Reduction of the nonspecific animal toxicity of immunotoxins by mutating the framework regions of the Fv to lower the isoelectric point Number:7,521,054 from the United States Patent and Trademark Office (PTO) owispatent

Title: Reduction of the nonspecific animal toxicity of immunotoxins by mutating the framework regions of the Fv to lower the isoelectric point

Abstract: The invention provides recombinant immunotoxins that have been modified from a parental immunotoxin to lower liver toxicity. The immunotoxins are created by specifically mutating charged residues in the framework regions of the heavy chain, the light chain, or both, of the antibody portion or antigen-binding fragment thereof of the parental immunotoxin to reduce the pI of the antibody or fragment. In preferred forms, the antibody portion of the parental is an anti-Tac, anti-mesothelin, or anti-LewisY antigen antibody or antigen-binding fragment, and in particularly preferred forms the antibody portion is an M16 dsFv, a St6 dsFv or a Mt9 dsFv, or a sequence that has at least 90% sequence identity to one of these molecules but retain the particular mutations that lower pI without affecting antibody activity. The invention further provides nucleic acids encoding the recombinant immunotoxins of the invention, expression cassettes comprising the nucleic acids, and host cells comprising the expression cassettes. The invention also provides a method for killing a cell comprising an antigen on the surface of the cell, the method comprising contacting the cell with a recombinant immunotoxin of the invention that has an antibody or antigen-binding fragment thereof that binds specifically to the antigen on the surface of the cell, and uses of immunotoxins of the invention for the manufacture of medicaments.

Patent Number: 7,521,054 Issued on 04/21/2009 to Pastan,   et al.

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Inventors:	Pastan; Ira H. (Potomac, MD), Onda; Masanori (Rockville, MD), Nagata; Satoshi (Rockville, MD), Tsutsumi; Yasuo (Mino, JP), Vincent; James J. (Takoma Park, MD), Kreitman; Robert J. (Potomac, MD), Vasmatzis; George (Rochester, MN), Lee; Byungkook (Potomac, MD)
Assignee:	The United States of America as represented by the Department of Health and Human Services (Washington, DC) N/A (
Appl. No.:	10/416,129
Filed:	November 16, 2001
PCT Filed:	November 16, 2001
PCT No.:	PCT/US01/43602
371(c)(1),(2),(4) Date:	December 18, 2003
PCT Pub. No.:	WO02/40545
PCT Pub. Date:	May 23, 2002

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	60249805	Nov., 2000

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Current U.S. Class:	424/178.1 ; 435/328; 435/69.7; 530/391.1
Current International Class:	A61K 47/48 (20060101); C07K 16/18 (20060101); C07K 16/28 (20060101)

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References Cited [Referenced By]

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Other References

Onda et al. (J. Immunol. 163:6072-6077 (Dec. 1, 1999)). cited by examiner . Search output from ATCC website for SS1 antibody/hybridoma (pp. 1-2). cited by examiner . Search output from ATCC website for B3 antibody/hybridoma (pp. 1-2). cited by examiner . Fundamental Immunology 242 (William E. Paul, M.D. ed., 3d ed. 1993). cited by examiner . Lederman et al (Molecular Immunology 28:1171-1181, 1991). cited by examiner . Li et al (Proc. Natl. Acad. Sci. USA 77:3211-3214, 1980). cited by examiner . Katz et al. (J. Exp. Med. 180:925-932 (1994). cited by examiner . Colcher et al., (Quarterly J. Nucl. Med. 42(4):225-241 (1998). cited by examiner . Pavlinkova et al. (Nuc. Med. & Biol. 26:27-34 (1999). cited by examiner . Idziorek et al. (Infect. Immun. 58(5): 1415-1420 (1990)). cited by examiner . Onda et al. (J. Immunol. 163:6072-6077 (1999)). cited by examiner . Chowdhury et al. (PNAS 95:669-674 (1998)). cited by examiner . Brinkman et al. (PNAS 90:7538-7542 (1993)). cited by examiner . Vajdos et al. (2002) J. Mol. Biol. 320, 415-428. cited by examiner . Wu et al. J. Mol. Biol. (1999) 294, 151-162. cited by examiner . Coleman (Research in Immunol. 145:33-36 (1994)). cited by examiner . Studnicka (Prot. Engineer. 7(6):805-814 (1994)). cited by examiner . Brinkmann, U. et al.; "A Recombinant Immunotoxin Containing a Disulfide-Stabilized Fv Fragment"; 1993, PNAS, vol. 90, pp. 7538-7542. cited by other . Chaudhary, Vijay K. et al.; "" Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity; 1998, PNAS, vol. 95, pp. 669-674. cited by other . Kobayashi, Hisataka et al; "The Pharmacokinetic Characteristics of Glycolated Humanized Anti-Tac Fabs Are Determined by Their Isoelectric Points"; 1999, Cancer Research, vol. 59, pp. 422-430. cited by other . Kreitman, Robert J. et al.; "Immunotoxins for targeted cancer therapy"; 1998, Advanced Drug Delivery Reviews, vol. 31, pp. 53-88. cited by other . Ll, Tianyong et al.; "Coexpression of cyt1Aa of Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus Binary Toxin Gene in Acrystalliferous Strain of B. thuringiensis"; 2000, Current Microbiology, vol. 40, pp. 322-326. cited by other . Onda, Masanori et al.; "Reduction of the Nonspecific Animal Toxicity of Anti-Tac(Fv)-PE38 by Mutations in the Framework Regions of the Fv Which Lower the Isoelectric Point"; 1999, The Journal of Immunology, pp. 6072-6077. cited by other . Pastan, Ira et al.; "Recombinant Toxins for Cancer Treatment"; 1991, Science, vol. 254, pp. 1173-1177. cited by other . Reiter, Yoram et al.; "Stabilization of the Fv Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions"; 1994, Biochemistry, vol. 33, pp. 5451-5459. cited by other . Yokota, Takashi et al.; "Rapid Tumor Penetration of a Single-Chain Fv and Comparison with Other Immunoglobulin Forms"; 1992, Cancer Research, vol. 52, pp. 3402-3408. cited by other . Bird, Robert E. et al.; "Single-Chain Antigen-Binding Proteins"; 1998, Science, vol. 242, pp. 423-426. cited by other . Chaudhary, Vijay K. et al.; "A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin"; 1989, Nature, vol. 339, pp. 394-397. cited by other . Chowdhury, Partha S. et al.; "Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity"; 1998, PNAS, vol. 95, pp. 669-674. cited by other . Kim, L.S. et al.; "Acylation with Glycolate Lowers pl of Anti-Tac dsFv and Reduces Renal Uptake of Its Tc-99 Label"; 1997, J. Label. Compd XL, vol. 40, pp. 422-425. cited by other.

