CA2071946A1 - Toxin for construction of immunotoxins - Google Patents
Toxin for construction of immunotoxinsInfo
- Publication number
- CA2071946A1 CA2071946A1 CA002071946A CA2071946A CA2071946A1 CA 2071946 A1 CA2071946 A1 CA 2071946A1 CA 002071946 A CA002071946 A CA 002071946A CA 2071946 A CA2071946 A CA 2071946A CA 2071946 A1 CA2071946 A1 CA 2071946A1
- Authority
- CA
- Canada
- Prior art keywords
- lyspe40
- cells
- molecule
- tumor
- tumors
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention described LysPE40-antibody immunotoxin which selectively kills cells bearing appropriate antigens or receptors.
Description
W09l/09965 ~ 7 ~ ~ ~ 6 P~T/VS9o/074~
AN IMPROVED TOXIN FOR CONSTRUCTION OF IMMUNOTOXIN~
The parent application ~06/911,227) teaches the production of recombinant pro~eins from modified Pseudomo~
nas exotoxin (PE) gene fused with DNA sequences encoding a recognition protein for which a sp~cific receptor exists on the cells. This was exemplified in the parent applica-tion with the construction and expression of an IL-2-PE
fusion gene. The present application relates to the production of improved toxins which are useful in con-structing more effective immunotoxin~. Particularly, the invention provides an altered PE molecule, designated hexei~ as Lys-PE40, that can be easily conjugated to an antibody with effective cytotoxicity ~oward target cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Various objects, features ~nd many of the atten-dant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the acoompanying drawings wherein:
Figure l schematically illustrates the structure of plasmid pVC85L showing the presence of a T7 promoter, and OmpA signal sequence and domains II (translocation domain) and III (ADP ribosylation domain) o Pseudomonas exotoxin.
Figure 2A shows the results of SDS-PAGE analysis of I,ysPE40 pools at various stages of purification. 12.5%
gels were stained with Coomassie blue. Lane 1, QMA
concentrated culture medium proteins; ~ane 2, Q Sepharose pool; Lane 3, Flow throug~ material frsm Mono S; ~ane 4, Mono S pool; L~ne 5,-TSK 250 pool. The positions of molecular weight standards are indicated. ~B: SDS-PAGE
analysis of anti-TFR-LysPE40. S~ples were applied on a 10% reducing polyacrylamide gel. Lane 1, TFR-LysPE40;
Lane 2, LysPE40; Lane 3, anti-TFR. Gels were stained with Coomassie blue.
Figure 3A demonstrates the inhibition of protein synthesis in A431 cells by anti-TFR LysPE40. Immunotoxin was added to the cells in the absence (*) and presence (o~
.... .
.
WO91/0996~ PCT/US90/074~
of excess anti-TFR (tp ~g/ml) for 18-24 hrs at 374C. B:
Cytotoxic activity of anti-Tac-LysPE40 on HUT102 cells.
Anti-Tac-LysPE40 was added at various concentrations to the indicated cell lines. For competition experiment 70 ~g/ml anti-Tac was added before '-he addition of the immunotoxin. 3H leucine incorporation was measured as described in the text.
Figure 4 shows the blood levels of anti-TFR-LysPE40. BALB/C mice or nude mice with A431 tumors were injected I.P. with 100 ~g or 50 ~g of anti-TFR-LysPE40.
Immunotoxin levels were measured in serum at different time periods. Results are average of two different experiments.
Figure 5 shows the effect of anti-TFR-LysPE40 on the growth of A431 tumors in nude mice. Mice were inject-ed with 3 x 106 A431 cells and treatad with the immunotoxin as indicated. Mice were given four doses of S0 ~g each on the indicated days: ~) days 2, 4, 6, 8, (A ) days 9, 11, 13 and 15; (~) no treatment. ~-B. Mice gi~en four doses on days 5, 7, 9 and 11; (~) no treatment;
(~) 5~g; (o); 20 ~g; (- ), 50 ~g; (~), single dose of 150 ~g on day 5.
Figure 6 shows the gross appearance of subcutane-ous A431 tumors in nude mice with and without treatment with HB21-PE40.
Nude mice were injected su~cutaneously with 3 x 1 o6 A431 cells on day 0. Without ~urther treatment, the tumors were apparent as small bumps under the skin on day (A; arrow~). ` By day 15, tumors were large, often erupting through the skin surface (B). Mice treated with HB21-PE40 on days 2, 4, 6 and 8 usually showed no develop-ment of tumor, demonstrated here as the absence of gross tumor on day 26 (C). When mice with large tumors were treated with HB21-PE40 (on days 9, 11, 13 and 15), the tumors often began to shrink, collapsing inward on their necrotic centers (D arrows)~
Figure 7 shows the histological appearance of typical treated and control A431 subcutaneous tumors.
:
-:
. .
. ., . ' .
w~l/09965 2 0 7 1 9 ~ 6 PCT/U~90/0746~
A431 tumors were removed at necropsy, fixed on formaldehyde and processed for routine paraffin embedding and sectioning, and staining with hema~oxylin and eosin.
A section from a tumor removed from a mouse at day 15 is shown in A-4. A similar section of a tumor removed from a mouse on day 19 that had been treated on days 9, 11, 13 and 15 with anti~TFR-LysPE40 is shown in B-B. The cross-section shown in A shows that the majority of the untreat-ed tumor contains viable tumor cells (VT) with only small areas of necrosis (arrow). In contrast, the tumor from a treated mouse ( B) shows ~hat a large percentage of the tumor is necrotic (NT), with only a small rim of viable tumor (VT) remaining (s=skin). (~, A") and (B'B") are higher magnification fields from the margins of tumors demonstrating that the untreated tumor is mostly composed of homogeneous viable tumors (VT), whereas the treated tumor shows a rim of viable cells of varia~le thickness lying ad~acent to a connective tissue c~psula (ct). The majority of the treated tumor shows large areas of central necrosis, and in some areas of the tumor margin all tumor cells are nec~otic, whereas in other areas a viable tumor rim only a few cells thicX remains (B", double arrow).
