of the (x)D(y) and/or a (x)D(y)T tri- or quatra-peptide sequence, and/or at least one native flanking sequence. The terms “removed” or “lacks” may be used interchangeably.

[0085] For example, it is contemplated that mutations including but not limited to at least one insertion or substitution of at least one amino acid selected from the group phenylalanine (F); cysteine/cystine (C); methionine (M); alanine (A); threonine (T); serine (S); tryptophan (W); tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine (O); aspartic acid (D); asparagine (N); lysine (K); and arginine (R), and including, but not limited to, those shown in Table 1 at the position (x) of one or more (x)D(y) and/or (x)D(y)T sequences would reduce its ability to promote VLS. Table 5 below lists exemplary, but not limiting, modified or unusual amino acids that are contemplated as useful in certain aspects of the invention.

TABLE 5
Modified and Unusual Amino Acids
Abbr. Amino Acid
Aad   2-Aminoadipic acid
Baad  3-Aminoadipic acid
Bala  β-alanine, β-Amino-propionic acid
Abu   2-Aminobutyric acid
4Abu  4-Aminobutyric acid, piperidinic acid
Acp   6-Aminocaproic acid
Ahe   2-Aminoheptanoic acid
Aib   2-Aminoisobutyric acid
Baib  3-Aminoisobutyric acid
Apm   2-Aminopimelic acid
Dbu   2,4-Diaminobutyric acid
Des   Desmosine
Dpm   2,2′-Diaminopimelic acid
Dpr   2,3-Diaminopropionic acid
EtGly N-Ethylglycine
EtAsn N-Ethylasparagine
Hyl   Hydroxylysine
Ahyl  Allo-Hydroxylysine
3Hyp  3-Hydroxyproline
4Hyp  4-Hydroxyproline
Ide   Isodesmosine
Aile  Allo-Isoleucine
MeGly N-Methylglycine, sarcosine
MeIle N-Methylisoleucine
MeLys 6-N-Methyllysine
MeVal N-Methylvaline
Nva   Norvaline
Nle   Norleucine
Orn   Ornithine

[0086] It is also contemplated that mutations including but not limited to at least one insertion or substitution of at least one amino acid selected from the group isoleucine (I); valine (V); leucine (L); phenylalanine (F); cysteine/cystine (C); methionine (M); alanine (A); glycine (G); threonine (T); serine (S); tryptophan (W); tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine (O); asparagine (N); lysine (K); and arginine (R), and including, but not limited to, those shown at Table 1 at the position (D) of one ore more (x)D(y) and/or (x)D(y)T sequences would reduce its ability to promote VLS.

[0087] It is contemplated that mutations including but not limited to at least one insertion or substitution of at least one amino acid selected from the group isoleucine (I); phenylalanine (F); cysteine/cystine (C); methionine (M); alanine (A); glycine (G); threonine (T); tryptophan (W); tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine (O); aspartic acid (D); asparagine (N); lysine (K); and arginine (R), and including, but not limited to, those shown at Table 1 at the position (y) of one or more (x)D(y) and/or (x)D(y)T sequences would reduce its ability to promote VLS.

[0088] Amino acids that flank either the (x) or (y) residue of the (x)D(y) sequence may also contribute to its ability to promote VLS. For example, is it contemplated that mutations including but not limited to at least one insertion or substitution of at least one amino acid selected from the group isoleucine (I); valine (V); leucine (L); phenylalanine (F); cysteine/cystine (C); methionine (M); alanine (A); glycine (G); serine (S); tryptophan (W); tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine (O); aspartic acid (D); asparagine (N); lysine (K); and arginine (R), and including, but not limited to, those shown at Table 1 at the position T of one or more (x)D(y)T sequences would reduce its ability to promote VLS.

[0089] It is further contemplated that at least one mutation, chemical modification, movement or other alteration in the N- or C-terminal flanking sequences of the (x)D(y) and/or (x)D(y)T sequence would also produce proteins, polypeptides or peptides that have a reduced ability to promote VLS. Preferably, such mutations or alterations would occur in one or more residues which will not effect the active site. In other embodiments, the mutations or alterations would occur in one or more residues of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or more N-terminal and/or C-terminal to the (x)D(y) tripeptide sequence. In other aspects, one or more residues that are not adjacent to the (x)D(y) tripeptide may contribute to the function of the (x)D(y) motif. Such residues may be identified by their proximity to the tripeptide sequence in a 3-dimentional model, as described herein and as would be known to one of ordinary skill in the art, and are contemplated for alteration as part of a flanking sequence. Such alterations may include any of those described above for altering the (x)D(y) and (x)D(y)T sequences, as long as one or more “wild type” flanking residues are altered, removed, moved, chemically modified, etc.

[0090] Proteins, polypeptides and peptides produced using the methods of the present invention that have a reduced ability to induce VLS would have application in serving as protective agents against VLS produced by compositions containing the (x)D(y) and/or (x)D(y)T sequence. It is contemplated that such proteins, polypeptides and peptides may serve as inhibitors that block the activity of the (x)D(y) and/or (x)D(y)T sequence. Additionally, such proteins, polypeptides and peptides may be used in the creation of ITs with a reduced ability to produce VLS.

[0091] 1. Mutagenesis

[0092] In certain aspects, mutagenesis of nucleic acids encoding peptides, polypeptides or proteins may be used to produce the desired mutations to enhance or reduce a composition's ability to promote VLS, apoptosis or other effects associated with the (x)D(y) and flanking sequences. Mutagenesis may be conducted by any means disclosed herein or known to one of ordinary skill in the art.

[0093] One particularly useful mutagenesis technique is alanine scanning mutagenesis in which a number of residues are substituted individually with the amino acid alanine so that the effects of losing side-chain interactions can be determined, while minimizing the risk of large-scale perturbations in protein conformation (Cunningham et al., 1989).

[0094] As specific amino acids may be targeted, site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the mutation site being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

[0095] In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

[0096] In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. Alternatively, a pair of primers may be annealed to two separate strands of a double stranded vector to simultaneously synthesize both corresponding complementary strands with the desired mutation(s) in a PCR™ reaction.

[0097] The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

[0098] 2. Recombinant Vectors, Host Cells and Expression

[0099] The term “expression vector or construct” means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of an RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense constructs.

[0100] Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein, polypeptide or smaller peptide, is positioned under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively positioned”, “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid coding for the gene product to control RNA polymerase initiation and expression of the gene.

[0101] The promoter may be in the form of the promoter that is naturally associated with a gene, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein (PCR™ technology is disclosed in U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, each incorporated herein by reference).

[0102] In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a gene in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell, and/or promoters made by the hand of man that are not “naturally occurring,” i.e., containing difference elements from different promoters, or mutations that increase, decrease, or alter expression.

[0103] Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., (1989), incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.

[0104] At least one module in a promoter generally functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0105] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0106] The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.

[0107] In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0108] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0109] It is contemplated that proteins, polypeptides or peptides may be co-expressed with other selected proteins, wherein the proteins may be co-expressed in the same cell or a gene(s) may be provided to a cell that already has another selected protein. Co-expression may be achieved by co-transfecting the cell with two distinct recombinant vectors, each bearing a copy of either of the respective DNA. Alternatively, a single recombinant vector may be constructed to include the coding regions for both of the proteins, which could then be expressed in cells transfected with the single vector. In either event, the term “co-expression” herein refers to the expression of both the gene(s) and the other selected protein in the same recombinant cell.

[0110] As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding a protein has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.

[0111] To express a recombinant protein, polypeptide or peptide, whether mutant or wild-type, in accordance with the present invention one would prepare an expression vector that comprises a wild-type, or mutant protein-encoding nucleic acid under the control of one or more promoters. To bring a coding sequence “under the control of” a promoter, one positions the 5′ end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein. This is the meaning of “recombinant expression” in this context.

[0112] Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcription/translational control sequences in order to achieve protein, polypeptide or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.

[0113] Certain examples of prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F—, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

[0114] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.

[0115] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM™-11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as E. coli LE392.

[0116] Further useful vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with ≢-galactosidase, ubiquitin, and the like.

[0117] Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.

[0118] The following details concerning recombinant protein production in bacterial cells, such as E. coli, are provided by way of exemplary information on recombinant protein production in general, the adaptation of which to a particular recombinant expression system will be known to those of skill in the art.

[0119] Bacterial cells, for example, E. coli, containing the expression vector are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein may be induced, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.

[0120] The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selective speed.

[0121] If the recombinant protein is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents, such as β-mercaptoethanol or DTT (dithiothreitol).

[0122] It is contemplated that the proteins, polypeptides or peptides produced by the methods of the invention may be “overexpressed”, i.e., expressed in increased levels relative to its natural expression in cells. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific protein, polypeptides or peptides in relation to the other proteins produced by the host cell and, e.g., visible on a gel.

[0123] 3. Proteins, Polypeptides, and Peptides

[0124] The present invention also provides purified, and in preferred embodiments, substantially purified, proteins, polypeptides, or peptides. The term “purified proteins, polypeptides, or peptides” as used herein, is intended to refer to an proteinaceous composition, isolatable from mammalian cells or recombinant host cells, wherein at least one protein, polypeptide, or peptide is purified to any degree relative to its naturally-obtainable state, i.e., relative to its purity within a cellular extract. A purified protein, polypeptide, or peptide therefore also refers to a wild-type or mutant protein, polypeptide, or peptide free from the environment in which it naturally occurs.

[0125] The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or by any technique that would be know to those of ordinary skill in the art. Additionally, peptide sequences may be synthesized by methods known to those of ordinary skill in the art, such as peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, Calif.).

[0126] Generally, “purified” will refer to a specific protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as described herein below, or as would be known to one of ordinary skill in the art for the desired protein, polypeptide or peptide.

[0127] Where the term “substantially purified” is used, this will refer to a composition in which the specific protein, polypeptide, or peptide forms the major component of the composition, such as constituting about 50% of the proteins in the composition or more. In preferred embodiments, a substantially purified protein will constitute more than 60%, 70%, 80%, 90%, 95%, 99% or even more of the proteins in the composition.