Primary Examiner: Blanchard; David J.
Assistant Examiner: Bristol; Lynn
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP

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Parent Case Text

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CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Patent Application No. 60/249,805, filed Nov. 17, 2000, the contents of which are incorporated herein by reference.

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Claims

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What is claimed is:

1. A recombinant immunotoxin comprising an antibody or antigen-binding fragment thereof comprising SEQ ID NO:3 and SEQ ID NO:10 and a toxin moiety.

2. A recombinant immunotoxin comprising an antibody or antigen-binding fragment thereof comprising SEQ ID NO:5 and SEQ ID NO:12 and a toxin moiety.

3. A recombinant immunotoxin comprising an antibody or antigen-binding fragment thereof comprising SEQ ID NO:7 and SEQ ID NO:14 and a toxin moiety.

4. A recombinant immunotoxin of any one of claims 1, 2 or 3, wherein the toxin moiety is selected from the group consisting of Pseudomonas exotoxin A ("PE") or a cytotoxic fragment or mutant thereof, Diphtheria toxin or a cytotoxic fragment or mutant thereof, ricin or a cytotoxic fragment thereof, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof, and bryodin 1 or a cytotoxic fragment thereof.

5. A recombinant immunotoxin of claim 4, wherein the toxin moiety is selected from the group consisting of PE38, PE35, PE40, PE4E, and PE38QQR.

6. A composition comprising a recombinant immunotoxin of claim 1 in a pharmaceutically acceptable carrier.

7. A composition comprising a recombinant immunotoxin of claim 2 in a pharmaceutically acceptable carrier.

8. A composition comprising a recombinant immunotoxin of claim 3 in a pharmaceutically acceptable carrier.

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Description

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STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Recombinant toxins are chimeric proteins in which a cell targeting moiety is fused to a toxin (Pastan et al., Science, 254: 1173-1177 (1991)). If the cell targeting moiety is the Fv portion of an antibody, the molecule is termed a recombinant immunotoxin (Chaudhary et al. Nature, 339: 394-397 (1989)). The toxin moiety is genetically altered so that it cannot bind to the toxin receptor present on most normal cells. Recombinant immunotoxins selectively kill cells which are recognized by the antigen binding domain. Fv fragments are the smallest functional modules of antibodies. When used to construct immunotoxins, Fv fragments are better therapeutic reagents than whole IgGs because their small size facilitates better tumor penetration (Yokota et al., Cancer Res., 52: 3402-3408 (1992)). Initially, Fvs were stabilized by making recombinant molecules in which the Variable Heavy (VH) and Variable Light (VL) domains are connected by a peptide linker so that the antigen binding domain site is regenerated in a single protein (a single chain Fv, or "scFv") (Bird R., et al., Science, 242:423-426 (1988)). Many Fvs, however, could not be stabilized by this approach.

An alternative approach is to stabilize the Fv by a disulfide bond that is engineered between framework regions of the two Fv domains. The disulfide-bond stabilized Fv (termed a "dsFv") is fused to the toxin through either of the Fv domains (Brinkmann et al., Proc Natl Acad Sci (USA), 90: 7538-7542 (1993)). One striking difference between scFv immunotoxins and dsFv immunotoxins is that dsFv immunotoxins are more stable. Also, dsFv immunotoxins can often be produced with higher yields than scFv immunotoxins (Reiter et al., Biochem, 33: 5451-5459 (1994)).

During the past several years, a number of recombinant toxins have been made using different antibodies ("Abs") (Reiter and Pastan, Trends Biotechnol., 16:513 (1998)). Several of these recombinant immunotoxins have now been evaluated in phase I trials in patients with cancer. All the recombinant immunotoxins that have been brought to clinical trials have been shown to reduce the size of human cancer xenografts growing in nude mice and to be relatively well tolerated by mice and monkeys (Reiter and Pastan, supra). In a phase I trial, eight partial responses were observed in patients with hematopoietic malignancies treated with an immunotoxin, called anti-Tac(Fv)-PE38 (also known as LMB-2), directed against the .alpha. subunit of the IL-2 receptor. Side effects have been observed, however, that cannot be attributed to targeting IL-2R positive cells. These side effects limit the amount of immunotoxin that can be given to humans.

The toxic side effects of recombinant immunotoxins are of two types. One type of toxicity results from specific targeting of normal cells which display the same antigen ("Ag") as the cancer cell. The second type of toxicity is nonspecific and usually is characterized by damage to liver cells; this increases the serum levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvate transaminase (Kreitman and Pastan, Semin. Cancer Biol., 6:297 (1995)), although other toxic effects may occur (Kreitnan and Pastan, Adv. Drug Delivery Rev., 31:53 (1998)).

Some attempts have been made to alter the toxicity of immunoconjugates such as immunotoxins. Morgan, Jr. et al., U.S. Pat. No. 5,322,678, states that the charge of antibodies can be modified to alter their uptake by the kidneys and hence affect their serum half-life. It indicates that antibodies with high positive charges at physiological pH, such as antibodies with highly basic isoelectric points (pI), are likely to undergo charge interaction with negatively charged patches in the glomeruli of the kidney and to be rapidly cleared from the circulation. The patent states that acidic shifts can be are made by reacting antibodies or antibody fragments with succinic anhydride to modify lysine residues.

Kim et al., J. Label. Compd. Radiopharm. XL:422-430 (1997) states that anti-Tac disulfide stabilized immunoconjugates acylated with glycolate to lower their pI had decreased renal uptake without altering tumor uptake.

Kobayashi et al., Cancer Res. 59:422-430 (1999) states that glycolated, unconjugated anti-Tac antibody fragments known as Fabs which were more anionic had less renal clearance and higher tumor accumulation.

SUMMARY OF THE INVENTION

The invention provides recombinant immunotoxins which exhibit reduced liver toxicity compared to their parental immunotoxins. The recombinant immunotoxins comprise an antibody or antigen-binding fragment thereof and a toxin moiety, the toxin moiety of the parental immunotoxin having a pI less than 8, the recombinant immunotoxin comprising an antibody or antigen-binding fragment thereof and an toxin moiety, wherein the antibody or antigen-binding fragment of the recombinant immunotoxin comprises at least one substitution of a negatively charged amino acid for a uncharged or positively charged amino acid on a surface of a framework region of the parental antibody or antigen-binding fragment thereof, and further wherein the recombinant immunotoxin has a pI below about 8.0 resulting from the substitution of amino acids in the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof of said recombinant immunotoxin has a pI, which pI is at least 4 units less than the pI of the parental antibody or antigen-binding fragment thereof.