(~ags: A,B = 5, bar - 1 mm; A', B' = x26, bar = 200 ~m;
A", B" = x260; bar = ~m).
DETAILED DESCRIPTION OF THE INVh'NTION
The above and various other objects and advantages of the present invention are achieved by an improved Pseudomonas exotoxin (PE) of the type including a deletion in the receptor ~inding domain Ia of the native toxin, wherein the improvement comprises a recombinant PE mole-cule possessing at least one lysine residue in domain Ia of the PE molecule, which otherwise will be devoid of a lysine residue when having a deletion in the receptor ` binding dom~in Ia, said lysine residue providing an essential linkage for effective coupling of the recombi-nant PE to other molecules, such as antibodies and the like.
- : : ' ,: , WO91/0996~ ~ PCr/US90/074 When PE is chemically attached to a~tibodies or other targeting molecules, lysine residues are required for coupling PE to other molecules. Since all 12 lysin~
residues of domain I are lost when domain I is deleted from PE, one of the three lysine residues in the other part of the molecule (for instance, domain III), must be used to couple PE40 to an antibody or another targeting molecule. However, when one of these three lysine resi-dues are used, a conjugate with low activity is obtained (Kondo et al, J. Biol. Chem. 263:9470, 1988). In order to overcome this problem and to obtain a conjugate with high activity, a new PE molecule was created by deleting most of domain I (residues 6-252) bu~ maintaining one of the 12 lysine residues originally present in domain I. This new 15 molecule, in accordance with the present invention, is designated herein as LysPE40.
In order to demonstrate the effect of the new molecule on immunotoxin cons~ructs, two novel immunotoxins were made, first with LysPE40 conjugated to antiTac antibody and second conjugated to an antibody specific for the human transferrin receptor ~anti-TFR).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalen~ to those described herein can be used in the practice or ~esting of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only illus-trative and not limiting.
The term "antibody" as used herein means a portion of an immunoglobulin molecule ~see W.E. Paul, ed., ~Funda-mental Immunolosy," Raven Press, N.Y., 1984, pp. 131-165) aapable of binding to an antigen. According to this WO9l/09965 2 o ~ ~ 9 4 6 PCT/~90/07~64 definition, the term "antibody" includes various f orms of modified or altered antibodies, such as an intact immuno-globulin, an Fv fragment containing only the light and heavy chain variable regions, an Fab or (Fab) 12 fragment containing th~ variable regions and parts of the constant regions, a single-chain antibody (Bird et al., lg88, Scisnce 242, 424-426; Huston et al., 1988, Proc. Natl.
Acad. Sci. USA 85, 5879-5883), and the like. The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., 1984, Proc._ Nat.
Acad. Sci. USA 81, 6851-6855) or humanized (Jones et al., 1986, Nature 321, 522-525, and published UK patent appli-cation #8707252). Methods of producing antibodies suit-able for use in the present invention are well known to those skilled in the art and can be found described in such publications as Harlow & Lane, Antibodies: A Labora-tory Manual, Cold Spring Harbor Laboratory, 1988. The genes encoding the antibody chains may be cloned in cD~A
or in genomic form by any cloning procedure known to those skilled in khe art. See for example Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.
The term "without significant cytotoxici~y" as used herein means that the fusion protein of the present invention does not affect the normal functions of the untargeted cells to any appreciable degree or to any abnormal level.
The recombinant antibody-LysPE40 ~usion protein may contain one polypeptide chain or two chains. The example disclosed herein relates to the one chain case.
To produce two chains, no amino acid linker would be inserted be~ween the VB and VL sequences. Instead a termination codon would be inserted after the V~ sequence, and an initiation codon and ribosome binding sequence would be inserted before the VL sequence. In another embodiment of the invention, the VL and V8 sequences will be followed respectively by part or all of the light and heavy chain constant regions, e.g., the whole kappa light ,, :,~: . : .......................... .
. .
, .
W09l/09965 PCT/U59~/074~
~ ~ 9 ~L6 6 --chain constant region and the CH1 domain of the heavy chain constant region, with or without the heavy chain hinge domain. The VL, V~ and PE40 genes may occur in any order on the plasmid, hence the PE40 gene may be attached to either the 5' or 3' end of either the light or heavy chain gene. Those skilled in the art will realize that additional modifications, deletions~ inser~ions and the like may be made to the antibody and LysPE40 genes.
Especially, deletions or changes may be made in LysPE40 or in the linker connecting the antibody gene to LysPE40, in order to increase cytotoxicity of the fusion protein toward target cells or to decrease cytotoxicity toward - cells without antigen for the antibody. All such con-structions may be made by methods of genetic engineering well known to those skilled in the art (see, generally, Maniatis et al., su~ra) and may produce proteins that have differing properties of affinity, specificity, stability and toxicity that make them particularly suitable for various clinical or biological applications.
AN IMPROVED TOXIN FOR CONSTRUCTION OF IMMUNOTOXIN~
The parent application ~06/911,227) teaches the production of recombinant pro~eins from modified Pseudomo~
nas exotoxin (PE) gene fused with DNA sequences encoding a recognition protein for which a sp~cific receptor exists on the cells. This was exemplified in the parent applica-tion with the construction and expression of an IL-2-PE
fusion gene. The present application relates to the production of improved toxins which are useful in con-structing more effective immunotoxin~. Particularly, the invention provides an altered PE molecule, designated hexei~ as Lys-PE40, that can be easily conjugated to an antibody with effective cytotoxicity ~oward target cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Various objects, features ~nd many of the atten-dant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the acoompanying drawings wherein:
Figure l schematically illustrates the structure of plasmid pVC85L showing the presence of a T7 promoter, and OmpA signal sequence and domains II (translocation domain) and III (ADP ribosylation domain) o Pseudomonas exotoxin.