[0128] A peptide, polypeptide or protein that is “purified to homogeneity,” as applied to the present invention, means that the peptide, polypeptide or protein has a level of purity where the peptide, polypeptide or protein is substantially free from other proteins and biological components. For example, a purified peptide, polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully.

[0129] Various methods for quantifying the degree of purification of proteins, polypeptides, or peptides will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific protein activity of a fraction, or assessing the number of polypeptides within a fraction by gel electrophoresis.

[0130] To purify a desired protein, polypeptide, or peptide a natural or recombinant composition comprising at least some specific proteins, polypeptides, or peptides will be subjected to fractionation to remove various other components from the composition. In addition to those techniques described in detail herein below, various other techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.

[0131] Another example is the purification of a specific fusion protein using a specific binding partner. Such purification methods are routine in the art. As the present invention provides DNA sequences for the specific proteins, any fusion protein purification method can now be practiced. This is exemplified by the generation of an specific protein-glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and subsequent purification using Ni-affinity chromatography. However, given many DNA and proteins are known, or may be identified and amplified using the methods described herein, any purification method can now be employed.

[0132] Although preferred for use in certain embodiments, there is no general requirement that the protein, polypeptide, or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified protein, polypeptide or peptide, which are nonetheless enriched in the desired protein compositions, relative to the natural state, will have utility in certain embodiments.

[0133] Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. Inactive products also have utility in certain embodiments, such as, e.g., in determining immunogenicity via antibody generation.

[0134] 4. Antibodies

[0135] As used herein, the term “antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

[0136] The term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

[0137] Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. The invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.

[0138] However, “humanized” antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Methods for the development of antibodies that are “custom-tailored” to the patient's disease are likewise known and such custom-tailored antibodies are also contemplated.

[0139] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic protein composition or comprising a target epitope in accordance with the present invention and collecting antisera from that immunized animal.

[0140] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.

[0141] Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, y-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated. MHC antigens may even be used. Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

[0142] In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokines such as γ-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

[0143] MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.

[0144] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

[0145] The animals are injected with antigen, generally as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals.

[0146] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.

[0147] Often, a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.

[0148] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

[0149] Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp2/0-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

[0150] One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

[0151] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).

[0152] Fusion procedures usually produce viable hybrids at low frequencies, about 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused mycloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

[0153] The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

[0154] This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

[0155] The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

[0156] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.

[0157] It is also contemplated that a molecular cloning approach may be used to generate monoclonals. For this, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

[0158] Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.

[0159] C. ITS

[0160] Toxins and/or Mabs may be derived from natural sources or produced using recombinant DNA technology. At least one toxin and at least one antibody may be combined to form an immunotoxin (IT). ITs combine into a single molecule, the exquisite specificity of a ligand and the extraordinary toxicity of a toxin. Despite their conceptual simplicity, ITs are large and complex molecules that are continually undergoing improvements for optimal in vivo activity since each of their common components, e.g., one or more a binding moieties, one or more cross-linkers, and one or more toxins, introduces a different set of problems that must be addressed for the IT to function optimally in vivo.

[0161] Any antibody of sufficient selectivity, specificity or affinity may be employed as the basis for an IT. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in the antibody molecule, in addition to the canonical antigen binding sites, include sites that reside in the variable domain that can bind pathogens, B-cell superantigens, the T cell co-receptor CD4 and the HIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1991). In addition, the variable domain is involved in antibody self-binding (Kang et al., 1988), and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

[0162] The origin or derivation of the antibody or antibody fragment for use in the invention (e.g., Fab′, Fab or F(ab′)2) is not crucial to the practice of the invention, so long as the antibody or fragment that is employed has the desired properties for the ultimately intended use of the IT. Thus, where monoclonal antibodies are employed, they may be of human, murine, monkey, rat, hamster, chicken or even rabbit origin. The invention also contemplates the use of human antibodies, “humanized” or chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, single chain antibodies, Fv domains, as well as recombinant antibodies and fragments thereof. Of course, due to the ease of preparation and ready availability of reagents, murine Mabs will typically be preferred.

[0163] In certain therapeutic embodiments, one may use known antibodies, such as those having high selectivity for solid tumors, such as B72.3, PRBC5 or PR4D2 for colorectal tumors; HMFG-2, TAG 72, SM-3, or anti-p 185.sup.Her2 for breast tumors; anti-p 185.sup.Her2 for lung tumors; 9.2.27 for melanomas; MO v18 and OV-TL3 for ovarian tumors, and anti-Id, CD19, CD22, CD25, CD7 and CD5 for lymphomas and leukemias. Anti-CD2, anti-CD25, anti-CD4 and anti-CD45R° ITs may be purified according to the invention and used to kill malignant T cells or HIV-infected cells. Also, CD3-specific ITs as well as CD4 and CD25 specific ITs may be purified and used to prevent acute GVHD after bone marrow transplantation.

[0164] In other embodiments, one may use another immunogen and prepare a new Mab. The technique for preparing Mabs is quite straightforward, and may be readily carried out using techniques well known to those of skill in the art, as exemplified by the technique of Kohler & Milstein (1975). Generally, immunogens are injected intraperitoneally into mice. This process is repeated three times at two-weekly intervals, the final immunization being by the intravenous route. Three days later the spleen cells are harvested and fused with SP2/0 myeloma cells by standard protocols (Kohler & Milstein, 1975): Hybridomas producing antibodies with the appropriate reactivity are then cloned by limiting dilution.

[0165] The toxins that have been used to form ITs are derived from bacteria or plants and are inhibitors of protein synthesis. They are among the most powerful cell poisons known. Fewer than ten molecules will kill a cell if they enter the cytosol (although many times that number must bind to the cell surface because the entry process is inefficient). This extraordinary potency initially led to the concern that such poisons were too powerful to control. However, the toxins can be rendered innocuous (except when directed to the target cells) simply by removing or modifying their cell-binding domain or subunit. The remaining portion of the toxin (lacking a cell-binding domain) is then coupled to a ligand (e.g., an antibody) that targets the toxic portion to the target cell. By selecting an antibody lacking unwanted cross-reactivity, ITs are safer and have fewer non-specific cytotoxic effects than most conventional anticancer drugs. The other main attraction of toxins is that because they are inhibitors of protein synthesis, they kill resting cells as efficiently as dividing cells. Hence, tumor or infected cells that are not in cycle at the time of treatment do not escape the cytotoxic effect of an IT.

[0166] “Toxin” is employed herein to mean any anticellular agent, and includes but is not limited to cytotoxins and any combination of anticellular agents. In the case of chemotherapeutic agents, agents such as a hormone, a steroid for example; an antimetabolite such as cytosine arabinoside, fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycin C; a vinca alkaloid; demecolcine; etoposide; mithramycin; or an antitumor alkylating agent such as chlorambucil or melphalan, may be used.

[0167] However, preferred toxins will be plant-, fungus- or bacteria-derived toxins, which, by way of example, include various A chain toxins, particularly RTA; RIPs such as saporin or gelonin, α-sarcin, aspergillin or restrictocin; ribonucleases such as placental ribonuclease; angiogenin, diphtheria toxin, and Pseudomonas exotoxin, to name just a few. The exemplary toxins that can be mutated to remove or alter the placement of sequences that induce VLS, for example those residues listed in Table 1, those residues described herein and equivalent residues in other proteins.

[0168] Plant holotoxin often contain two disulfide-bonded chains, the A and B chains. The B chain carries both a cell-binding region (whose receptor is often uncharacterized) and a translocation region, which facilitates the insertion of the A chain through the membrane of an acid intracellular compartment into the cytosol. The A chain then kills the cell after incorporation. For their use in vivo, the ligand and toxin must be coupled in such a way as to remain stable while passing through the bloodstream and the tissues and yet be labile within the target cell so that the toxic portion can be released into the cytosol.

[0169] The most preferred toxin moiety for use in connection with the invention is RTA, and particularly toxin A chain which has been treated to modify or remove carbohydrate residues, so-called dgRTA. Recombinant A chain expressed in E. coli and also lacking carbohydrates can be used. In certain embodiments, RTA may be made as described herein below in Example 3.

[0170] However, it may be desirable from a pharmacological standpoint to employ the smallest molecule possible that nevertheless provides an appropriate biological response. One may thus desire to employ smaller A chain peptides or other toxins which will provide an adequate anti-cellular response. To this end, it has been discovered by others that RTA may be “truncated” by the removal of 30 N-terminal amino acids by Nagarase (Sigma), and still retain an adequate toxin activity. It is proposed that where desired, this truncated A chain may be employed in conjugates in accordance with the invention.

[0171] Alternatively, one may find that the application of recombinant DNA technology to the toxin moiety will provide additional significant benefits in accordance the invention. In that the cloning and expression of biologically active RTA and other VLS-inducing toxins have now been enabled through the publications of others (O'Hare et al., 1987; Lamb et al., 1985; Halling et al., 1985), it is now possible to identify and prepare smaller or otherwise variant peptides which nevertheless exhibit an appropriate toxin activity. Moreover, the fact that RTA and other VLS-inducing toxins have now been cloned allows the application of site-directed mutagenesis, through which one can readily prepare and screen for A chain and other VLS-inducing toxins, toxin-derived peptides and obtain additional useful moieties for use in connection with the present invention. Once identified, these moieties can be mutated to produce toxins with a reduced ability to promote VLS, apoptosis, disintegrin-like activity, EC damaging activity and other effects of such sequences described herein or known to one of skill in the art.

[0172] Fusion-ITs with PE, DT-A, etc. in any combination are made by recombinant DNA technology as would be known to one of ordinary skill in the art. Antibodies, cytokines or soluble receptor DNA may be used in such preparations.

[0173] The cross-linking of many, but not all toxins, of the conjugate with the binding agent region is an important aspect of the invention. In the case of RTA, if one desires a conjugate having biological activity, it is believed that a cross-linker which presents a disulfide function is required. The reason for this is unclear, but is likely due to a need for the toxin moiety to be readily releasable from the binding agent once the agent has “delivered” the toxin inside the targeted cells. Each type of cross-linker, as well as how the cross-linking is performed, will tend to vary the pharmacodynamics of the resultant conjugate. Ultimately, one desires to have a conjugate that will remain intact under conditions found everywhere in the body except the intended site of action, at which point it is desirable that the conjugate have good “release” characteristics. Therefore, the particular cross-linking scheme, including in particular the particular cross-linking reagent used and the structures that are cross-linked, will be of some significance.