In one set of preferred embodiments, the parental antibody or antigen-binding fragment thereof is an anti-IL2 receptor antibody or antigen-binding fragment thereof, an anti-mesothelin antibody or antigen-binding fragment thereof, or an anti-LewisY antigen antibody or antigen-binding fragment thereof. In a preferred subset of this set of embodiments, the parental antibody is an anti-Tac antibody or antigen-binding fragment thereof. In some embodiments, the parental antibody or antigen-binding fragment thereof is an M1 dsFv (SEQ ID NOS:2 and 9) or scFv. Further, in some embodiments, the recombinant antibody or antigen-binding fragment thereof has a percent sequence identity that is 90% or more identical to SEQ ID NOS:3 and 10 (the amino acid sequence of M16) and wherein V.sub.H chain positions 13 and 73 are occupied by negatively charged residues, V.sub.L chain positions 18, 45 and 77 are occupied by uncharged residues, and V.sub.L chain positions 103 and 107 are occupied by negatively charged residues, all positions being numbered according to FIG. 2. In some embodiments, these recombinant immunotoxins have an amino acid residue at least one position in a framework region, which position is determined according to FIG. 2, wherein said residue in said position of said framework region is an amino acid residue selected from the group consisting of amino acid residues set forth in a "Percent Frequency" Table of FIG. 2 for said position. In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof has the sequence of SEQ ID NOS:3 and 10 (antibody M16).

In another group of embodiments, the parental antibody or antigen-binding fragment thereof is selected from the group consisting of SS1 and B3. In a subgroup of this group, the parental antibody is SS1 and the recombinant antibody or antigen-binding fragment thereof has a percent sequence identity that is 90% or more identical SEQ ID NOS:5 and 12 (to the amino acid sequence of St6 dsFv), wherein V.sub.H chain position 1 is occupied by a negatively charged residue, and further wherein V.sub.L chain positions 7, 60, 80, and 107 are occupied by negatively charged residues, all positions being numbered according to FIG. 2. In some embodiments, these recombinant immunotoxins have an amino acid residue at least one position in a framework region, which position is determined according to FIG. 2, wherein said residue in said position of said framework region is an amino acid residue selected from the group consisting of amino acid residues set forth in a "Percent Frequency" Table of FIG. 2 for said position. In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof is an St6 dsFv (SEQ ID NOS:5 and 12) or scFv. In a second subgroup, the parental antibody is an anti-B3 antibody and the recombinant antibody or antigen-binding fragment thereof has a sequence identity that is 90% or more identical to SEQ ID NOS:7 and 14 (the amino acid sequence of Mt9 dsFv), wherein VL positions 3, 103, and 107 are occupied by negatively charged residues, all positions being numbered according to FIG. 2. In some embodiments, the recombinant immunotoxin has an amino acid residue at least one position in a framework region, which position is determined according to FIG. 2, wherein said residue in said position of said framework region is an amino acid residue selected from the group consisting of amino acid residues set forth in a "Percent Frequency" Table of FIG. 2 for said position. In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof is Mt9 dsFv (SEQ ID NOS:7 and 14) or scFv.

The recombinant immunotoxins described above preferably have a toxin moiety is selected from the group consisting of Pseudomonas exotoxin A ("PE") or a cytotoxic fragment or mutant thereof, Diphtheria toxin or a cytotoxic fragment or mutant thereof, ricin or a cytotoxic fragment thereof, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof, and bryodin 1 or a cytotoxic fragment thereof. In some preferred embodiments, the toxin moiety is selected from the group consisting of PE38, PE35, PE40, PE4E, and PE38QQR.

The invention further provides compositions comprising any of the above-described recombinant immunotoxins in a pharmaceutically acceptable carrier.

The invention further provides nucleic acid sequences encoding the recombinant immunotoxins described above, as well as expression cassettes comprising a promoter operably linked to a nucleic acid molecule encoding one of these recombinant immunotoxins. The invention further provides host cells comprising one or more of these expression cassettes.

In another set of embodiments, the invention provides methods of killing a malignant cell bearing an antigen, comprising contacting the cell with a recombinant immunotoxin of the invention, as described above, wherein the antibody or antigen-binding fragment of said immunotoxin specifically binds to said antigen. In preferred embodiments, the antigen is selected from the group consisting of an IL-2 receptor, mesothelin, and a Lewis.sup.Y antigen. In some preferred embodiments, the antigen is an IL-2 receptor and the antibody or antigen-binding fragment thereof is an anti-TAC antibody. In some of these embodiments, the antigen is an IL-2 receptor and the anti-TAC antibody or antigen-binding fragment thereof is a M16 dsFv (SEQ ID NO:3 and 10) or scFv. 40. In some preferred embodiments, the antigen is mesothelin and the antibody or antigen-binding fragment thereof is an anti-mesothelin antibody. In some of these embodiments, the anti-mesothelin antibody or antigen-binding fragment thereof is a St6 dsFv (SEQ ID NOS:5 and 12) or scFv. In other preferred embodiments, the antigen is a Lewis.sup.Y antigen and the and the antibody or antigen-binding fragment thereof is an anti-Lewis.sup.Y antibody. In some of these embodiments, the antigen is a Lewis.sup.Y antigen and the anti-Lewis.sup.Y antibody is a Mt9 dsfv (SEQ ID NOS:7 and 14) or scFv.

In another group of embodiments, the invention provides for the use of the recombinant immunotoxins of the invention for the manufacture of a medicament to inhibit the growth of a cancer cell, which cancer cell bears an antigen specifically bound by the antibody or antigen-binding fragment thereof of said immunotoxin. In some of these embodiments, the invention provides a use for the manufacture of a medicament to inhibit the growth of a cancer cell, wherein the parental antibody or antigen-binding fragment thereof is an anti-IL2 receptor antibody or antigen-binding fragment thereof, an anti-mesothelin antibody or antigen-binding fragment thereof, or an anti-LewisY antigen antibody or antigen-binding fragment thereof. In one preferred group of embodiments, the recombinant antibody or antigen-binding fragment thereof has an amino acid sequence that has a percent sequence identity that is 90% or more identical to SEQ ID NOS:3 and 10 (M16), wherein V.sub.H chain positions 13 and 73 are occupied by negatively charged residues, wherein V.sub.L chain positions 18, 45 and 77 are occupied by uncharged residues, and wherein V.sub.L chain positions 103 and 107 are occupied by negatively charged residues, all positions being numbered according to a Percent Frequency table of FIG. 2. In some of these embodiments, the recombinant antibody or antigen binding fragment thereof has amino acid residues at positions in framework regions which positions are determined according to a "Percent Frequency" table of FIG. 2, which antibody has an amino acid sequence selected from the group consisting of SEQ ID NOS:3 and 10 (M16), or a sequence in which residues at positions in framework regions of M16 have been mutated to be amino acid residues selected from the group consisting of residues set forth for that position in a "Percent Frequency" table of FIG. 2.

In particularly preferred embodiments, the antibody or antigen binding fragment thereof is M16 (SEQ ID NOS:3 and 10).