Figure 2A shows the results of SDS-PAGE analysis of I,ysPE40 pools at various stages of purification. 12.5%
gels were stained with Coomassie blue. Lane 1, QMA
concentrated culture medium proteins; ~ane 2, Q Sepharose pool; Lane 3, Flow throug~ material frsm Mono S; ~ane 4, Mono S pool; L~ne 5,-TSK 250 pool. The positions of molecular weight standards are indicated. ~B: SDS-PAGE
analysis of anti-TFR-LysPE40. S~ples were applied on a 10% reducing polyacrylamide gel. Lane 1, TFR-LysPE40;
Lane 2, LysPE40; Lane 3, anti-TFR. Gels were stained with Coomassie blue.
Figure 3A demonstrates the inhibition of protein synthesis in A431 cells by anti-TFR LysPE40. Immunotoxin was added to the cells in the absence (*) and presence (o~
.... .
.
WO91/0996~ PCT/US90/074~
of excess anti-TFR (tp ~g/ml) for 18-24 hrs at 374C. B:
Cytotoxic activity of anti-Tac-LysPE40 on HUT102 cells.
Anti-Tac-LysPE40 was added at various concentrations to the indicated cell lines. For competition experiment 70 ~g/ml anti-Tac was added before '-he addition of the immunotoxin. 3H leucine incorporation was measured as described in the text.
Figure 4 shows the blood levels of anti-TFR-LysPE40. BALB/C mice or nude mice with A431 tumors were injected I.P. with 100 ~g or 50 ~g of anti-TFR-LysPE40.
Immunotoxin levels were measured in serum at different time periods. Results are average of two different experiments.
Figure 5 shows the effect of anti-TFR-LysPE40 on the growth of A431 tumors in nude mice. Mice were inject-ed with 3 x 106 A431 cells and treatad with the immunotoxin as indicated. Mice were given four doses of S0 ~g each on the indicated days: ~) days 2, 4, 6, 8, (A ) days 9, 11, 13 and 15; (~) no treatment. ~-B. Mice gi~en four doses on days 5, 7, 9 and 11; (~) no treatment;
(~) 5~g; (o); 20 ~g; (- ), 50 ~g; (~), single dose of 150 ~g on day 5.
Figure 6 shows the gross appearance of subcutane-ous A431 tumors in nude mice with and without treatment with HB21-PE40.
Nude mice were injected su~cutaneously with 3 x 1 o6 A431 cells on day 0. Without ~urther treatment, the tumors were apparent as small bumps under the skin on day (A; arrow~). ` By day 15, tumors were large, often erupting through the skin surface (B). Mice treated with HB21-PE40 on days 2, 4, 6 and 8 usually showed no develop-ment of tumor, demonstrated here as the absence of gross tumor on day 26 (C). When mice with large tumors were treated with HB21-PE40 (on days 9, 11, 13 and 15), the tumors often began to shrink, collapsing inward on their necrotic centers (D arrows)~
Figure 7 shows the histological appearance of typical treated and control A431 subcutaneous tumors.
:
-:
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. ., . ' .
w~l/09965 2 0 7 1 9 ~ 6 PCT/U~90/0746~
A431 tumors were removed at necropsy, fixed on formaldehyde and processed for routine paraffin embedding and sectioning, and staining with hema~oxylin and eosin.
A section from a tumor removed from a mouse at day 15 is shown in A-4. A similar section of a tumor removed from a mouse on day 19 that had been treated on days 9, 11, 13 and 15 with anti~TFR-LysPE40 is shown in B-B. The cross-section shown in A shows that the majority of the untreat-ed tumor contains viable tumor cells (VT) with only small areas of necrosis (arrow). In contrast, the tumor from a treated mouse ( B) shows ~hat a large percentage of the tumor is necrotic (NT), with only a small rim of viable tumor (VT) remaining (s=skin). (~, A") and (B'B") are higher magnification fields from the margins of tumors demonstrating that the untreated tumor is mostly composed of homogeneous viable tumors (VT), whereas the treated tumor shows a rim of viable cells of varia~le thickness lying ad~acent to a connective tissue c~psula (ct). The majority of the treated tumor shows large areas of central necrosis, and in some areas of the tumor margin all tumor cells are nec~otic, whereas in other areas a viable tumor rim only a few cells thicX remains (B", double arrow).
(~ags: A,B = 5, bar - 1 mm; A', B' = x26, bar = 200 ~m;
A", B" = x260; bar = ~m).
DETAILED DESCRIPTION OF THE INVh'NTION
The above and various other objects and advantages of the present invention are achieved by an improved Pseudomonas exotoxin (PE) of the type including a deletion in the receptor ~inding domain Ia of the native toxin, wherein the improvement comprises a recombinant PE mole-cule possessing at least one lysine residue in domain Ia of the PE molecule, which otherwise will be devoid of a lysine residue when having a deletion in the receptor ` binding dom~in Ia, said lysine residue providing an essential linkage for effective coupling of the recombi-nant PE to other molecules, such as antibodies and the like.
- : : ' ,: , WO91/0996~ ~ PCr/US90/074 When PE is chemically attached to a~tibodies or other targeting molecules, lysine residues are required for coupling PE to other molecules. Since all 12 lysin~
residues of domain I are lost when domain I is deleted from PE, one of the three lysine residues in the other part of the molecule (for instance, domain III), must be used to couple PE40 to an antibody or another targeting molecule. However, when one of these three lysine resi-dues are used, a conjugate with low activity is obtained (Kondo et al, J. Biol. Chem. 263:9470, 1988). In order to overcome this problem and to obtain a conjugate with high activity, a new PE molecule was created by deleting most of domain I (residues 6-252) bu~ maintaining one of the 12 lysine residues originally present in domain I. This new 15 molecule, in accordance with the present invention, is designated herein as LysPE40.
In order to demonstrate the effect of the new molecule on immunotoxin cons~ructs, two novel immunotoxins were made, first with LysPE40 conjugated to antiTac antibody and second conjugated to an antibody specific for the human transferrin receptor ~anti-TFR).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalen~ to those described herein can be used in the practice or ~esting of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only illus-trative and not limiting.