[0174] Cross-linking reagents are used to form molecular bridges that tie together functional groups of two different proteins (e.g., a toxin and a binding agent). To link two different proteins in a step-wise manner, heterobifunctional cross-linkers can be used which eliminate the unwanted homopolymer formation. An exemplary heterobifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the crosslinker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., dgRTA).

[0175] The spacer arm between these two reactive groups of any cross-linkers may have various length and chemical composition. A longer spacer arm allows a better flexibility of the conjugate components while some particular components in the bridge (e.g., benzene group) may lend extra stability to the reactive group or an increased resistance of the chemical link to the action of various aspects (e.g., disulfide bond resistant to reducing agents).

[0176] The most preferred cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to its delivery to the site of action by the binding agent. The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to crosslink functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the heterobifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

[0177] Although the “hindered” cross-linkers will generally be preferred in the practice of the invention, non-hindered linkers can be employed and advantages in accordance herewith nevertheless realized. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane. The use of such cross-linkers is well understood in the art.

[0178] 1. IT Conjugates

[0179] The present invention provides ITs against target epitopes, such as epitopes expressed on a diseased tissue or a disease causing cell. In certain embodiments the IT comprises at least one toxin described herein. In other embodiments the IT or toxin further comprises at least a second agent. Such an agent may be a molecule or moiety. Such a molecule or moiety may comprise, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

[0180] Certain examples of at least a second agent comprises at least one detectable label. “Detectable labels” are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired.

[0181] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.

[0182] Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and may be used as antibody binding agents.

[0183] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0184] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.

[0185] 2. Methods for IT Preparation

[0186] Methods of making and preparing ITs are known to those of ordinary skill in the art. Such methods are disclosed in U.S. Pat. No. 5,686,072, U.S. Pat. No. 5,578,706, U.S. Pat. No. 4,792,447, U.S. Pat. No. 5,045,451, U.S. Pat. No. 4,664,911, and U.S. Pat. No. 5,767,072, each incorporated herein by reference). The toxin moiety of the IT may be any one of a variety of toxins that are commonly employed in the art. It may be an intact toxin, a toxin A chain, or a naturally occurring single-chain RIP. Toxins which are encompassed by the invention include, but are not limited to, diphtheria toxin (DT) and DT(CRM-45); pseudomonas endotoxin derived PE38; RTA and abrin and blocked forms of both of these; gelonin and saporin.

[0187] ITs comprising Mabs covalently bound to dgRTA by hindered disulfide linkers have recently entered clinical trials for the treatment of non-Hodgkin's (B cell) lymphoma, Hodgkin's lymphoma neoplasms or GVHD. These “second generation” ITs are stable, long lived and display potent cytotoxicity to target cells. Standardized procedures for rapid preparation of high yields of these ITs have been developed (Ghetie et al., 1991).

[0188] The procedure for preparation of the ITs with, for example, dgRTA comprises the derivatization of Mabs with SMPT and reduction of dgRTA with dithiothreitol (DTT), followed by the reaction of the two components to establish a hindered interchain disulfide bond. The chemical crosslinking reaction results in a mixture of antibody, toxin and ITs which are then purified, initially to remove the free antibody and free toxin molecules and subsequently to separate the different IT species which comprise one antibody molecule conjugated with one, two, three or more than three toxin molecules, respectively. The unreacted components of the crosslinking reaction may be removed by successive chromatographies on an affinity chromatography column such as activated dye/agarose to remove free antibody followed by gel filtration to remove high molecular weight material and free toxin.

[0189] The result of this procedure is a mixture of conjugates of various toxin/antibody ratios. An important embodiment of the present invention is the further purification of this mixture to obtain preparations essentially comprising ITs of a single toxin/antibody ratio separated from ITs of different toxin/antibody ratios. This purification is accomplished by further chromatographic separation which may be accomplished by affinity chromatography for example, using a salt gradient to elute the various species of ITs and gel filtration to separate the ITs from larger molecules.

[0190] Another important embodiment of the present invention is the ability to determine which of the toxin/IgG ratios is the most effective cytotoxic agent to be used in pharmacological preparations. The isolation and characterization of each of the single species of IT made possible by the present invention is of particular advantage in clinical applications as it allows the practitioner to exercise more precise control over the effective amount of IT to be administered in a particular situation.

[0191] 3. Gel Filtration

[0192] A gel to be used in the procedures of the present invention is a three dimensional network which has a random structure. Molecular sieve gels comprise cross-linked polymers that do not bind or react with the material being analyzed or separated. For gel filtration purposes, the gel material is generally uncharged. The space within the gel is filled with liquid and the liquid phase constitutes the majority of the gel volume. Materials commonly used in gel filtration columns include dextran, agarose and polyacrylamide.

[0193] Dextran is a polysaccharide composed of glucose residues and is commercially available under the name SEPHADEX (Phamacia Fine Chemicals, Inc.). The beads are prepared with various degrees of cross-linking in order to separate different sized molecules by providing various pore sizes. Alkyl dextran is cross-linked with N,N′-methylenebisacrylamide to form SEPHACRYL-S100 to S1000 which allows strong beads to be made that fractionate in larger ranges than SEPHADEX can achieve.

[0194] Polyacrylamide may also be used as a gel filtration medium. Polyacrylamide is a polymer of cross-linked acrylamide prepared with N,N′-methylenebisacrylamide as the cross-linking agent. Polyacrylamide is available in a variety of pore sizes from Bio-Rad Laboratories (USA) to be used for separation of different size particles.

[0195] The gel material swells in water and in a few organic solvents. Swelling is the process by which the pores become filled with liquid to be used as eluant. As the smaller molecules enter the pores, their progress through the gel is retarded relative to the larger molecules which do not enter the pores. This is the basis of the separation. The beads are available in various degrees of fineness to be used in different applications. The coarser the bead, the faster the flow and the poorer the resolution. Superfine is to be used for maximum resolution, but the flow is very slow. Fine is used for preparative work in large columns which require a faster flow rate. The coarser grades are for large preparations in which resolution is less important than time, or for separation of molecules with a large difference in molecular weights. For a discussion of gel chromatography, see Freifelder, Physical Biochemistry, Second Edition, pages 238-246, incorporated herein by reference.

[0196] The most preferred methods of gel filtration for use in the present invention are those using dextran gels, such as SEPHADEX, and those using dextran-polyacrylamide gels such as SEPHACRYL which are able to separate molecules in the 180 to 240 kilodalton range.

[0197] 4. Affinity Chromatography

[0198] Affinity chromatography is generally based on the recognition of a protein by a substance such as a ligand or an antibody. The column material may be synthesized by covalently coupling a binding molecule, such as an activated dye, for example to an insoluble matrix. The column material is then allowed to adsorb the desired substance from solution. Next, the conditions are changed to those under which binding does not occur and the substrate is eluted. The requirements for successful affinity chromatography are that the matrix must adsorb molecules, the ligand must be coupled without altering its binding activity, a ligand must be chosen whose binding is sufficiently tight, and it must be possible to elute the substance without destroying it.

[0199] A preferred embodiment of the present invention is an affinity chromatography method wherein the matrix is a reactive dye-agarose matrix. Blue-SEPHAROSE, a column matrix composed of Cibacron Blue 3GA and agarose or SEPHAROSE may be used as the affinity chromatography matrix. The most preferred matrix is SEPHAROSE CL-6B available as Reactive Blue 2 from Sigma Chemical Company, catalogue #R 8752. This matrix binds the ITs of the present invention directly and allows their separation by elution with a salt gradient.

[0200] 5. ELISA

[0201] ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incorporating toxin A chain sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0202] After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/TWEEN. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 2 to 4 hours, at temperatures preferably on the order of 25° C. to 37° C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/TWEEN, or borate buffer.

[0203] Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0204] After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

[0205] D. Vaccination

[0206] The present invention contemplates vaccines for use in immunization embodiments. It is contemplated that proteinaceous compositions that are less effective in promoting VLS or other toxic effects by alterations in one or more (x)D(y), (x)D(y)T and/or flanking region sequences may be useful as antigens. In particular embodiments, peptides comprising one or more (x)D(y), (x)D(y)T and/or flanking region sequences are contemplated as useful antigens. Preferably the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle. In other embodiments, it is also possible to use toxins lacking one or more active site residues (i.e., a toxoid) as vaccines.

[0207] 1. Immunomodulators

[0208] It is contemplated that immunomodulators can be included in the vaccine to augment the patient's response. Immunomodulators can be included as purified proteins or their expression engineered into the cells when cells are part of the composition. The following sections list examples of immunomodulators that are of interest.

[0209] a. Cytokines

[0210] Interleukins and cytokines, and vectors expressing interleukins and cytokines are contemplated as possible vaccine components. Interleukins and cytokines, include but not limited to interleukin 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, P-interferon, α-interferon, γ-interferon, angiostatin, thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumor necrosis factor, TGFβ, LT and combinations thereof.

[0211] b. Chemokines

[0212] Chemokines or genes that code for chemokines also may be used as vaccine components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.

[0213] The preparation of polyvalent vaccine is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables. Either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

[0214] Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously, intradermally or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral or nasal formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10 to about 95% of active ingredient, preferably about 25 to about 70%.

[0215] The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations. The intramuscular route may be preferred in the case of toxins with short half lives in vivo.

[0216] Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101° C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide monooleate (Arecel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed.

[0217] In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.

[0218] 2. Adjuvants

[0219] Immunization protocols have used adjuvants to stimulate responses for many years. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. Other adjuvants, for example, certain organic molecules obtained from bacteria, act on the host rather than on the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. The effects of MDP, as with most adjuvants, are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly. The effects of adjuvants, therefore, are not antigen-specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.

[0220] Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). This has been attempted particularly in the treatment of cancer. For many cancers, there is compelling evidence that the immune system participates in host defense against the tumor cells, but only a fraction of the likely total number of tumor-specific antigens are believed to have been identified to date.