In a further set of preferred embodiments, the invention provides uses of the recombinant immunotoxins of the invention for the manufacture of a medicament to inhibit the growth of a cancer cell, in which the recombinant antibody or antigen-binding fragment thereof of the immunotoxin has an amino acid sequence with a percent sequence identity 90% or more identical to the amino acid sequence of St6 dsFv (SEQ ID NOS:5 and 12), and wherein V.sub.H chain position 1 is occupied by a negatively charged residue and V.sub.L chain positions 7, 60, 80, and 107 are occupied by negatively charged residues, all positions being numbered according to a "Percent Frequency" table of FIG. 2. In some of these embodiments, the recombinant antibody or antigen binding fragment thereof has amino acid residues at positions in framework regions which positions are determined according to a "Percent Frequency" table of FIG. 2, which antibody has an amino acid sequence selected from the group consisting of a sequence identical to that of St6 (SEQ ID NOS:5 and 12), or a sequence in which residues at positions in framework regions of St6 have been mutated to be amino acid residues selected from the group consisting of residues set forth for that position in a "Percent Frequency" section of FIG. 2. In a particularly preferred embodiment, the antibody or antigen binding fragment thereof is St6 (SEQ ID NOS:5 and 12).

In another set of embodiments, the invention provides uses of the recombinant immunotoxins of the invention for the manufacture of a medicament to inhibit the growth of a cancer cell, in which the recombinant antibody or antigen-binding fragment thereof of the immunotoxin has an amino acid sequence with a percent sequence identity that is 90% or more identical to the amino acid sequence of SEQ ID NOS:7 and 14 (Mt9 dsFv) and wherein V.sub.L chain positions 3, 103 and 107 are occupied by negatively charged residues, all positions being numbered according to a "Percent Frequency" table of FIG. 2. In some of these embodiments, the recombinant antibody or antigen binding fragment thereof has amino acid residues at positions in framework regions which positions are determined according to a numbering system of a "Percent Frequency" table of FIG. 2, which antibody has an amino acid sequence selected from the group consisting of a sequence identical to that of Mt9 (SEQ ID NOS:7 and 14), or a sequence in which residues at positions in framework regions of Mt9 have been mutated to be amino acid residues selected from the group consisting of residues set forth for that position in the "Percent Frequency" table of FIG. 2. In a particularly preferred embodiment, the antibody or antigen binding fragment thereof is Mt9 (SEQ ID NOS:7 and 14).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Anti-tumor effect of M1(Fv)-PE38 in nude mice. Open circles: animals treated with 0.025 mg/kg, triangles: animals treated with 0.075 mg/kg, open squares: animals treated with 0.3 mg/kg of M1(Fv)-PE38 in Dulbecco's modified PBS containing 0.2% HSA. Filled squares: control animals receiving carrier only.

FIG. 2. Sequences of Fvs aligned with frequency table. Legend: VL denotes amino acid sequence of variable light chain; VH denotes amino acid sequence of variable heavy chain. "Kabat" denotes amino acid residue number numbered by system as set forth in Kabat et al. (1987). "FR"=framework region; "CDR"=complementarity determining region. "dsAT"=disulfide stabilized anti-Tac Fv (V.sub.L sequence: SEQ ID NO:1; V.sub.H sequence: SEQ ID NO:8); M1=Fv portion of anti-Tac antibody in which selected neutral residues are mutated to acidic residues (V.sub.L sequence: SEQ ID NO:2; V.sub.H sequence: SEQ ID NO:9);

M16=Fv portion of anti-Tac antibody in which neutral and basic residues are mutated to acidic residues (V.sub.L sequence: SEQ ID NO:3; V.sub.H sequence: SEQ ID NO:10); SS1=Fv portion of an anti-mesothelin antibody (V.sub.L sequence: SEQ ID NO:4; V.sub.H sequence: SEQ ID NO:11); ST6=Fv portion of antibody SS1 in which neutral and basic residues have been mutated to acidic residues (V.sub.L sequence: SEQ ID NO:5; V.sub.H sequence: SEQ ID NO:12); dsB3=disulfide stabilized B3 Fv (V.sub.L sequence: SEQ ID NO:6; V.sub.H sequence: SEQ ID NO:13); "Mt9"=Fv portion of antibody B3 in which neutral and basic residues have been mutated to acidic residues (V.sub.L sequence: SEQ ID NO:7; V.sub.H sequence: SEQ ID NO:14). Vertical shaded areas identify positions at which residues were mutated from that of the amino acid of the parental antibody. All amino acids are designated by standard single letter code. The lower portion of each panel, labeled "Percent Frequency," is a table of amino acids sorted into bands denoting, for each position of the Kabat sequence, the percentage of antibodies in the Kabat database in which the amino acids appears at that position of the framework regions of the VL and VH chains, respectively. For example, in the top panel, reading vertically down from Kabat position 1, the amino acid "D" (aspartic acid) occurs in from 50-100% of all antibodies, "Q" (glutamine) occurs in 10-20% of all antibodies, "E" (glutamic acid) occurs in 5-10% of all antibodies, and "N" (asparagine) occurs in only 2-3% of antibodies. Dashes (--) in the lines corresponding to the individual Fvs, refers to the absence of residues present in the Kabat numbering sequence. CDRs are regions that vary among antibodies since the CDRs determine binding specificity. The dashes in the 50-100% line for the CDRs denote that no frequency determination was made for residues in the CDRs.

FIG. 3. Polyacrylamide gel electrophoresis of purified recombinant immunotoxins. The purified proteins were run on 4-20% gradient SDS-polyacrylamide electrophoresis gels under non-reducing conditions (A), and under reducing conditions (B). The gels were stained with Coomasie Blue. Lane 1, M1(dsFv)-PE38; lane 2, M16(dsFv)-PE38; lane 3, SS1(dsFv)-PE38; lane 4, St6(dsFv)-PE38; lane 5, B3(dsFv)-PE38; lane 6, Mt9(dsFv)-PE38; M, molecular mass standards are (top to bottom) 204, 120, 80, 50, 34, 29, 21.6 and 7 kDa, respectively.

FIG. 4. Isoelectric focusing of immunotoxins. M, pI standard marker; lane 1, M1(dsFv)-PE38; lane 2, M16(dsFv)-PE38; lane 3, SS1(dsFv)-PE38; lane 4, St6dsFv)-PE38; lane 5, B3(dsFv)-PE38; lane 6, Mt9(dsFv)-PE38, respectively.