The term "antibody" as used herein means a portion of an immunoglobulin molecule ~see W.E. Paul, ed., ~Funda-mental Immunolosy," Raven Press, N.Y., 1984, pp. 131-165) aapable of binding to an antigen. According to this WO9l/09965 2 o ~ ~ 9 4 6 PCT/~90/07~64 definition, the term "antibody" includes various f orms of modified or altered antibodies, such as an intact immuno-globulin, an Fv fragment containing only the light and heavy chain variable regions, an Fab or (Fab) 12 fragment containing th~ variable regions and parts of the constant regions, a single-chain antibody (Bird et al., lg88, Scisnce 242, 424-426; Huston et al., 1988, Proc. Natl.
Acad. Sci. USA 85, 5879-5883), and the like. The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., 1984, Proc._ Nat.
Acad. Sci. USA 81, 6851-6855) or humanized (Jones et al., 1986, Nature 321, 522-525, and published UK patent appli-cation #8707252). Methods of producing antibodies suit-able for use in the present invention are well known to those skilled in the art and can be found described in such publications as Harlow & Lane, Antibodies: A Labora-tory Manual, Cold Spring Harbor Laboratory, 1988. The genes encoding the antibody chains may be cloned in cD~A
or in genomic form by any cloning procedure known to those skilled in khe art. See for example Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.
The term "without significant cytotoxici~y" as used herein means that the fusion protein of the present invention does not affect the normal functions of the untargeted cells to any appreciable degree or to any abnormal level.
The recombinant antibody-LysPE40 ~usion protein may contain one polypeptide chain or two chains. The example disclosed herein relates to the one chain case.
To produce two chains, no amino acid linker would be inserted be~ween the VB and VL sequences. Instead a termination codon would be inserted after the V~ sequence, and an initiation codon and ribosome binding sequence would be inserted before the VL sequence. In another embodiment of the invention, the VL and V8 sequences will be followed respectively by part or all of the light and heavy chain constant regions, e.g., the whole kappa light ,, :,~: . : .......................... .
. .
, .
W09l/09965 PCT/U59~/074~
~ ~ 9 ~L6 6 --chain constant region and the CH1 domain of the heavy chain constant region, with or without the heavy chain hinge domain. The VL, V~ and PE40 genes may occur in any order on the plasmid, hence the PE40 gene may be attached to either the 5' or 3' end of either the light or heavy chain gene. Those skilled in the art will realize that additional modifications, deletions~ inser~ions and the like may be made to the antibody and LysPE40 genes.
Especially, deletions or changes may be made in LysPE40 or in the linker connecting the antibody gene to LysPE40, in order to increase cytotoxicity of the fusion protein toward target cells or to decrease cytotoxicity toward - cells without antigen for the antibody. All such con-structions may be made by methods of genetic engineering well known to those skilled in the art (see, generally, Maniatis et al., su~ra) and may produce proteins that have differing properties of affinity, specificity, stability and toxicity that make them particularly suitable for various clinical or biological applications.
2 0 ~TERIALS AND METHODS
~he following example is offered by way of illus-tration, not limitation.
Construction of a vector that encodes LysPE40 and secretes it into the medium.
Plasmid pVC4 (derived from p~H4 by trea~in~ pJH4 with SphI and Tthllll and relegating the large fragment containing ~he PE gene) was joined ~o an OmpA signal sequence as described by Chaudhary et al., PNAS 85, 2929-2943, 1988, to produce pVC45. Site directed mutagenesis is a con~enient way to make deletions and it-was used to create plasmid pJY85L which produces LYSPE4 0 as shown in Figure 1. A HindIII/SalI fragment of the PE gene in pVC45 was cloned into M13, mpl9 and uracil containing single stxanded DNA prepared by the method of Kunkel (kunkel, T.A., PNAS 82, 488-492, 1985). An oligonucleotide 36 nucleotides in length with the structure 5'CCAGGCTGCCGC-CCTTGAAAGCTTGGCGTAATCATG3' was synthesized which generated a large deletion in PE of amino acids 6 through 252 and wosl/o9965 ~CT/US90/0746q 7 2~7~946 retained a lysine residue between amino acids 5 and 253 to give a molecule ~ith the sequence:
1 2 3 4 5 x 253 254 255 256 Ala-Glu-Glu Ala-Phe-Lys-Gly~Gly-Ser-L~u A 176 bp HindIIX/SalI fragment was excised from the replicati~e form of the mutant DNA in M13 and ligated with a 3.3 kb HindIII/SalI fragment of pVC45 to give pJY85L which encodes a protein with the following struc-ture:
Me~-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Yal-Ala-Leu-Ala-Gly-Phe Ala-Thr-Val-Ala-Gln-Ala-Ala-Asn-Leu-Glu-Glu-Ala-Phe-Lys Gly-Gly-Ser ....
This protein is processed at the (l) and the proressed product secreted into the medium. When the plasmid was expressed in E. coli BL21 (ADE3 ) cells, LysPE40 was found in and purified from the medium wi~h a yield of greater than 1 mg per liter. The se~uence of amino acids at its amino terminus was found to be that predicted from the DNA
sequence:
1 2 3 4 ~ 253 254 255 H2N Ala-Asn-Leu-~la-Glu-Glu-Ala-Phe-Lys-Gly-Gly-Ser (the numbers indica~e ~he location of the amino acids in the native PE structure).
A deposit o~ plasmid pJY85L has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., on December 18, 1989 under the accession number 68189. The deposit shall be viably maintained, replacing it if it becomes non-viable, for the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public upon issuance of a patent from this application, without re-striction, in accordance with the provisions of the law.
The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
WO 91/~)9g65 Pcr/uS9O/07464 Ex~ression of LysPE40 BL21 ( DE3) cells were transformed with plasmid pVC85L and grown in LB medium containing 100 ~g/ml ampi-cillin at 37C. At absorbance 0.6 (650 nm), isopropyl-1-- 5 thio-~-D-galactopyranoside was added ~:o a final concentra-tion of 1 mM. Cells were harvested 90 min later. The culture medium was used as the source of LysPE40 because most of the protein was secreted into the medium.