[0221] The present invention contemplates that a variety of adjuvants may be employed in the membranes of cells, such as tumor cells, resulting in an improved immunogenic composition. The only requirement is, generally, that the adjuvant be capable of incorporation into, physical association with, or conjugation to, the cell membrane of the cell in question.

[0222] Those of skill in the art will know the different kinds of adjuvants that can be conjugated to cellular vaccines in accordance with this invention and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram− cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the invention (Takada et al., 1995).

[0223] Hemocyanins and hemoerythrins may also be used in the invention. The use of hemocyanin from keyhole limpet (KLH) is particularly preferred, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.

[0224] Various polysaccharide adjuvants may also be used. For example, Yin et al., (1989) describe the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice. Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.

[0225] A further preferred group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.

[0226] U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide which is proposed for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. The compounds of U.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, which have not previously been suggested for use with cellular carriers, are now proposed for use in the present invention.

[0227] A preferred adjuvant in the present invention is BCG. BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cell wall skeleton (CWS) may also be used as adjuvants in the invention, with or without trehalose dimycolate. Trehalose dimycolate may be prepared as described in U.S. Pat. No. 4,579,945.

[0228] Cell wall extracts of BCG have proven to have excellent immune adjuvant activity. Recently developed molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990). BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively (Luelmo, 1982; Lotte et al., 1984). An exemplary BCG vaccine is sold as TICE® BCG (Organon Inc., West Orange, N.J.). In a typical practice of the present invention, cells of Mycobacterium bovis-BCG are grown and harvested by methods known in the art (e.g., Dubos et al., 1947; Rosenthal, 1937).

[0229] Amphipathic and surface active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of preferred adjuvants for use with the immunogens of the present invention. Nonionic block copolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) may also be employed. Oligonucleotides, as described by Yamamoto et al., (1988) are another useful group of adjuvants. Quil A and lentinen complete the currently preferred list of adjuvants. Although each of the agents, and the endotoxins described below, are well-known as adjuvants, these compounds have not been previously incorporated into the membrane of a target cell, as shown herein.

[0230] One group of adjuvants particularly preferred for use in the invention are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals.

[0231] The detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant-incorporated cells. Combination of detoxified endotoxins with trehalose dimycolate is contemplated, as described in U.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Pat. No. 4,520,019.

[0232] Various adjuvants, even those that are not commonly used in humans, may still be employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.

[0233] E. Pharmaceutical Preparations

[0234] Pharmaceutical aqueous compositions of the present invention comprise an effective amount of one or more IT, VLS inhibitory peptide or polypeptide, VLS stimulatory peptide or polypeptide and/or cytokine dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0235] The following buffers and reagents are particularly contemplated for use in the preparation of pharmaceutical preparations of the present invention: dgRTA, deglycosylated ricin A chain; DMF, dimethylformamide (Pierce, Rockford, Ill.); DTT (Pierce); PBE, 0.05M sodium phosphate, pH 7.5 with 1 mM EDTA; PBES, 0.05M sodium phosphate, pH 7.5 with various concentrations of NaCl (such as 0.1M, 0.2M, 0.3M, 0.4M and 0.5M NaCl) and 1 mM EDTA; PBSE, 0.01M sodium phosphate, pH 7.5 with 0.17M NaCl and 1 mM EDTA; SMPT, N-succinimidyl-oxycarbonyl-α-methyl-a(2-pyridyldithio)toluene (Pierce). All buffers may be prepared with endotoxin-free distilled water using enzyme grade salts (Fisher Biotec, Springfield, N.J.).

[0236] The ITs, peptides or polypeptides and/or cytokines may be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular or sub-cutaneous routes, though other routes such aerosol administration may be used. The preparation of an aqueous composition that contains at least one IT, proteinaceous material and/or cytokine as an active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated herein by reference. Moreover, for human administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0237] Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified. The compositions will be sterile, be fluid to the extent that easy syringability exists, stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

[0238] Although it is most preferred that solutions of ITs, peptide or polypeptides and/or cytokines be prepared in sterile water containing other non-active ingredients, made suitable for injection, solutions of such active ingredients can also be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose, if desired. Dispersions can also be prepared in liquid polyethylene glycols, and mixtures thereof and in oils. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0239] The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0240] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0241] It is particularly contemplated that suitable pharmaceutical IT, peptide or polypeptide compositions will generally comprise, but are not limited to, from about 10 to about 100 mg of the desired IT conjugate, peptide or polypeptide admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a final concentration of about 0.25 to about 2.5 mg/ml with respect to the conjugate, in, for example, 0. 15M NaCl aqueous solution at pH 7.5 to 9.0. The preparations may be stored frozen at −10° C. to −70° C. for at least 1 year.

[0242] F. Kits

[0243] In still further embodiments, the present invention concerns kits for use with the IT or vaccination methods described above. Toxins, cytokines or antigenic compositions with reduced VLS promoting or toxic effects may be provided in a kit. Such kits may be used to combine the toxin with a specific antibody to produce an IT, provide cytokines with reduced toxicity, or provide antigens for vaccination in a ready to use and storable container. Additionally, peptide inhibitors of VLS producing sequences or proteinaceous enhancers of extravasation may be included in a kit. However, kits including combinations of such components may be provided. The kits will thus comprise, in suitable container means, a proteinaceous composition with reduced or enhanced VLS promoting activity. The kit may comprise an antibody or IT in suitable container means.

[0244] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the at least one protein, polypeptide or peptide may be placed, and/or preferably, suitably aliquoted. The kits of the present invention may include a means for containing at least one antibody, IT and/or any other reagent containers in close confinement for commercial sale. Such containers may include injection and/or blow-molded plastic containers into which the desired vials are retained.

EXAMPLES

[0245] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Structural Motif for Initiating Vascular Leak Syndrome

[0246] This example demonstrates that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 is responsible for damaging vascular ECs. Short (<20 amino acid) (x)D(y) motif-containing peptides from RTA or IL-2 which contained flanking glycines and a cysteine, as well as peptides with deleted or mutated sequences were generated. These peptides were attached via the cysteine to a mouse IgG1 Mab (RFB4) not reactive with HUVECs. The VLS-inducing ability of this IgG-peptide conjugate (IgG-RTA) in three VLS model systems was compared. The first was in vitro damage to human umbilical endothelial cells, (HUVECs) (Soler-Rodriguez et al., 1993); the second was in vivo fluid accumulation in mouse lungs (Baluna and Vitetta, 1996); and the third was in vivo human skin xenografts in SCID mice (Baluna and Vitetta, 1999).

[0247] Peptide Synthesis. A peptide representing 13 amino acids (residues 69-81, SEQ ID NO: 1) from RTA, with added N- and C-terminal glycine residues to improve solubility (Table 6), was synthesized. The peptides containing the x(D)y motif were difficult to solubilize even with the additional three flanking glycines on each end of the peptide. For this reason, they were conjugated to a soluble carrier protein. The MAb RFB4 was chosen because the RFB4-dgRTA is a prototypic IT, and therefore RFB4-peptides should “mimic” ITs.

[0248] An N-terminal cysteine was added to couple the peptide to the RFB4 MAb. Two RTA control peptides (Table 6) were synthesized. A peptide of 9 amino acids representing residues 15-23 from IL-2 as well as a control peptide (Table 6) was also synthesized. Again, flanking glycines and a cysteine were added. All peptides were synthesized on an Applied Biosystems Model 430A Solid-phase Peptide Synthesizer.

[0249] Conjugation of the peptides to RFB4. All peptides contained an N-terminal cysteine residue to facilitate conjugation with maleimide-derivatized RFB4. RFB4 was treated with a 25-fold molar excess of succinimidyl 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate and excess reagent was removed by gel filtration. The number of maleimide groups introduced into each molecule of RFB4 was determined by the back-titration of 2-mercaptoethylamine using Ellman's reagent (Husain and Bieniarz, 1994). The derivatized RFB4 was reacted with a 10-fold excess of the SH-peptide at room temperature for 4 hr and excess peptide was removed by dialysis against PBS. The maleimide reaction allowed the formation of the IgG1-C—S-peptide conjugate in which the number of peptide groups attached was similar to that of free maleimide groups.

TABLE 6
Peptides from RTA and IL-21
Origin	Designation	Type	Peptide Sequence
RTA	LDV +	Native	CysGlyGlyGlySerValThrLeuAla73Leu74Asp75Val76Thr77Asn
	SEQ ID NO: 4		AlaTyrValGlyGlyGly
	LDV −	Deleted	CysGlyGlyGlySerValThrLeuAla73Thr77AsnAlaTyrValGlyGl
	SEQ ID NO: 5		yGly
	GQT +	Mutant	CysGlyGlyGlySerValThrLeuAla73Gly74Glu75Thr76Thr77Asn
	SEQ ID NO: 6		AlaTyrValGlyGlyGly
IL-2	LDL +	Native	CysGlyGlyGlyGluHisLeuLeu18Leu19Asp20Leu21Glu22Met
	SEQ ID NO: 7		GlyGlyGly
	LDL	Deleted	CysGlyGlyGlyGluHisLeuLeu18Glu22MetGlyGlyGly
	SEQ ID NO: 8
1Each peptide was conjugated to the mouse MAb, RFB4 as described.

[0250] As determined by both HPLC and radiolabeling, the RFB4-peptide conjugates (Table 6) contained 6 to 9 maleimide groups per molecule of IgG1 and these groups formed stable thioether bonds by reaction with the cysteine-containing peptides.

[0251] Effect of the RFB4-peptides on the morphology of HUVEC monolayers. To determine whether the LDV sequence in RTA and the LDL sequence in IL-2 damage HUVECs, monolayers were incubated with different concentrations of RFB4-RTA-peptides, RFB4-IL-2-peptides or controls. HUVECs were isolated, cultured and studied microscopically (Baluna et al., 1996; Soler-Rodriguez et al., 1993).