FIG. 5. Antitumor activities of M16(dsFv)-PE38 (shown in panel A), St6(dsFv)-PE38 (panel B), and Mt9(dsFv)-PE38 (panel C) in nude mice bearing human cancer cells which have antigen expression (ATAC4 cells in panel A, A431-K5 cells in panel B, and A431 cells in panel C). Groups of five animals were injected subcutaneously with 3.times.10.sup.6 human cancer cells on day 0. Tumors approximately 0.05 cm.sup.3 in size developed in animals by day 4 after tumor implantation. Starting on day 4, animals were treated with intravenous injections of one of the immunotoxins, diluted in 0.2 ml of PBS/0.2% HAS, or of carrier alone, as a control. Animals administered immunotoxins were divided into three cohorts; each cohort received a different dosage of the immunotoxin. The particular dosages administered of each immunotoxin are set forth in the legend to the right of each panel. Therapy was given once every other day (on day 4, 6, and 8, Arrow head). No death or toxicity was observed at these doses.

FIG. 6. Pharmacokinetics of M1(dsFv)-PE38 and M16(dsFv)-PE38 (shown in panel A), SS1(dsFv)-PE38 and St6(dsFv)-PE38 (panel B), and B3(dsFv)-PE38 and Mt9(dsFv)-PE38 (panel C) in mice. NIH Swiss mice were injected intravenously with 5 .mu.g of M1(dsFv)-PE38, M16(dsFv)-PE38, SS1(dsFv)-PE38, or St6(dsFv)-PE38, or 10 .mu.g of B3(dsFv)-PE38 or Mt9(dsFv)-PE38. Blood samples were drawn at different times. The level of immunotoxin in blood was measured by a bioassay in which serum samples were incubated with human cancer antigen positive cells (ATAC4 cells for A, A431-K5 cells for B, or A431 cells for C), and the ability of the serum sample to inhibit protein synthesis was measured. Results are the average of 4 or 5 animals for each time point.+-.SE.

FIG. 7. Correlation between pI and mice toxicity (LD.sub.50).

TABLE-US-00001 Symbols indicate the following: .smallcircle. M1(dsFv)-PE38, .circle-solid. M16(dsFv)-PE38, .DELTA. SS1(dsFv)-PE38, .tangle-solidup. St6(dsFv)-PE38, .quadrature. B3(dsFv)-PE38, .box-solid. Mt9(dsFv)-PE38 n = 6, r = -0.9691, p = 0.0014.

DETAILED DESCRIPTION

Introduction

It has now been discovered that the nonspecific toxicity of immunotoxins which use antibodies or fragments thereof as targeting moieties can be reduced by mutating specific residues of the Fv of the antibody to reduce the isoelectric point ("pI") of the Fv portion of the immunotoxin, without affecting the toxicity of the immunotoxin to the targeted cells or other desirable characteristics. Moreover, a method has been developed which permits the rational selection of residues of an antibody to be mutated, permitting the predictable development of immunotoxins which will retain the selective cytotoxicity and stability of the starting immunotoxin, but which have lower toxicity than the starting immunotoxin. Surprisingly, the pI of the Fv portion of the immunotoxin can be reduced by more than four units when the reduction is performed according to the methods taught herein. Since animal toxicity is a limiting factor in administering immunotoxin therapeutically, the immunotoxins of the invention, which have lower toxicity, can be administered in higher doses than current immunotoxins. The higher doses, in turn, facilitate the killing of a higher percentage of the target cells, and an improved therapeutic outcome.

The Fv regions of the immunotoxin are mutated in a manner which succeeds in decreasing the pI without causing a substantial decrease either in the targeting capability of the immunotoxin or the toxic effect of the immunotoxin on the targeted cells. Conversely, it is generally not necessary to make changes to the toxic moiety of the immunotoxin to reduce pI. Modifications to the toxic moiety may, however, be made to reduce undesirable characteristics, such as non-specific binding of the toxin, or to enhance desirable characteristics, such as internalization of the toxin into a target cell or the translocation of the toxin into the cytosol of the cell. Such modifications are known in the art and are discussed further below.

Any immunotoxin of interest can predictably have its non-specific toxicity reduced by rationally mutating specific residues chosen according to the following protocol, without interfering antigen binding, stability, or normal antibody folding. The protocol achieves this goal, in part, by homing in on particular candidate residues, and by determining whether the candidate residue can be mutated by comparing that residue to the ones most commonly found at the same position in hundreds, if not thousands, of other antibodies. In essence, the method uses natural evolution as a guide to whether mutation of the candidate residue will be advantageous or contraindicated by looking at thousands of antibodies to determine if a negative charge can be placed at particular positions in the structure of the antibody. If evolution has indicated a negative charge can be placed at the position of the candidate residue, than the method permits mutation of the residue to one containing a negative charge. If the candidate residue is at a position where a negatively charged is never found, it is considered likely that a negative charge at that position is disadvantageous, and a mutation is not made.

The protocol identifies residues that are candidates for mutation as follows. First, the heavy and light chains of the Fv region of the antibody of interest are aligned with the Kabat numbering system for amino acid residues in the V.sub.H and V.sub.L chains. (Kabat, E., et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Department of Health and Human Services, (5.sup.th Ed., 1991)) (hereafter, "Kabat"). The Kabat numbering system is the most widely used system in the art for numbering residues of antibody chains consistently so that one of skill can determine, for example, which residues are within complementarity determining regions ("CDRs") and which are within framework regions, and persons of skill in the art are well familiar with determining Kabat positions for antibody chains. The Kabat database is now too large to be maintained in print and is available online at http://immuno.bme.nwu.edu/. if the amino acid sequences of the antibody chains are not known, the sequence should first be determined to permit the alignment to be made. Conveniently, this can be done by cloning the gene and determining the amino acid sequence encoded by the nucleic acid sequence. Alternatively, the sequence of the amino acids of the antibody can be determined chemically.

FIG. 2 shows an alignment with the Kabat numbering of the amino acid sequences of the light and the heavy chains of six exemplary Fvs: a disulfide stabilized anti-Tac Fv (denoted as "dsAT") (the amino acid sequence of the V.sub.L chain ("V.sub.L sequence") is SEQ ID NO:1; the amino acid sequence of the V.sub.H chain ("V.sub.H sequence") is SEQ ID NO:8), a mutant of dsAT in which certain neutral residues of the CDRs were mutated to acidic residues ("M1") (V.sub.L sequence: SEQ ID NO:2; V.sub.H sequence: SEQ ID NO:9), a mutant of M.sub.1 in which certain neutral and basic residues of the CDRs were mutated to acidic residues ("M16") (V.sub.L sequence: SEQ ID NO:3; V.sub.H sequence: SEQ ID NO:10), an anti-mesothelin Fv known as SS1 (V.sub.L sequence: SEQ ID NO:4; V.sub.H sequence: SEQ ID NO:11), a mutant of SS1 in which certain neutral and basic residues of the CDRs were mutated to acidic residues ("ST6") (V.sub.L sequence: SEQ ID NO:5; V.sub.H sequence: SEQ ID NO:12), a disulfide stabilized anti-LewisY antigen Fv ("dsB3") (V.sub.L sequence: SEQ ID NO:6; V.sub.H sequence: SEQ ID NO:13), and a mutant of dsB3 in which certain neutral and basic residues of the CDRs were mutated to acidic residues ("Mt9") (V.sub.L sequence: SEQ ID NO:7; V.sub.H sequence: SEQ ID NO:14).