Purification of LysPE40 Clarified culture medium, 25 liters, containing LysPE40 was diluted four-fold with chilled deionized water and applied on a 10 x 5.5 cm silica-based anion exchange column (QMA, Waters) at a flow rate of 100 ml/min. The column was washed with 0.05 M sodium phosphate buffer, pH7.0, and proteins were eluted with two liters of 0.25 M
NaCl in the equilibration buffer. LysPE40 from the QMA
column was concentrated further by using Amicon YM30 membranes to 150 ml, dialyzed for 12-16 hours against 0.02 M Tris-HCl, pH7.6, centrifuged at 10,000 x g for 20 minutes and applied on a 2.5 x 14 cm Q-Sepharose column equilibrated with 0.02 M Tris-HCl, pH 7.6. Proteins were eluted with a 500 ml linear gradient of 0-0.5 M NaC1;
LysPE40-containing fractions were detected by SDS PAGE, pooled, diluted 10-~old with 0.02 M Mes, pH 5.5, and loaded onto an ~PLC Mono S 16/10 colu~n equilibrated with O.02 M Mes, pH 5.5. LysPE40 bound to the column and was eluted by a 100 ml linear gradient of 0-0.5 M NaC1. It was further purified on TSX250 (22.5 x 600 mM) column (BioRad) using 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM ED~A.
Construction of immunoconiuqates LysPE40 (2.5 mg/ml) in 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA was mixed with a 3-fold molar excess of SMCC and incubated at room temperature 3S (about 22-24C) for 30 minutes. Protein was separated from the unreacted cross linker on a PD10 column. MoAb (~B21 or ~Tac) (5-10 mg/ml) was mixed with a 3-fold molar excess of 2-iminothiolane-HCl in 0.2 M sodium phosphate WO91/09965 PCT/US90/074~4 2~71~6 g buffer pH 8.0 containing 1 mM EDTA and incubated at 37C
for 1 hr. The deri~atized antibody was separa~ed from the reactants on a PD10 column. To make immunotoxins, deri~atized LysPE40 and derivatized antibody were mixed in 4:1 molar ratio and incubated at room tempera~ure for 16-hrs. Immunotoxin was then purified by successive chromatography on a mono Q and TSK 253 columns.
In order to determine the efficacy, two different immunotoxins were prepared and used. One is anti-TFR-LysPE40, which binds to human transferrin receptor (TFR),and the other is anti-Tac-LysPE40, which binds to 55 kD a subunit of human IL2 receptor.
SDS-PAGE and immunoblottinq SDS-PAGE described by (Reference).
Protein synthesis inhibition assay Activities of the conjugates were tested on A431, KB, HUT102, HT29, OVCAR2, OVCAR3~ OVCAR4, CEM and ~O~T4 cells by measuring 3H-leucine incorporation (Kondo et al., Biol. Chem. 263, 9470, 1988). HUT102 cells were washed two times with RPM1640 and used immediately. Immunotoxins were diluted with 0.2% human serum albumin in PBS prior to addition to cells.
Assav of blood levels of anti-TFR-LysPE40 in Mice BALB/C mice or nude mice bearing A431 su~cutaneous tumors were injected with 100 ~g or 50 ~g of immunotoxin I.P. Blood was drawn at different time points and the level of the IT was assayed by incubating serum with A431 cells and measuring its effect on protein synthesis. A
standard curve was made using anti TFR-LysPE4~.
An,t~tumor activity of anti-TFR-LvsPE40 in nude mice bearinq a human e~idermoid carcinoma ~ 431 cells ( c x 106) were injected subcu~aneously on day O into nude mice: this injection produced detect-able tumors in all mice by day fi~e. Treatment of mice was started either 2 days, 5 days or 9 days after tumor implantation. Each treatment group consisted of 4-6 animals. Tumors were measured using a caliper every fourth day and the volume of the tumor was calculated .
.
WO91/099~5 PCT/US90/07464 9~ lo using the formula: tumor volume in mm3 = length x (width)2 x O.4. Tests with anti-Tac-LysPE40 were per~ormed simi-larly.
As mentioned before, the amino end of PE40 was s altered so that it contained a lysine residue and an OmpA
signal se~uence. (The OmpA signal sequence was added primarily to direct the export of LysPE40 to the growth medium.) The structure of LysPE40 and the plasmid encod-ing LysPE40 which is under the control of a T7 promoter is shown in Figure 1. Similar to PE40, LysPE40 was also secreted in the culture medium in large amounts. The purity of the protein at each purification step is shown in Figure 2. Material from the TSK 250 column, which was used as the final step, was found to be homogeneous when analyzed by SDS PAGE (Fig. 2, lane 5) as well as by immunoblotting with an antibody to PE (data not shown).
Typically, 2 mg of pure LysPE40 was obtained from one liter of culture.
The N-terminal sequence of the purified protein (LysPE40) was found to be Ala-Asn-heu-~la-Glu-Ala-Phe-Ly~-Gly-Gly-Ser-Leu. The purified protein had the exact sequence expected from the DNA sequence and processing occurred within the OmpA sequence (Figure 1). In con-trast, the N-terminal sequence of PE40 is Ala-Asn-Leu-Ala-Glu-Glu-Gly-Gly.
Construction of immunotoxins with LysPE40 LysPE40 was chemically coupled to two different monoclonal antibodies, HB21 which binds to the human transferrin receptor (anti-TFR) (Haynes et ~l, J. mmunol 127:347, 1981) and anti-Tac which binds to the 5~ kDa subunit of human interleukin 2 receptor (Uchiyama et al, J. Immuno., 126:1393, 1981). After the conjugation, ~he immunotoxins (ITs) were puri~ied on MonoQ and TSK 250 col~mns. Purified conjugates contain LysPE40 coupled ~o hoth the light and hea~y chain and do not contain any free LysPE40 (Figure 3). For comparison, immunotoxin~ were also made with native PE.
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WO9~/0~96~ PCT/VS901074~
2~7~ 946 Activity of immunotoxins made with LvsPE40 The activity of anti-TFR-LysPE40 was assayed on a variety of human cell lines and it inhibited protein synthesis in all the human cell lines studied (Table I ) .