[0252] HUVEC monolayers were incubated at 37° C. for 18 hr with 10−6 M RFB4-LDV+, RFB4-LDV−, RFB4-GQT, RFB4-LDL+, RFB4-LDL− or medium-only and then examined by phase-contrast microscopy (magnification 20×). Normal monolayers consisted of highly packed cells with elongated shapes whereas damaged cells rounded up and detached from the plate. Untreated HUVECs consisted of tightly packed elongated cells. Treatment with 10−6M RFB4-LDV+ or RFB4-LDL+ caused cell rounding after 2 hr of incubation and the formation of gaps in the monolayer after 18 hr. Toxic effects on HUVECs were not observed using RFB4-LDV−, RFB4-GQT, or RFB4-LDL−. The toxic effect of RFB4-peptides containing LDV or LDL were dose-dependent and comparable to the effects observed using RFB4-dgRTA (Table 7). These results indicate that the LDV sequence in RTA and its LDL homologue in IL-2 are involved in the EC toxicity of these agents.

TABLE 7
Effect of different concentrations of the RFB4-peptide
constructs on the morphology of HUVEC monolayers1
	Concentration (M)2
	10−6	10−7	10−8	0
	RFB4-RTA-derived peptides
	RFB4-LDV+	++	++	+	−
	RFB4-LDV−	−	−	−	−
	RFB4-GGT+	−	−	−	−
	RFB4-IL-2-derived peptides
	RFB4-LDL+	++	++	+	−
	RFB4-LDL−	−	−	−	−
	RFB4-dgRTA	++	+	+	−
	RFB4	−	−	−	−
	1HUVECs were grown to confluence in 96-well tissue culture plates and cells were treated for eighteen hours with different concentrations of RTA-derived peptide-constructs in M199 with 2% PCS.
	2The morphological changes were score as “−” no changes, “+” rounding up of cells and “++” disruption and detachment of cells from the cell monolayer.

[0253] In vivo effect of the RFB4-peptides. Although the vascular toxicity of IL-2 has been observed in experimental animals (Orucevic and Lala, 1995; Puri et al., 1989; Puri and Rosenberg, 1989; Rosenstein et al., 1986), it has been difficult to induce dgRTA-IT-mediated systemic manifestations of VLS in mice, rats or monkeys (Soler-Rodriguez, 1992). A model has been developed to study the effect of ITs on human endothelium in vivo by grafting vascularized human skin onto SCID mice, injecting the mice with dgRTA-ITs and measuring fluid accumulation in the graft as the wet/dry weight ratio (Baluna et al., 1998). Fluid accumulation in the human skin was measured by weighing punch biopsies of the skin grafts before and after freeze drying. This model was used to evaluate the effect of RFB4-LDV+, RFB4-GQT+, and RFB4-dgRTA in vivo (FIG. 1A).

[0254] The fluid accumulation in the lungs of normal SCID mice was also evaluated as IL-2 induces fluid accumulation in the lungs of mice (Orucevic and Lala, 1995). The water content of the lungs or skin grafts was calculated as the wet/dry weight ratio.

[0255] Although it has been difficult to demonstrate systemic manifestations of VLS in mice injected with RTA-ITs, vascular leak occurs in human skin xenografts in SCID mice. Increases in the wet/dry weight ratio of the human skin grafts after injection of RFB4-LDV+ and RFB4-dgRTA were found but not after injection of RFB4-GQT+. The fluid accumulation in these xenografts was comparable using either RFB4-dgRTA or RFB4-LDV+. Comparable results were obtained using SCID mouse lungs (FIG. 1B). It should be noted that although the difference in the Figures may appear small, they are statistically significant and consistent with reports using IL-2 (Orucevic and Lala, 1995).

[0256] Flow cytometric analysis of the binding of dgRTA and RFB4-peptides to HUVECs. The fact that RFB4-LDV+ and RFB4-LDL+ damage HUVECs, implies that these peptides interact with a binding site on HUVECs, although, in the intact IL-2 or RTA molecules, the (x)D(y) motif may not be the primary binding site for ECs. To address these issues with toxins, a series of binding and binding/inhibition studies were carried out.

[0257] The proteins were coupled to fluorescein isothiocyanate (FITC) (Sigma, St. Louis, Mo.). 105 HUVECs were washed twice in cold PBS containing 1% bovine serum albumin (BSA) and 0.01% sodium azide (PBS/BSA/AZ), resuspended in 100 μl of the same buffer and incubated with FITC-reagents for 30 minutes on ice in the dark, washed three times with PBS/BSA/AZ, fixed in 0.5 ml of 1% paraformaldehyde PBS/AZ, and analyzed using a FACScan (Becton Dickinson, Mountain View, Calif.) and the CytoQuest software.

[0258] The binding of dgRTA, PE38-lys and RFB4-peptides to HUVECs was examined. 105 HUVECs were incubated on ice for 30 min with FITC-reagents in 100 μl PBS/BSA/AZ at varying, concentrations, washed, fixed in 1% paraformaldehyde and analyzed by flow cytometry. The values represent the mean ±SD of three experiments using FITC-dgRTA, FITC-PE38-lys, and FITC-carbonic anhydrase as a control. Histograms of flow cytometric analyses of the binding of dgRTA, PE38-lys and carbonic anhydrase to HUVECs were made. The binding of FITC-RFB4-LDV+ (▴), FITC-RFB4-LDV−, FITC-RFB4-GQT+, FITC-RFB4, FITC-RFB4-LDL+, and FITC-RFB4-LDL− were also evaluated. Histograms of RFB4-LDV+, RFB4-LDL+ and RFB4 were also made. The results of this study demonstrated that the 50% of maximal binding of FITC-dgRTA and FITC-PE38-lys required 0.035 μg and >100 μg/105 cells, respectively, demonstrating that dgRTA has a >3 log higher relative binding affinity for HUVECs than PE38-lys. This may be due to the fact that the LDV receptor on HUVECs has a lower affinity for homologous sequences in PE38-lys and/or that LDV in RTA, is more exposed. It is also possible that other non-homologous sequences in RTA (but not in PE38-lys) bind to HUVECs. The difference between the relative binding affinity of FITC-dgRTA (0.035 μg/105 cells/100 μl) and FITC-RFB4-LDV+(0.5 μg/105 cells/100 μl) was only 2-fold if calculated on molar basis. Since the RFB4-peptide conjugates with deleted or mutated LDV sequences did not bind to HUVECs, the (x)D(y) motif is clearly involved in the binding.

[0259] Inhibition of the binding of dgRTA and RFB4-peptides to HUVECs. To provide further evidence for the role of the (x)D(y) motif in the binding of RTA to HUVECs, a series of binding inhibition studies were carried out. FITC-dgRTA or FITC-RFB4-LDV+ at concentrations representing 20-50% of maximal binding (0.035 μg/105 cells for dgRTA and 1.0 μg/105 cells for RFB4-LDV+) were incubated with HUVECs in the presence or absence of a 100-fold excess of dgRTA (Inland Laboratories, Austin, Tex.), RFB4-LDV+, RFB4, Fn (GIBCO Laboratories, Grand Island, N.Y.) or PE38-lys (NCI, Bethesda) for 30 min on ice in the dark. Washed cells were fixed in 1% paraformaldehyde and analyzed on the FACS.

[0260] It was found that the binding of FITC-dgRTA to HUVECs was inhibited by >90% by dgRTA and by >60% by RFB4-LDV+ indicating that the binding of dgRTA is specific and that it involves, at least in part, the LDV sequence (FIG. 2A). The fact that the homologue-containing PE38-lys could not inhibit the binding of dgRTA (FIG. 2A) may be due to the fact that its relative affinity for HUVECs is more than three logs lower. In addition, dgRTA may have additional non-homologue binding sites for HUVECs, as suggested by the fact that RFB4-LDV+ inhibited its binding by 60% and not 100%. Furthermore, in the reverse studies, both dgRTA and RFB4-LDV+ inhibited the binding of FITC-RFB4-LDV+ to HUVECs to a similar extent (FIG. 2B), further indicating that the LDV sequence in RTA is involved in binding to HUVECs. Surprisingly, PE38-lys very effectively inhibited the binding of FITC-LDV+ to HUVECs (FIG. 2B) indicating that one or more of its LDV homologue sequences can compete with the LDV motif for binding of an LDV-containing peptide. It is contemplated that one or more homologue sequences in PE38-lys (GDL-348-350; GDV-430-432; or GDL-605-607) bind to and damage HUVEC. Fn also inhibited the binding of both FITC-dgRTA (FIG. 2A) and FITC-RFB4-LDV+ (FIG. 2B) to HUVECs, but it did so less effectively. In this regard, although Fn also contains the LDV motif, it has different flanking residues which may play a role in the availability of its LDV motif.

[0261] The data described above demonstrates that peptides containing the LDV motif in RTA and the LDL motif in IL-2, when attached to the RFB4 MAb specifically bind to and damage HUVECs in vitro. The IgG-peptide conjugates and the IgG-RTA IT were equally effective in inducing endothelial cell damage and increased vascular permeability in all three models.

[0262] The LDV sequence in RTA may be responsible for the initiation of events leading to VLS-like symptoms in vivo since injection of RFB4-RTA-peptides containing the native, but not mutated or deleted, LDV sequence caused vascular leak in lungs and in human skin xenografts in a manner analogous to that of the RFB4-dgRTA IT. dgRTA utilizes its LDV sequence, at least in part, to bind to HUVECs since peptides or proteins containing this motif inhibited the dose-dependent, saturable binding of RTA to HUVECs.

[0263] The stereoviews of LDV in RTA and LDL in IL-2 indicate that these motifs are partially exposed and should interact with cells. For RTA, this is supported by its dose dependent, saturable binding to HUVECs in vitro. Since the binding of RFB4-LDV+ to HUVECs could be partially inhibited not only by dgRTA but aiso by proteins containing LDV or LDV-homologues, i.e. Fn and PE38-lys, this further indicates a functional conservation in the (x)D(y) motif in several divergent molecules. Deletions or mutations in this sequence or the use of non-damaging blocking peptides may increase the therapeutic index of both IL-2 as well as ITs prepared with a variety of plant or bacterial toxins.

Example 2

Production and Purification of dgRTA

[0264] Inland Laboratories has produced dgRTA. However, the following example describes a procedure for producing dgRTA for use in the present invention.