Second, the frequency in which particular residues are found at each location in the framework regions of Fvs are noted. This information is presented in tabular form in FIG. 2, and presents an analysis of the current Kabat database of the residues found at each position of thousands of antibodies of all classes, as set forth in the Kabat database website, http://immuno.bme.nwu.edu. The top panel of FIG. 2 presents this information for positions of the variable light ("V.sub.L" or "VL") chain of the Fv, while the bottom panel of the Figure presents this information for positions of the variable heavy ("V.sub.H") chain. The frequency with which a particular residue is found at a particular position in the framework region of the Fv is set forth in the portion of the panel marked on the left side as "Percent Frequency," which is sometimes referred to herein as the Percent Frequency Table. The part of the panel denoted by the arrows is broken into percentage bands showing the percent of antibodies in which a particular residue appears at the particular position.

The Table is read vertically, starting from the Kabat position number at the top of the panel, and may be most easily understood with reference to an example. Turning to the top panel, which describing the V.sub.L portion of the Fv, the line setting forth the Kabat position numbers is read horizontally until the residue of interest is reached. In this example, it will be assumed the first residue of interest is found at Kabat position 1, which appears just to the right of the word "Kabat." Proceeding down vertically, the residue which appears at that position in the V.sub.L of the dsAT Fv is "Q", the standard single letter code for glutamine; the V.sub.Ls of the other five Fvs listed have the residue "D" (aspartic acid) at position 1. Proceeding down into the "Percent Frequency" Table below the aligned Fv regions, the reader finds that 50-100% of all antibodies have a "D" (aspartic acid) at position 1, that 10-20% of all antibodies have a "Q" (glutamine) at that position, that 5-10% of all antibodies have an "E" (glutamic acid) at position 1, and that 2-3% of antibodies have a and "N" (asparagine) at position 1. It therefore appears that, if desired, position 1 can be mutated to a "D," "Q," "E," or "N" without affecting the ability of the Fv to function, while other residues at that position would probably not be desirable. In contrast, position 23 of the light chain is always occupied by a "C" (cysteine) residue, as can be seen by the fact that a "C" appears in the "50-100%" band, while no other residue appears in any other percentage frequency bands. Similarly, position 88 of the V.sub.L is also always a cysteine. Since positions 23 and 88 of the VL are occupied by a cysteine residue in every antibody in the Kabat database, it is very likely that a cysteine at this position is required for correct function of the Fv. Thus, the Percentage Frequency table set forth in FIG. 2 can be used to determine which residues can be considered permissible substitutions at any given position, and which residues should be considered invariant.

Returning to the protocol, the third step is to identify residues in the alignment which are in a complementarity determining region ("CDR"). CDRs are considered to be involved in antigen recognition and binding. Residues in the CDRs are therefore likely to affect antigen recognition and are excluded from consideration as candidates for mutation. This exclusion is symbolized on the "Percentage Frequency" table in FIG. 2 by a dash (-) in the 50-100% band in each position representing a residue in a CDR (to assist in interpreting the Figure, it is noted that the alignments for the individual Fvs also contain dashes at some positions in the CDRs. A dash in the alignment of any particular the Fvs represents a position in the Kabat numbering system for which there is not a corresponding residue in the Fv when the Fv is aligned. Most commonly, one or more of the CDRs of the particular Fv is shorter than the maximum length permitted for CDRs in the Kabat database. For example, the V.sub.L of Fv dsAT does not have a residue corresponding to positions 27A-28 of the Kabat sequence, while the V.sub.L of Fv Mt9 lacks a residue corresponding to Kabat position 27E.).

Fourth, acidic residues in the Fv are excluded as candidates for mutation, since they are already negatively charged. Fifth, residues which show 100% conservation in the Kabat Percent Frequency table of FIG. 2 are excluded since they are likely to be important to antibody folding or to contribute to other functional characteristics of the molecule. Sixth, positively charged residues which are at positions in the Kabat sequence at which neutral or negative residues never occur on the "Percent Frequency" Table are excluded, in part because that implies that evolution has determined that a positive charge at that position is likely to be advantageous. Similarly, neutral residues which are at positions at which an acidic residue never appears on the Percentage Frequency Table are excluded from consideration as candidates for mutation.

The seventh and eighth factors are based on creating a model of the Fv structure based on the crystal structure of the antibody, if one is available, or of other antibodies in the Kabat class or family in which the Fv of interest is categorized. Many crystal structures of antibodies are now known. As is well known in the art, a model of the crystal structure of the antibody of interest, or an antibody of the same Kabat class, and the position of the candidate residue when aligned with the Kabat numbering system, permits the determination of whether the residue in question is exposed at the surface of the Fv and whether the residue is predicted to interact with other residues in the Fv (for example, by forming hydrogen bonds).

Determinations of whether a particular residue is likely to be exposed at the surface of the Fv and of whether the residue is predicted to interact with other residues in the Fv (by, for example, forming hydrogen bonds), can be performed by building a model structure of the antibody of interest from the sequence alignment to an antibody of the same Kabat class whose structure is known and inspecting the model structure. Modeling from known structures is taught by, e.g., Eigenbrot et al., J. Mol. Biol., 229:969 (1993) and Barry et al., J. Biol. Chem., 269:3623 (1994). Detailed information on modeling antibodies is available at http://antibody.bath.ac.uk. This website is styled "Web Antibody Modeling," or "WAM," and provides software for aligning residues as well as WAM modeling software based on the modeling algorithm "AbM" developed by Martin et al., Meth Enzymol. 203:121 (1991), Martin et al., Proc Natl Acad Sci USA 86:9268 (1989).

Candidate residues which have less than 30% of their surface area exposed are excluded from mutation, since the limited amount of exposure would effectively reduce amount of effect mutating the residue would have on reducing the pI. Residues which are predicted from the crystal structure or by modeling to interact with others are excluded from mutation since it is assumed that a change in charge would change the interaction and consequent function of the Fv. Ninth, the C-terminal residue is considered never to be involved in interactions with other residues, folding, or antigen binding and is always considered a candidate for mutation. This freedom to mutate can be considered to extend to the C-terminal residue and as many as the next four residues most proximal to the C-terminus.