Anti-~FR-LysPE40 was most active on A431 cells with an ID50 of 4.0 ng/ml. Specificity was demonstrated by showing that excess unconjugated antibody prevented the cytotoxic effect of anti-~FR-LysPE40 (Fig. 3A). Anti-TFR-LysPE40 was not cytotoxic to murine Swiss 3T3 cells even at 2 ~g/ml (data not shown).
The cytotoxic activity of anti-Tac-LysPE40 was determined on HUT102 cells, a human T cell leukemia line containing IL2 receptors. As shown in Figure 5, anti-Tac-hysPE~0 inhibited protein synthesis in HUT102 cells with an ID50 of 2.5 ng/ml and excess anti-Tac blocked ~his effect demonstrating the specificity of the immunotoxin (Fig. 3B). Anti-Tac-LysPE40 did not inhibit protein synthesis on IL2 receptor negative cells, even at 2000 ng/ml, ~urther showing the specificity of the IT.
. 20 BloQd levels of anti-~FR-LysP~40 in mice Balb~C mice were injected I.P. with a single dose of 100 ~g of anti-TRF-LysPE40 and blood was drawn at different times after the injection to assay for immuno-toxin activity. As shown in Fig. 4, a peak blood level of 78 ~g/ml was obtained 4 hrs after the injection and a le~el of 10 ~g/ml was still present 24 hrs after the injection. A similar experiment was performed in arthymic mice bearing A431 tumors. After injecting 50 ~g of anti-TFR-LysPE40, a blood level of 27 ~g/ml was detected 4 hrs after injection and 8 ~g/ml after 24 hrs (Fig. 4).
Effect of anti-TFR~LysPE40 on A431 tumors in mice Anti-TF~-LysPE40 was assayed fvr its ability to inhibit the growth of A431 cells as subcutaneous xeno-graf~s in nude mice. To produce tumors, 3 x 106 A431 cells were injected subcutaneously on day 0. In the control group treated only with diluent, the tumors grew very rapidly and the anLmals were sacrificed on day 19 with very large tumors that were penetrating the skin , ~
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., ~ , .
.
, . . ~, .
WO 91/Og965 PCr/~S90/074~4 '~L9 ~6 ( Fig . 5, 6 ) . In a group that received anti-TFR-LysPE40 on days 2, 4, 6, 8, no tumors were evident on day 24 whan the experiment was terminated ~ Fiy . 5A and 6 ) . In another group of animals, treatment was delayed until the tumors were about 125 mm3 in volume and gi~en on days 9, 11, 13 and 15. As soon as the treatment was initiated, the tumors stopped increasing in size (Figure 5A and 6) and then developed soft centers. His~ological examination showed that almost all of the cells in the center of ~he tumor were nonviable (Figure 7). However, at the rim of the tumor many via~le cells were observed.
To determine if the antitumor effect was dose related, treatment with various amoun~s of anti-TFR-LysPE40 was begun on day 5 when small tumors were evident (Figure 5B). Even the 5 ~g dose produced a delay in tumor growth but after the treatment was stopped on day 11, the tumors began to grow rapidly. As the dose was increased, (20 or 50 ~g), some or all of the tumors became undetect-able by day 11, but with time began to reappear and grow.
One group of animals received a single dose on day 5 of 150 ~g, which is close to the ID50. Two animals died, but in the other three animals the tumor regressed and did not reappear. When antibody alone or an immunotoxin composed of a~ antibody that did not react with the tumor (MOPC-LysPE40) was administered at 50 ~g doses, no antitumorresponse was observed.
In summary, the results indicate that LysPE40 can be efficiently coupled to antibodies yielding an immuno-toxin with high cytotoxic activity against cultured cell lines bearing the appropriate antigen, and no detectable cytotoxicity against cultured cells to which the antibody does not bind. Furthermore, an immunotoxin composed of an antibody to the human transferrin coupled to LysPE40 (anti-TFR-LysPE40~ could be administered safely in large amounts to mice and caused regression of a rapidly growing human epidermoid carcinoma implanted subcutaneously.
Nhen administered intraperi~oneally in mice, anti-TFR-LysPE40 appeared rapidly in the blood. A dose of 50 .
Wogl/09965 PCT/US90/07~64 7194~
~g gave a peak blood level 4 hrs post injection of about 30 ~g/ml, and blood levels were still 8 ~g/ml at 24 hrs ( Fig . 3) indicating that this immunotoxin has a relatively long half-life. A single dose of 100 ~g gave a peak blood level of 80 ~g/ml. since a 10 g mouse has a blood volume of about 1 ml, almost all the immunotoxin is found in the blood four hours after intraperitoneal administration.
In the treatment protocols described herein, the tumor cells were allowed to grow to form a detectable solid tumor before treatment was initiated. Under these conditions, a treatment consisting of four injections given over eight days caused obvious tumor regression (Figs. 5-8). At the lower dose levels with small tumors, or even at the high dose level with large tumors, viable tumor cells remained. By continuing the treatment for lonyer periods, a larger antitumor effect could most likely be achieved. Nevertheless, the protocol caused marked regression of a solid tumor resistant to standard chemotherapy, thereby indicating that hysPE40 when at-tached to target-specific antibodies acts as a potent immunotoxin.
A therapeutic composition in accordan~e with the present invention comprises an effective amount of the LysPE40-coupled to a target-specific ligand to kill target cells, and a pharmaceutically acceptable carrier, the target cells being those that are desired to be selective-ly killed and carry a binding site to which said ligand speciflcally binds.
Of course, ligands other than antibodies, such as receptors, growth factors and molecules which selectively recognize target cells are coupled to the LysPE40 follow-ing the methodology similar to that described herein to obtain target-specific cytotoxic entities.
It is understood that the embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggest-ed to persons skilled in the art and are to be included .
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., wo91/09g6~ 9~6 PCr~US9~/07464 within the spirit and pur~iew of this application and scope of the appended claims.