[0265] A powdered acetone extract of castor beans is the starting material. Ricin is extracted from this powder, the ricin is deglycosylated, separated into A and B chains, and the dgRTA is purified.

[0266] Bulk raw material, powdered acetone extract of castor beans, can be purchased from Sigma Bulk Chemical Sales. The material arrives in plastic bottles which have a 1.0 L capacity and contain 200 grams of powder. The bottles are stored in a locked cabinet until the procedure is initiated.

[0267] Table 8 lists the composition of the buffers and solutions needed for the procedure and Table 9 lists the specifications for the buffers and solutions. Table 10 lists the equipment used for the production and purification of dgRTA and Table 11 provides a summary of the steps and columns used in the procedure.

TABLE 8
COMPOSITION OF BUFFERS
AA                   200 mM acetic acid adjusted to pH 3.5
                     with 1 M NaOH
BB                   50 mM boric acid adjusted to pH 8.0
                     with 1 M concentrated NaOH
BB + 1 M NaCl        BB + 1 M NaCl, pH 8.0
BB + 0.2 M galactose BB + 0.2 M galactose, pH 8.0
Deglycosylation      AA + 40 mM NalOQ, +80 mM NaCNBH3,
buffer               pH 3.5
PBS                  50 mM H2NaP04 + 150 mM NaCl adjusted
                     to pH 7.2 with 1 M NaOH
Reducing BB          BB + 4% mercaptoethanol, pH 8.0
Tris                 2 M Tris-HCI, pH 10.8

[0268]

TABLE 9
BUFFER SPECIFICATIONS
Buffer	Sterility	PH	Conductivity	Endotoxin
AA	Sterile	3.5	0.9 mΩ	Zero
BB	Sterile	8.0	5.0 mΩ	Zero
BB + 1 M NaCl	Sterile	8.0	60 mΩ	Zero
BB + 0.2 M	Sterile	8.0	4.6 mΩ	Zero
galactose
Deglycosylation	Sterile	3.5	8.0 mΩ	Zero
buffer
PBS	Sterile	7.2	11.5 mΩ	Zero
Reducing BB	Sterile	8.0	5.0 mΩ	Zero
Tris	Sterile	10.8	25 mΩ	Zero

[0269]

TABLE 10
EQUIPMENT
Amicon concentrator, Model #CH2
Autoclave, Model Hirayama
Biological safety cabinet (hood), Model NuAire #NU-425-400
Circulating shaker, Model Fisher #631
1μ Centrifuge; Model, Jovan #GR-412
Centrifuge tubes description, IEC 750 ml
Gravity Connection Owen, Model Precision
Negative pressure refrigerator (a negative pressure, refrigerated,
chromatography box), Model EJS #CR-84
Pharmacia spectro ultrospec III
Technicloth TX 609 (from Tex Wipe)
Whatmann 90377A (1.0 p.m) capsule filter

[0270]

TABLE 11
COLUMN SPECIFICATIONS
			EQUILI-		VOLUME
	SIZE	BED	BRATION	PROTEIN	ELUTION
COLUMN	(CM)	VOLUME	BUFFER	LOADED	BUFFER	FLOW RATE
Acid-Treated	25 × 15	7.36 L	BB	˜100,000	ODU	4 L	BB + 0.2 M galactose	4.5 L/h
Sepharose 4B
Sephacryl S-	25 × 90	44.2 L	AA	˜7,000	ODU	1.5 L	None	4.5 L/h
200
Columns run	11 × 30	2.8 L	BB	8,000	mg ricin	3.5 L	Reducing BB	4.5 L/h
in tandem:	25 × 30	14.7 L		toxin	(RCA2)
DEAE-
Sepharose
And
Acid-Treated
Sepharose 4B
Blue	25 × 15	7.36 L	BB	3,000	mg dgA	30 L	BB + 1 M NaCl	1.5 L/h
Sepharose
CL-4B
Aslalofetuin-	11 × 30	2.8 L	BB	2,000	mg dgA	10 L	None	1.5 L/h
Sepharose
*All columns are kept in the negative refrigerator at 4° C.

[0271] Extraction of bulk ricin: Each extraction batch may consist of two bottles of the castor bean powder. These are opened in the biological safety cabinet (hood), and 1.0 L of PBS is added to each bottle. Each bottle is capped with the original lid, placed on the circular shaker in the hood, and shaken for 1 hour at 200 cycles/minute, at room temperature (RT). The bottles are placed at 4° C. for 30 min to allow for sedimentation of particulates. The supernatant from each bottle is poured into an intermediate vessel which has a cover and spout, and from that vessel, the liquid is poured into 750 ml plastic centrifuge bottles, both steps being performed in the hood. The centrifuge bottles are capped and wiped clean with moist paper towels before they are removed from the hood. The centrifuge bottles are placed in carriers in the centrifuge and centrifuged at 3,700 rpm for 20 minutes at 4° C. The centrifuge bottles are removed, taken to the hood and the supernatants are decanted and filtered through Technicloth TX609 paper into a 6 L Erlenmeyer flask.

[0272] A second extraction is performed. The sediment in the two factory bottles is resuspended with 1.0 L PBS and the extraction procedure including 1 h of shaking at RT followed by centrifugation and filtration is repeated.

[0273] Purification of ricin: All of the following procedures are performed in the negative pressure chromatography box at 4° C.

[0274] Both the first and second extractions are pooled and filtered through a Whatman 90377A (1.0 pm) capsule filter. The absorbance at 280 nm is determined and the total protein extracted from the powder is calculated and recorded.

[0275] The clarified supernatant is then pumped from the flask onto the acid-treated Sepharose 4B column. The unbound fraction is collected, autoclaved and discarded. The bound fraction is eluted with BB+0.2 M galactose (FIG. 3). The eluate is collected directly into a CH2 Amicon concentrator and concentrated to 1.5 L. The concentrate is transferred to a flask and the volume is measured. An aliquot is used to measure the absorbance at 280 nm and the total protein in ODU is recorded.

[0276] The protein is pumped from the flask onto the Sephacryl S-200 column, equilibrated with AA. Separation of peak 1 (RCA1=ricin agglutinin) and peak 2 (RCA2=ricin toxin) is determined by the lowest absorbance at 280 nm between peak 1 and peak 2 (see FIG. 4).

[0277] The first peak contains ricin agglutinin and is discarded. The second peak contains ricin, it is collected directly into the Amicon concentrator and concentrated to 2.5 mg/ml. The volume, OD 280 and total amount of protein in mg is recorded. The ricin is pumped from the concentrator into a capped vessel, which is then wiped with moist paper towels and removed from the chromatography refrigerator.

[0278] Deglycosylation of ricin: The vessel containing the ricin solution is opened in the hood. An equal volume of deglycosylation buffer is added. The vessel is capped, wiped, and placed in the chromatography refrigerator for a 4 h incubation at 4° C. The vessel is then placed in the hood, opened, and glycerol is added to a final concentration of 1% in order to stop the reaction. The vessel is again capped, wiped, placed in the chromatography refrigerator and kept overnight at 4° C.

[0279] Separation of ricin A and B grains and purification of dgRTA: After overnight incubation, the vessel is removed from the refrigerator and placed in the hood. The vessel is opened and Tris is added until the pH reaches 8.0. The neutralized ricin solution is pumped into 2 columns connected together in tandem in the chromatography refrigerator. The first column contains DEAF-Sepharose and the second contains acid-treated Sepharose 4B. Both columns ate equilibrated with BB. The purpose of the DEAE—Sepharose column is to bind endotoxin. Once the absorbance at 280 nm of the effluent from the acid-treated Sepharose 413 column falls to 0.05 ODU (end of peak 1, FIG. 5), the ricin has gone through the DEAE Sepharose column and into the acid-treated Sepharose 4B column, where it is bound.

[0280] Peak 1 represents a small amount of dgRTA that does not bind to acid-treated Sepharose 4B. The columns are then separated and reducing BB is pumped into the acid-treated Sepharose 4B column until the absorbance at 280 nm equals the absorbance of the reducing BB (0.12 ODU). The pump is then stopped and the column is incubated for 4 hours at 4° C. During this time, the S—S bond between the ricin A and B chains is reduced which results in the separation of the two chains.

[0281] The ricin B chain remains bound to the acid-treated-Sepharose 413 column. The column is then washed with reducing BB and the free dgRTA is collected (peak 2, FIG. 5). The collection is stopped when the absorbance at 280 nm returns to the absorbance of reducing BB. The OD 280 of the collected effluent (dgRTA solution) is measured and the volume and protein concentration in ODU is recorded.

[0282] The acid-treated Sepharose 4B column is eluted with BB+0.2 M galactose and the eluted ricin B chain is discarded.

[0283] The dgRTA solution in reducing BB is then pumped over a column of Blue-Sepharose CL-4B equilibrated with BB and held at 4° C. The column is washed with BB until the absorbance at 280 nm drops to baseline. The column is then washed with BB+0.2 M galactose until the small peak is eluted (Peak 2) and the absorbance at 280 nm returns to the baseline value. This procedure is applied to remove all traces of whole ricin toxin (RCA2) or B chain that can interact with the Sepharose matrix. dgRTA is eluted from the Blue-Sepharose column with BB+1 M NaCl as shown in FIG. 6, Peak 3 and the volume and absorbance at 280 nm is measured and recorded.

[0284] The eluate is then pumped onto the Asialofetuin-Sepharose column equilibrated with BB and held at 4° C. The non-bound fraction, starting when the absorbance is over 0.05 ODU and ending when it returns 0.05 ODU, as shown in FIG. 7, is diafiltered in the Amicon concentrator to decrease the NaCl concentration to 0.25 M and to increase the protein concentration to 2 mg/ml. DTT is then added to a final concentration of 10 mM and the solution is filtered through a 0.22 μm filter. The reduced dgRTA solution is mixed with an equal volume of glycerol and stored at −20° C. The column is eluted with BB+0.2 M galactose and the eluate is discarded.

[0285] Testing and specifications: Table 12 lists the tests and specifications for dgRT.