The residues remaining after these exclusions are candidates for mutation. If the residue is basic, it can be replaced with either an acidic or a neutral residue. Guidance as to whether to substitute an acidic or a neutral residue for the basic residue is provided by the Percent Frequency table. If the most common residue found at the position under consideration is neutral, then the first choice for mutation of the existing residue is to substitute a neutral residue at that position. If the most common residue at the position is acidic, then the residue used to replace the existing residue should be acidic. As to the particular neutral or acidic residue to substitute, it is preferable to use the residue most commonly found at that position. Similarly, if the residue which is a candidate for mutation is neutral, an acidic residue can be substituted. Generally, one refers to the Percent Frequency table appearing on FIG. 2 and replaces the candidate residue with the most common acidic residue appearing at the position occupied by the candidate residue.

The value of following some or all of this protocol can be seen by the exemplary studies reported in the Examples. To ensure that the results of the studies have relevance to treatment of humans, the immunotoxins chosen as the parental immunotoxins to be improved are ones that are already showing positive results in human clinical trials. Since immunotoxins which are in clinical trials first underwent extensive pre-clinical animal testing, the positive results achieved in the human clinical trials to date show that the positive results in the pre-clinical animal testing are indeed correlated with like results in humans. Further, to reduce variables irrelevant to larger purposes of the study, all of the immunotoxins used as a toxic moiety a Pseudomonas exotoxin A (PE) mutated to remove non-specific binding but to retain cytotoxic capability when directed into cells by a targeting moiety of an immunotoxin. The particular mutant PE used has a molecular weight of 38 kD, and is known as PE38. (Hwang et al., Cell, 48: 129-136 (1987)). Other toxic moieties suitable for use in humans as part of recombinant immunotoxins can, however, be used with like results.

As previously noted, six Fvs are set forth on FIG. 2. The first Fv listed is dsAT, used to construct anti-Tac(dsFv)-PE38, which targets the .alpha. chain of the IL-2 receptor and which is directed at CD25-expressing hematologic malignancies. dsAT is an anti-Tac Fv which was used as the starting, or "parental" antibody for studies of mutations to reduce pI and, it was hoped, non-specific toxicity. One of the dose limiting toxicities of recombinant immunotoxins is liver damage due to cytokine release. Liver damage due to TNF.alpha. release is also a dose limiting toxicity in mice given anti-Tac(scFv)-PE38, the scFv version of the anti-Tac immunotoxin also known as LMB-2. Molecular modeling and site directed mutagenesis was used to lower the pI of the Fv of anti-Tac without decreasing its binding activity. In studies conducted before development of all the steps of the protocol set forth above, the mutations were restricted to mutating neutral residues to acidic amino acids. The resulting Fv was termed "M1." An immunotoxin was constructed containing this mutated Fv, and was termed M1(scFv)-PE38. The toxicity in mice of M1(scFv)-PE38 is more than 3-fold lower than that of anti-Tac(scFv)-PE38. The pI of the Fv of anti-Tac Fv is 10.21. This was lowered to 6.82 in M1 scFv, yet its ability to kill CD25 positive target cells when used in an immunotoxin with the same toxic moiety (PE38) was undiminished. See, Examples 1-3, below.

Other immunotoxins employed in the exemplary studies were: SS1(dsFv)-PE38, an immunotoxin targeted at ovarian cancers and other epithelial cancers expressing the protein mesothelin (Chowdhury et al., Proc Natl Acad Sci (USA, 95: 669-674 (1998); Chowdhury et al., Nature Biotechnol, 17: 568-572 (1999)), B3(dsFv)-PE38 (LMB9), an immunotoxin targeted at epithelial cancers that express Lewis.sup.Y (Pastan et al., Cancer Research, 51: 3781-3787(1991)), and RFB4(dsFv)-PE38(BL-22), which is directed at CD22-expressing hematologic malignancies, and which is very well tolerated in mice. Anti-Tac(scFv)-PE38 and RFB4(dsFv)-PE38 have both shown good antitumor activity in patients (Kreitman et al., Blood, 94: 3340-334 (1999); Kreitman et al., J. Clinical Oncology, 18: 1622-1636 (2000); Kreitman et al., Clinical Cancer Research, 6: 1476-1487 (2000)).

As the protocol was developed for mutating antibodies to reduce pI, the initial studies were extended by mutating basic residues to acidic residues. Further, the Fv portion of an immunotoxin was stabilized by replacing the peptide linking the V.sub.H and V.sub.L chains with a disulfide bond introduced into the framework region. Therefore, M1(scFv)-PE38 was first converted into M1(dsFv)-PE38, and then selected basic residues were mutated to acidic residues to form a further mutant, termed M16(dsFv)-PE38. Conversion of scFvs to dsFvs can be performed by published techniques, such as that set forth in Brinkmann et al., Proc Natl Acad Sci (USA) 90:7538-7542 (1993). Similarly, B3(dsFv)-PE38 was mutated by the protocol set forth above to form a mutant known as Mt9(dsFv)-PE38, and SS1(dsFv)-PE38 was mutated to form a mutant known as St6(dsFv)-PE-38. The sequences of the parental and mutated antibodies (SEQ ID NOS:1-14) are set forth in FIG. 2.

The results show that animal toxicity is markedly diminished by lowering the pI of the Fv, while the full antitumor activity of the parental immunotoxin is retained. Further, each of the mutants was as stable at physiological temperature as its parental immunotoxin. Interestingly, in in vivo studies, the pharmacokinetics of the immunotoxins showed that each of the immunotoxins engineered to have a lower pI than that of its parental immunotoxin also had a shorter half-life in the serum than that of its parent in an animal model. See, Example 6, infra. But, the studies also showed that the lower pI mutated immunotoxins had tumor-reducing effects as great as that of the parental immunotoxin. Without wishing to be bound by theory, it is possible that the lower pI of the Fv facilitates specific recognition of the target antigen or enhances cell uptake of the immunotoxin. Thus, while a shorter half-life might normally be correlated with a reduced effect of the immunotoxin, immunotoxins mutated by the methods of the invention surprisingly retain the full activity of the parental immunotoxin against targeted cells.

The results obtained for these three different immunotoxins indicate that the protocol set forth above can be used to reduce the non-specific toxicity of any antibody, or fragment thereof which retains antigen-binding capability, which has not already been the subject of pI-reducing alterations. In general, any Fv can be modified by the procedures set forth above to improve its utility as the targeting portion of an immunotoxin.

In general, disulfide-stabilized Fvs (dsFvs) are more useful in clinical use, and in preferred embodiments, the targeting moiety of the immunotoxin is a dsFv. scFvs can, however, be employed if desired and can be modified to reduce pI and toxicity in the manner set forth above.