TABLE I
ACTIVITY OF ANTI -TFR-LYSPE4 0 ON ~ARIOUS HU~N CELL LINES
CELLS ;E~soa ( ng!m~
anti-TFR-L~sPE40 L~sPE40 A431 4 . 0 >2000 KB 14 . 0 >2000 Hl!296 . 6 >2000 HUT10221. 0 >2000 CEM 22 . 5 ~2000 OVCAR2135.0 ND
OVCAR3280.O ND
OVCAR430.O ND
MOLT4 32.0 ND
aID50 is described as concentration of the immunotoxin needed for 50% inhibition of protein synthesis.
N.D. - ~ot done.
TABLE II
INCIDENCE OF TUMOR IN NUDE NICE TREATED WITH
Athymic mice were injected subcutaneously with 3 x 106 A431 cells. On days 5, 7, 9 and 11, the animals were injected I.P. with anti-TFR-LysPE40 at the indicated dose.
Number of animals with tumors/total animals is shown on different days after tumor transplantation.
Treatment _ _DAYS POST TUMOR IMPhANTATION _ None 5/5 5/5 5/5 - - ~
5 ~g 5/6 6/6 6/6 6/6 6/6 20 ~ 1/5 1/5 3/5 3/5 3/5 4/5 50 ~ 0/5 0/5 lJ5 1/5 1/5 2/5 lS0 uq~ 0/3_ 0/3 0/3 _ 0/3 0/3 0/3 ~Single dose on day 5 . ~
~he following example is offered by way of illus-tration, not limitation.
Construction of a vector that encodes LysPE40 and secretes it into the medium.
Plasmid pVC4 (derived from p~H4 by trea~in~ pJH4 with SphI and Tthllll and relegating the large fragment containing ~he PE gene) was joined ~o an OmpA signal sequence as described by Chaudhary et al., PNAS 85, 2929-2943, 1988, to produce pVC45. Site directed mutagenesis is a con~enient way to make deletions and it-was used to create plasmid pJY85L which produces LYSPE4 0 as shown in Figure 1. A HindIII/SalI fragment of the PE gene in pVC45 was cloned into M13, mpl9 and uracil containing single stxanded DNA prepared by the method of Kunkel (kunkel, T.A., PNAS 82, 488-492, 1985). An oligonucleotide 36 nucleotides in length with the structure 5'CCAGGCTGCCGC-CCTTGAAAGCTTGGCGTAATCATG3' was synthesized which generated a large deletion in PE of amino acids 6 through 252 and wosl/o9965 ~CT/US90/0746q 7 2~7~946 retained a lysine residue between amino acids 5 and 253 to give a molecule ~ith the sequence:
1 2 3 4 5 x 253 254 255 256 Ala-Glu-Glu Ala-Phe-Lys-Gly~Gly-Ser-L~u A 176 bp HindIIX/SalI fragment was excised from the replicati~e form of the mutant DNA in M13 and ligated with a 3.3 kb HindIII/SalI fragment of pVC45 to give pJY85L which encodes a protein with the following struc-ture:
Me~-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Yal-Ala-Leu-Ala-Gly-Phe Ala-Thr-Val-Ala-Gln-Ala-Ala-Asn-Leu-Glu-Glu-Ala-Phe-Lys Gly-Gly-Ser ....
This protein is processed at the (l) and the proressed product secreted into the medium. When the plasmid was expressed in E. coli BL21 (ADE3 ) cells, LysPE40 was found in and purified from the medium wi~h a yield of greater than 1 mg per liter. The se~uence of amino acids at its amino terminus was found to be that predicted from the DNA
sequence:
1 2 3 4 ~ 253 254 255 H2N Ala-Asn-Leu-~la-Glu-Glu-Ala-Phe-Lys-Gly-Gly-Ser (the numbers indica~e ~he location of the amino acids in the native PE structure).
A deposit o~ plasmid pJY85L has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., on December 18, 1989 under the accession number 68189. The deposit shall be viably maintained, replacing it if it becomes non-viable, for the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public upon issuance of a patent from this application, without re-striction, in accordance with the provisions of the law.
The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
WO 91/~)9g65 Pcr/uS9O/07464 Ex~ression of LysPE40 BL21 ( DE3) cells were transformed with plasmid pVC85L and grown in LB medium containing 100 ~g/ml ampi-cillin at 37C. At absorbance 0.6 (650 nm), isopropyl-1-- 5 thio-~-D-galactopyranoside was added ~:o a final concentra-tion of 1 mM. Cells were harvested 90 min later. The culture medium was used as the source of LysPE40 because most of the protein was secreted into the medium.
Purification of LysPE40 Clarified culture medium, 25 liters, containing LysPE40 was diluted four-fold with chilled deionized water and applied on a 10 x 5.5 cm silica-based anion exchange column (QMA, Waters) at a flow rate of 100 ml/min. The column was washed with 0.05 M sodium phosphate buffer, pH7.0, and proteins were eluted with two liters of 0.25 M
NaCl in the equilibration buffer. LysPE40 from the QMA
column was concentrated further by using Amicon YM30 membranes to 150 ml, dialyzed for 12-16 hours against 0.02 M Tris-HCl, pH7.6, centrifuged at 10,000 x g for 20 minutes and applied on a 2.5 x 14 cm Q-Sepharose column equilibrated with 0.02 M Tris-HCl, pH 7.6. Proteins were eluted with a 500 ml linear gradient of 0-0.5 M NaC1;
LysPE40-containing fractions were detected by SDS PAGE, pooled, diluted 10-~old with 0.02 M Mes, pH 5.5, and loaded onto an ~PLC Mono S 16/10 colu~n equilibrated with O.02 M Mes, pH 5.5. LysPE40 bound to the column and was eluted by a 100 ml linear gradient of 0-0.5 M NaC1. It was further purified on TSX250 (22.5 x 600 mM) column (BioRad) using 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM ED~A.