TABLE 12
Tests and Specifications for dgRTA
TEST
MATERIAL	TEST	SPECIFICATION
DgRTA	Protein concentration	2-3 mg/ml
	Sterility	Sterile
	Endotoxin by LAL	<5 EU/mg
	Purity by SDS gel	>95% of protein @ 28-33
		Kda MW peak
	Con A binding	<20% bound
	IC50 (reticulocyte assay)	1-5 × 1011 M
	IC50 (Daudi cell assay)	0.5-5 × 107 M
	LD50 (BALB/c mice)	15-30 μg/g

Example 3

Alteration in the Flanking Regions that Reduce or Eliminate VLS

[0286] A panel of recombinant (r)RTA mutants with single residue changes in either the LDV sequence or in one of two residues adjacent to this sequence in its three dimensional structure (for example R48 or N97) were generated. The candidates, typically had to meet the following criteria: 1) high yield production and long-term stability; 2) full enzymatic activity in a cell-free assay; 3) good cytotoxicity as an IT; 4) failure to induce pulmonary vascular leak (PVL) when used as an IT in SCID mice; 5) an LD50 higher than that of wild type (wt) rRTA; 6) pharmacokinetics (PK) in mice which are similar to those of the ITs containing wt rRTA; and 7) efficacy of the RFB4-rRTA IT in our SCID/Daudi tumor model. Generally, the material and methods used in this example are as described above.

[0287] Plasmids and mutagenesis. The pKK223 plasmid with wt rRTA under IPTG-inducible control was kindly provided by J. Michael Lord, Department of Biology Sciences, University of Warwick, Coventry, U.K. (O'Hare et al., 1987 and Simpson et al., 1995). All DNA manipulations were performed using standard techniques (Sambrook et al, 1989). Mutations were introduced using QuikChange® (Stratagene, LaJolla, Calif.) and pairs of mutagenic primer as follows (only one primer per complementary pair is listed, mutation(s) are underlined):

TABLE 13
Mutagenic primers.
(SEQ ID NO:20)
R48A 5′-AGTGTTGCCAAACGCAGTTGGTTTGCCTATAAA
     CC-3′;
(SEQ ID NO:15)
L74A 5′-CTTTCTGTTACATTAGCCGCGGATGTCACCAATG
     CATATG-3′;
(SEQ ID NO:21)
L74M 5′-GCTTTCTGTTACATTAGCCATGGATGTCACCAA
     TGC-3′;
(SEQ ID NO:16)
D75A 5′-GTTACATTAGCCCTGGCTGTCACCAATGCATAT
     G-3′;
(SEQ ID NO:17)
D75E 5′-CTGTTACATTAGCCCTGGAAGTCACCAATGCAT
     ATG-3′;
(SEQ ID NO:18)
D75N 5′-CTGTTACATTAGCCCTGAACGTCACCAATGCAT
     ATGTGG-3′;
(SEQ ID NO:19)
V76A 5′-GTTACATTAGCCCTGGATGCTACCAATGCATAT
     GTGGTC-3′;
(SEQ ID NO:22)
V76M 5′-GTTACATTAGCCCTGGATATGACCAATGCATAT
     GTGGTC-3′;
(SEQ ID NO:23)
N97A 5′-TCTTTCATCCTGACGCTCAGGAAGATGCAGAA
     GC-3′.

[0288] Pulmonary Vascular Leak. SCID mice (Taconic, Germantown, N.Y.) were injected i.p. with a total of 15 μg IT/g body weight over the course of three days. Following the final IT injection, they were injected i.v. with 5-7 μCi/mouse of 125I-labeled albumin and 24 hours later the 125I content of the right lung was measured and compared with a saline-treated control. Levels of radioactivity in the whole body and blood were determined prior to sacrifice (data not shown).

[0289] Therapeutic protocols. SCID mice with disseminated Daudi lymphoma were treated as described previously with 100 μg/mouse of each IT, MAb or with PBS. Mice were monitored and sacrificed when hind leg paralysis occurred. Comparison of survival curves was carried out using log-rank and Wilcoxon tests (Shah et al., 1993). The median survival time of mice was calculated by the log-rank test at the 5% significance level.

[0290] Pharmacokinetics. SCID mice of 18 to 22 g were given 0.05% Lugol's solution in sweetened drinking water throughout the experiment. Radiolabeled proteins were injected i.v. in the tail vein in a maximum volume of 100 μL with a radioactive load of 1-5×107 cpm and a dose of 5 pg. Whole body radioactivity was counted immediately after injection and on a daily basis for six days using an AtomLab 100 dose calibrator (Atomic Products Corporation, Shirley, N.Y.). Results were expressed relative to the initial whole body radioactivity (%). The pharmacokinetic parameters were determined using a non-compartmental model with the PKCALC program (Schumaker, 1986).

[0291] The in vitro activity of rRTAs and ITs prepared with mutant rRTAs. The plasmid containing wt rRTA (in vector pKK233) was used as the starting material. Several mutants were generated with conservative changes in the LDV sequence, including L74A, L74M, D75N, D75A, D75E, V76A, and V76M, as well as to surface residues adjacent to the LDV sequence and distal from the active site, R48A and N97A (FIG. 8). Purified rRTA preparations were >90% pure (data not shown). The enzymatic activities of these mutant rRTAs, wt rRTA, and dgRTA were then analyzed in a cell free rabbit reticulocyte assay (Press et al., 1986). They were also conjugated to the mouse IgG, anti-human CD22 MAb, RFB4 (Shen et. al., 1988), and were again evaluated in the cell-free reticulocyte assay after reduction. Finally, the ITs were tested on CD22-positive Daudi cells in a standard in vitro cytotoxicity assay, as described herein.

[0292] As shown in Table 14, when tested in the reticulocyte assay, the wt rRTA and dgRTA had very similar activities although the wt rRTA was slightly more active. Following coupling to the RFB4 MAb, both retained their activity in the reticulocyte assay and they had the same relative activity in the Daudi cell cytotoxicity assay. Six RTA mutants, R48A, L74A, L74M, V76A, V76M, and N97A, were as active or more active than dgRTA in the reticulocyte assays. They retained this activity after they were coupled to RFB4, reduced and retested. With the exception of L74A, the same ITs were highly active in the Daudi cell cytotoxicity assay.

TABLE 14:
The enzymatic activity of rRTAs and ITs prepared with these rRTAs
	Cell Free Reticulocyte Assay
	RTA before coupling to	RTA after coupling to	Daudi Cell Cytotoxicity Assay
	RFB4	RFB4	RFB4-RTA
	DgRTA (IC50)
	1.7 ± 0.6 × 10−11a	1.3 ± 0.5 × 10−11b	8.7 ± 8.6 × 10−13c
RTA	Fold reduction	Fold reduction	Fold reduction
WT	0.7 ± 0.6d,c	0.3 ± 0.1	0.7 ± 0.6
L74A	0.8 ± 0.3	0.4 ± 0.2	18 ± 14
M	0.6 ± 0.2	0.2 ± 0.1	1.3 ± 0.6
D75A	1.6 ± 1.3	1.7 ± 1.1	210 ± 150
E	2.5 ± 1.1	4.1 ± 3.4	390 ± 310
N	8.8 ± 5.3	1.8 ± 0.5	480 ± 190
V76A	2.7 ± 1.9	0.8 ± 0.5	6.1 ± 4.5
M	1.0 ± 0.4	0.4 ± 0.1	2.0 ± 1.7
R48A	3.8 ± 1.7	0.7 ± 0.2	3.4 ± 0.8
N97A	0.8 ± 0.1	0.2 ± 0.1	0.9 ± 0.6
a12 experiments
b6 experiments
c>20 experiments
d<1 indicates an increase in activity
e3 to 12 experiments per mutant

[0293] In contrast to these six mutants, rRTA mutants containing D75A, D75E and D75N were two to nine-fold less active in the reticulocyte assay and >200 fold less active as ITs in the Daudi cell cytotoxicity assay. Based on these results, the R48A, L74M, V76A, V76M and N97A mutants were selected for further testing.

[0294] The ability of ITs containing RTAs to induce PVL in vivo. Unlike humans, mice injected with RTA-containing ITs do not manifest systemic VLS but they do show PVL (Baluna et al., 1999 and Soler-Rodriguez et al., 1992). Therefore SCID mice were injected with the ITs prepared with the mutant rRTAs which “passed” the Daudi cell screening assay and monitored PVL (Rosenstein et al., 1986). When mice were injected with 15 μg IT/g of body weight of the ITs containing wt rRTA, dgRTA, L74M, V76A or V76M, PVL was observed (FIG. 9). In contrast, the ITs containing R48A or N97A did not induce PVL. In light of the inventors previous observations that prior radiotherapy can exacerbate VLS in patients (Schindler et al., 2001), mice were pre-irradiated with 150 cGy prior to IT therapy but observed no PVL for the ITs prepared with either R48A or N97A (data not shown).

[0295] The LD50 of RFB4-ITs. It was determined whether the two RFB4-coupled mutant rRTAs, R48A and N97A, were toxic to mice. Groups of 10 mice were injected i.p. with different doses of each IT. Animals were weighed daily for a week. When animals lost 20% of their body weight, they were sacrificed, since this amount of weight loss always leads to death. The LD50s of RFB4-dgRTA and RFB4-wt rRTA were 10 μg/g while the LD50s of RFB4-R48A or RFB4-N97A were >100 μg/g (highest dose tested). Hence, R48A and N97A ITs are as active as RFB4-dgRTA on Daudi cells in vitro but are at least 10-fold less toxic to mice and do not cause PVL at the same dose as the RFB4-dgRTA.

[0296] PK of mutant rRTA. PK and in vivo stability of both the RFB4-coupled mutants and the wt rRTA-IT were examined. The various PK parameters for ITs prepared with N97A and R48A compare well with those observed for the IT containing the wt rRTA (Table 15). Sera from these animals was collected at the end of the experiment, and examined by SDS-PAGE and autoradiography. The ITs were intact without obvious aggregation after seven days in the mice (data not shown).