In the most preferred embodiments, the V.sub.L and the V.sub.H of the Fv portion of immunotoxins mutated by the methods taught herein have the amino acid sequence of the V.sub.L and V.sub.H chains of M16 (SEQ ID NOS: 3 and 10), of St6 (SEQ ID NOS: 5 and 12), of Mt9 (SEQ ID NOS:7 and 14), or of M1 (SEQ ID NOS:2 and 9). In other preferred embodiments, the V.sub.L and V.sub.H chains of the Fv have about 90% or greater sequence identity amino acid sequences of the V.sub.L and the V.sub.H, respectively, M16, St6, Mt9, or M1 (but, of course, do not have the sequence of the parental immunotoxin), and have reduced toxicity compared to the parental immunotoxin. It is desirable that the V.sub.L and the V.sub.H chains of the antibodies or antigen binding fragments thereof retain the pI-lowering mutations of the residues made according to the protocol set forth above. That is, if the protocol indicates that a positively charged residue can be mutated to a negatively charged residue, it is desirable that that mutation be retained in any variation of the sequence. Similarly, if the protocol indicates that a positively charged residue can be mutated to a residue with neutral charge, it is desirable that that mutation also be retained in any variation of the amino acid sequence.

It should be noted that the Percent Frequency Table of FIG. 2 shows which residues vary in thousands of antibodies, and what substitutions occur at various Kabat positions. Thus, while it is generally desirable to retain the mutations of the antibodies to reduce their pI, if desired residues at other positions in the antibody varied by substituting residues according to the Percent Frequency Table of FIG. 2 for the variable light chain or for the variable heavy chain, respectively.

In preferred embodiments, the V.sub.L and V.sub.H of the Fv of the immunotoxins have 91%, 92%, 93%, 94%, 95%, or greater sequence identity to the V.sub.L and V.sub.H, respectively, of M16, St6, Mt9, or M1, respectively. The immunotoxins preferably retain at least 75% of the cytotoxicity to targeted cells of the parental immunotoxin, and more preferably 80%, 85%, 90%, 95% or even more of the cytotoxicity of the parental immunotoxin to cells bearing an antigen to which the antibody or antigen-binding fragment thereof specifically binds.

The liver enzyme alanine aminotransferase (ALT), formerly known as sGPT, is considered an indicator of liver inflammation. ALT levels are routinely measured in patients, for example, to monitor the course of chronic hepatitis and of treatments such as the administration of prednisone. ALT levels can conveniently be used as a marker for the overall toxicity of an immunotoxin in animal models by determining whether the level of in the serum is increased by more than 5, 6, 7, 8, 9, 10, or more times 24 hours after administration of an immunotoxin, compared to the level of the liver enzyme in the serum at 3 hours after administration. Immunotoxins which induce a lower level of the enzyme or enzymes at 24 hours are considered to have lower toxicity than immunotoxins which induce higher levels of such enzymes. Conveniently, the toxicity is determined using a mouse model. In preferred embodiments, the immunotoxin does not cause the upper level of the enzyme (even taking into account the error limits of the measure) in the serum of the mice to which it is administered to exceed 220 U/L. In more preferred embodiments, the immunotoxin does not cause the upper level in the serum of the mice to exceed 210 U/L. In even more preferred embodiments, it does not cause the upper level of the serum level to exceed 200 U/L, and in the most preferred embodiments, does not cause it to exceed 190 U/L. A number of devices which analyze ALT levels are commercially available, such as the Boehringer Mannheim ("BM")/Hitachi 917 analyzer (Hitachi Instruments, Inc.), sold by Roche Diagnostics GmbH (Mannheim, Germany).

As noted above, mice are an art-recognized model for testing immunotoxins for their effect on human tumors, and the immunotoxins used as the parental immunotoxins in the exemplary studies herein are already in human clinical trials. Given the lower toxicity to mice shown by the immunotoxins comprising the Fvs mutated by the procedures set forth above, immunotoxins mutated according to the procedures taught herein will exhibit lower toxicity in humans compared to their parental immunotoxins, permitting higher doses to be employed. The higher doses that can be used result in even better killing of tumor cells targeted by these and other immunotoxins mutated according to the invention.

Definitions

Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

"Antibody" refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope (e.g., an antigen). This includes intact immunoglobulins and the variants and portions of them well known in the art such as, Fab' fragments, F(ab)'.sub.2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). An scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York (1997).

Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs have been defined (see, Kabat, E., et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Department of Health and Human Services, (1991), which is hereby incorporated by reference. The Kabat database is now maintained online at http://immuno.bme.nwu.edu/.). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V.sub.H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V.sub.L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

References to "V.sub.H" or a "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to "V.sub.L" or a "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab

The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.

The term "linker peptide" includes reference to a peptide within an antibody binding fragment (e.g., Fv fragment) which serves to indirectly bond the variable domain of the heavy chain to the variable domain of the light chain.

The term "parental antibody" means any antibody of interest which is to be mutated or varied to obtain antibodies or fragments thereof which bind to the same epitope as the parental antibody, but which have a lower pI.

A "targeting moiety" is the portion of an immunoconjugate, such as an immunotoxin, intended to target the immunoconjugate to a cell of interest. Typically, the targeting moiety is an antibody, a scFv, a dsFv, an Fab, or an F(ab')2.

A "toxic moiety" is the portion of a immunotoxin which renders the immunotoxin cytotoxic to cells of interest.

As used herein, "cytotoxicity" refers to the toxicity of an immunotoxin to the cells intended to be targeted by the immunotoxin, as opposed to the cells of the rest of an organism. Unless otherwise noted, in contrast, the term "toxicity" refers to toxicity of an immunotoxin to cells others than those that are the cells intended to be targeted by the targeting moiety of the immunotoxin, and the term "animal toxicity" refers to toxicity of the immunotoxin to an animal by toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.

The term "mesothelin" includes reference to a mesothelin protein and fragments thereof which may be present on the surface of one or more cells of a mammal, such as a rat, a mouse, a primate, or, in particular, a human. The preferred nucleic acid and amino acid sequences of mesothelin are as described in PCT published application WO 97/25,068, U.S. application Ser. No. 08/776,271 and U.S. Provisional Application 60/010,166. In addition, see, Chang, K. & Pastan, I., Int. J. Cancer 57:90 (1994); Chang, K. & Pastan, I., Proc. Nat'l Acad. Sci. USA 93:136 (1996); Brinkmann U., et al., Int. J. Cancer 71:638 (1997); and Chowdhury, P. S., et al., Mol. Immunol. 34:9 (1997).

As used herein, the term "anti-mesothelin" in reference to an antibody, includes reference to an antibody which is generated against mesothelin. In preferred embodiments, the mesothelin is a primate mesothelin such as human mesothelin. In a particularly preferred embodiment, the antibody is generated against human mesothelin synthesized by a nonprimate mammal after introduction into the animal of cDNA which encodes human mesothelin.

The term "anti-Tac" refers to a monoclonal antibody which binds to the IL-2 receptor. IL-2 is a polypeptide hormone which mediates activation of human T cells through binding to the IL-2 receptor (IL-2R). Anti-Tac has been known since at least 1982 (see, e.g., Depper et al., J. Immunol., 131:690-696 (1983)), and has been used for more than a decade in trials to trea