TABLE 15:
Pharmacokinetics in mice of ITs constructed with rRTAs
	Number	T ½	AUC1	FCR2	MRT3
rRTA-IT	of mice	(h)	(μg·h/mL)	(day−1)	(h)
Wt	8	60.9 ± 1.5	182.6 ±	0.272 ±	85.6 ±
			22.8	0.007	2.1
R48A	5	70.3 ± 1.6	238.0 ±	0.236 ±	99.8 ±
			6.5	0.005	2.4
N97A	5	56.4 ± 1.3	191.8 ±	0.293 ±	78.2 ±
			6.5	0.006	2.0
1Area under the curve;
2Fractional catabolic rate;
3Mean residence time

[0297] Mutant rRTAs in vivo. Since the ITs containing N97A and R48A had virtually full activity, good PK, low in vivo toxicity and did not induce PVL, they were assessed in vivo as RFB4-IT in a disseminated SCID/Daudi model (Ghetie et al., 1990). The SCID/Daudi mice injected with ITs prepared with R48A or N97A had mean paralysis times (MPTs) of 60 and 72 days respectively (FIG. 10). Hence RFB4-N97A was as effective as the RFB4-dgRTA used in previous studies (MPT of 73 days) (O'Hare et al., 1987) and RFB4-R48A showed a statistically significant improvement over RFB4. However, both are less effective than the RFB4-wt rRTA (MPT of 90 days) (FIG. 10). Since the LD50s of the ITs prepared with N97A and R48A are >10-fold higher, they should out-perform the dgRTA-ITs (and the wt rRTA-ITs) at higher doses.

[0298] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

1. A modified proteinaceous composition comprising a protein having a (x)D(y) sequence and at least one amino acid mutation that alters the ability of a (x)D(y) sequence to induce Vascular Leak Syndrome.

2. The modified proteinaceous composition of claim 1, wherein the protein comprises at least two amino acid mutations.

3. The modified proteinaceous composition of claim 1, wherein the at least one mutant amino acid is within approximately 10 angstroms of the aspartic acid in the (x)D(y) sequence of the native protein conformation.

4. The modified proteinaceous composition of claim 1, wherein the at least one mutant amino acid is located at a distance greater than approximately 6 angstroms from an active site residue of the native protein conformation.

5. The modified proteinaceous composition of claim 1, wherein the at least one mutant amino acid is at least partially exposed to the surface of the native protein conformation.

6. The modified proteinaceous composition of claim 1, wherein the protein is a toxin, cytokine, or a viral protein.

7. The modified proteinaceous composition of claim 6, wherein the protein is a toxin.

8. The modified proteinaceous composition of claim 7, wherein the toxin is a ricin A chain toxin, Abrin A chain, Diptheria Toxin (DT) A chain, Pseudomonas exotoxin, Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, or Barley Toxin.

9. The modified proteinaceous composition of claim 8, wherein the toxin is a ricin A chain toxin.

10. The modified proteinaceous composition of claim 9, wherein at least one mutant amino acid is R48.

11. The modified proteinaceous composition of claim 10, wherein R48 is substituted with an alanine residue.

12. The modified proteinaceous composition of claim 9, wherein at least one mutant amino acid is N97.

13. The modified proteinaceous composition of claim 12, wherein N97 is substituted with an alanine residue.

14. The modified proteinaceous composition of claim 9, wherein at least two mutant amino acids are R48 and N97.

15. The modified proteinaceous composition of claim 1, wherein the proteinaceous composition is a pharmaceutical composition.

16. A modified proteinaceous composition comprising a protein having a (x)D(y) sequence and at least one amino acid mutation in a flanking sequence that alters the toxicity of the protein.

17. The modified proteinaceous composition of claim 16, further comprising a mutation in the (x)D(y) sequence.

18. The modified proteinaceous composition of claim 16, wherein the at least one amino acid mutation is within approximately 10 angstroms of the aspartic acid in the (x)D(y) of the native protein conformation.

19. The modified proteinaceous composition of claim 16, wherein the at least one amino acid mutation is located at a distance greater than approximately 6 angstroms from an active site residue of the native protein conformation.

20. The modified proteinaceous composition of claim 16, wherein the at least one amino acid mutation is at least partially exposed to the surface of the native protein conformation.

21. The modified proteinaceous composition of claim 16, wherein the protein is a toxin, cytokine, or a viral protein.

22. The modified proteinaceous composition of claim 21, wherein the protein is a toxin.

23. The modified proteinaceous composition of claim 22, wherein the toxin is a ricin A chain toxin, Abrin A chain, Diptheria Toxin (DT) A chain, Pseudomonas exotoxin, Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, or Barley Toxin.

24. The modified proteinaceous composition of claim 23, wherein the toxin is a ricin A chain toxin.

25. The modified proteinaceous composition of claim 24, further comprising a mutation in the (x)D(y) sequence.

26. The modified proteinaceous composition of claim 24, wherein the ricin A chain toxin is enzymatically active.

27. The modified proteinaceous composition of claim 24, wherein the ricin A chain toxin is not enzymatically active.

28. The modified proteinaceous composition of claim 24, wherein the (x)D(y) sequence is LDV.

29. The modified proteinaceous composition of claim 24, wherein the at least one amino acid mutation is R48.

30. The modified proteinaceous composition of claim 29, wherein R48 is substituted with an alanine residue.

31. The modified proteinaceous composition of claim 24, wherein the at least one amino acid mutation is N97.

32. The modified proteinaceous composition of claim 31, wherein N97 is substituted with an alanine residue.

33. The modified proteinaceous composition of claim 24, wherein the amino acid mutations are R48 and N97.

34. The modified proteinaceous composition of claim 16, wherein the proteinaceous composition is a pharmaceutical composition.

35. A ricin A chain toxin with a reduced ability to promote toxicity in a patient, wherein at least one amino acid flanking the (x)D(y) sequence altered.

36. The ricin A chain toxin of claim 35, wherein toxicity in a patient is further defined as vascular leak syndrome, aphasia, myalgia, fatigue, hypotension or rhabdomyalysis.

37. The ricin A chain toxin of claim 35, wherein toxicity in a patient is further defined as vascular leak syndrome.

38. The ricin A chain toxin of claim 35, wherein the at least one amino acid flanking the (x)D(y) sequence is within approximately 10 angstroms of the aspartic acid in the (x)D(y) of the native protein conformation.

39. The ricin A chain toxin of claim 36, wherein the at least one amino acid flanking the (x)D(y) sequence is located at a distance greater than approximately 6 angstroms from an active site residue of the native protein conformation.

40. The ricin A chain toxin of claim 35, wherein the at least one amino acid flanking the (x)D(y) sequence is located at a distance greater than approximately 6 angstroms from an active site residue of the native protein conformation.

41. The ricin A chain toxin of claim 35, wherein the at least one amino acid flanking the (x)D(y) sequence is at least partially exposed to the surface of the native protein conformation.

42. The ricin A chain toxin of claim 35, wherein the at least one amino acid flanking the (x)D(y) sequence is R48.

43. The ricin A chain toxin of claim 42, wherein R48 is substituted with an alanine residue.

44. The ricin A chain toxin of claim 35, wherein the mutant amino acid is N97.

45. The ricin A chain toxin of claim 44, wherein N97 is substituted with an alanine residue.

46. The ricin A chain toxin of claim 35, wherein the mutant amino acids are R48 and N97.

47. The ricin A chain toxin of claim 41, wherein the (x)D(y) sequence is LDV.

48. The ricin A chain toxin of claim 35, wherein the proteinaceous composition is a pharmaceutical composition.

49. A method of reducing the ability of a proteinaceous composition to reduce induction of VLS, comprising the steps of: a) identifying a protein comprising at least one amino acid sequence of (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine; and b) altering at least one amino acid residue flanking the (x)D(y) sequence, wherein the ability to induce VLS is reduced.

50. The method of claim 49, wherein the at least one amino acid residue flanking the (x)D(y) sequence is within approximately 10 angstroms of the aspartic acid in the (x)D(y) sequence of the native protein conformation.

51. The method of claim 49, wherein the at least one amino acid residue flanking the (x)D(y) sequence is located at a distance greater than approximately 6 angstroms from an active site residue of the native protein conformation.

52. The method of claim 49, wherein the at least one amino acid residue flanking the (x)D(y) sequence is at least partially exposed to the surface of the native protein conformation.

53. The method of claim 49, wherein the protein is ricin A chain toxin.

54. The method of claim 53, wherein the at least one amino acid residue flanking the (x)D(y) sequence is R48.

55. The method of claim 54, wherein R48 is substituted with an alanine residue.

56. The method of claim 53, wherein the at least one amino acid residue flanking the (x)D(y) sequence is N97.

57. The method of claim 56, wherein N97 is substituted with an alanine residue.

58. The method of claim 53, wherein the at least one amino acid residue flanking the (x)D(y) sequence are R48 and N97.

59. A method of preparing an immunotoxin with a reduced ability to induce VLS comprising the steps of: a) identifying a toxin comprising at least one amino acid sequence of (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine; and b) altering at least one amino acid residue flanking the (x)D(y) sequence, wherein the ability to induce VLS is reduced; and c) conjugating said toxin to a composition comprising at least one antibody to produce an immunotoxin, wherein the immunotoxin produced possesses a reduced ability to promote VLS when compared to a like immunotoxin wherein the flanking amino acid sequence was not altered from the toxin.

60. The method of claim 59, wherein the at least one amino acid residue flanking the (x)D(y) sequence is within approximately 10 angstroms of the aspartic acid in the (x)D(y) sequence of the native protein conformation.

61. The method of claim 59, wherein the at least one amino acid residue flanking the (x)D(y) sequence is located at a distance greater than approximately 6 angstroms from an active site residue of the native protein conformation.

62. The method of claim 59, wherein the at least one amino acid residue flanking the (x)D(y) sequence is at least partially exposed to the surface of the native protein conformation.

63. The modified proteinaceous composition of claim 59, wherein the at least one amino acid residue flanking the (x)D(y) sequence is R48.

64. The method of claim 63, wherein R48 is substituted with an alanine residue.

65. The modified proteinaceous composition of claim 59, wherein the at least one amino acid residue flanking the (x)D(y) sequence is N97.

66. The method of claim 65, wherein N97 is substituted with an alanine residue.

67. The modified proteinaceous composition of claim 59, wherein the at least one amino acid residue flanking the (x)D(y) sequence are R48 and N97.