Construction of immunoconiuqates LysPE40 (2.5 mg/ml) in 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA was mixed with a 3-fold molar excess of SMCC and incubated at room temperature 3S (about 22-24C) for 30 minutes. Protein was separated from the unreacted cross linker on a PD10 column. MoAb (~B21 or ~Tac) (5-10 mg/ml) was mixed with a 3-fold molar excess of 2-iminothiolane-HCl in 0.2 M sodium phosphate WO91/09965 PCT/US90/074~4 2~71~6 g buffer pH 8.0 containing 1 mM EDTA and incubated at 37C
for 1 hr. The deri~atized antibody was separa~ed from the reactants on a PD10 column. To make immunotoxins, deri~atized LysPE40 and derivatized antibody were mixed in 4:1 molar ratio and incubated at room tempera~ure for 16-hrs. Immunotoxin was then purified by successive chromatography on a mono Q and TSK 253 columns.
In order to determine the efficacy, two different immunotoxins were prepared and used. One is anti-TFR-LysPE40, which binds to human transferrin receptor (TFR),and the other is anti-Tac-LysPE40, which binds to 55 kD a subunit of human IL2 receptor.
SDS-PAGE and immunoblottinq SDS-PAGE described by (Reference).
Protein synthesis inhibition assay Activities of the conjugates were tested on A431, KB, HUT102, HT29, OVCAR2, OVCAR3~ OVCAR4, CEM and ~O~T4 cells by measuring 3H-leucine incorporation (Kondo et al., Biol. Chem. 263, 9470, 1988). HUT102 cells were washed two times with RPM1640 and used immediately. Immunotoxins were diluted with 0.2% human serum albumin in PBS prior to addition to cells.
Assav of blood levels of anti-TFR-LysPE40 in Mice BALB/C mice or nude mice bearing A431 su~cutaneous tumors were injected with 100 ~g or 50 ~g of immunotoxin I.P. Blood was drawn at different time points and the level of the IT was assayed by incubating serum with A431 cells and measuring its effect on protein synthesis. A
standard curve was made using anti TFR-LysPE4~.
An,t~tumor activity of anti-TFR-LvsPE40 in nude mice bearinq a human e~idermoid carcinoma ~ 431 cells ( c x 106) were injected subcu~aneously on day O into nude mice: this injection produced detect-able tumors in all mice by day fi~e. Treatment of mice was started either 2 days, 5 days or 9 days after tumor implantation. Each treatment group consisted of 4-6 animals. Tumors were measured using a caliper every fourth day and the volume of the tumor was calculated .
.
WO91/099~5 PCT/US90/07464 9~ lo using the formula: tumor volume in mm3 = length x (width)2 x O.4. Tests with anti-Tac-LysPE40 were per~ormed simi-larly.
As mentioned before, the amino end of PE40 was s altered so that it contained a lysine residue and an OmpA
signal se~uence. (The OmpA signal sequence was added primarily to direct the export of LysPE40 to the growth medium.) The structure of LysPE40 and the plasmid encod-ing LysPE40 which is under the control of a T7 promoter is shown in Figure 1. Similar to PE40, LysPE40 was also secreted in the culture medium in large amounts. The purity of the protein at each purification step is shown in Figure 2. Material from the TSK 250 column, which was used as the final step, was found to be homogeneous when analyzed by SDS PAGE (Fig. 2, lane 5) as well as by immunoblotting with an antibody to PE (data not shown).
Typically, 2 mg of pure LysPE40 was obtained from one liter of culture.
The N-terminal sequence of the purified protein (LysPE40) was found to be Ala-Asn-heu-~la-Glu-Ala-Phe-Ly~-Gly-Gly-Ser-Leu. The purified protein had the exact sequence expected from the DNA sequence and processing occurred within the OmpA sequence (Figure 1). In con-trast, the N-terminal sequence of PE40 is Ala-Asn-Leu-Ala-Glu-Glu-Gly-Gly.
Construction of immunotoxins with LysPE40 LysPE40 was chemically coupled to two different monoclonal antibodies, HB21 which binds to the human transferrin receptor (anti-TFR) (Haynes et ~l, J. mmunol 127:347, 1981) and anti-Tac which binds to the 5~ kDa subunit of human interleukin 2 receptor (Uchiyama et al, J. Immuno., 126:1393, 1981). After the conjugation, ~he immunotoxins (ITs) were puri~ied on MonoQ and TSK 250 col~mns. Purified conjugates contain LysPE40 coupled ~o hoth the light and hea~y chain and do not contain any free LysPE40 (Figure 3). For comparison, immunotoxin~ were also made with native PE.
, .
. . , ,~ . .
WO9~/0~96~ PCT/VS901074~
2~7~ 946 Activity of immunotoxins made with LvsPE40 The activity of anti-TFR-LysPE40 was assayed on a variety of human cell lines and it inhibited protein synthesis in all the human cell lines studied (Table I ) .
Anti-~FR-LysPE40 was most active on A431 cells with an ID50 of 4.0 ng/ml. Specificity was demonstrated by showing that excess unconjugated antibody prevented the cytotoxic effect of anti-~FR-LysPE40 (Fig. 3A). Anti-TFR-LysPE40 was not cytotoxic to murine Swiss 3T3 cells even at 2 ~g/ml (data not shown).
The cytotoxic activity of anti-Tac-LysPE40 was determined on HUT102 cells, a human T cell leukemia line containing IL2 receptors. As shown in Figure 5, anti-Tac-hysPE~0 inhibited protein synthesis in HUT102 cells with an ID50 of 2.5 ng/ml and excess anti-Tac blocked ~his effect demonstrating the specificity of the immunotoxin (Fig. 3B). Anti-Tac-LysPE40 did not inhibit protein synthesis on IL2 receptor negative cells, even at 2000 ng/ml, ~urther showing the specificity of the IT.
. 20 BloQd levels of anti-~FR-LysP~40 in mice Balb~C mice were injected I.P. with a single dose of 100 ~g of anti-TRF-LysPE40 and blood was drawn at different times after the injection to assay for immuno-toxin activity. As shown in Fig. 4, a peak blood level of 78 ~g/ml was obtained 4 hrs after the injection and a le~el of 10 ~g/ml was still present 24 hrs after the injection. A similar experiment was performed in arthymic mice bearing A431 tumors. After injecting 50 ~g of anti-TFR-LysPE40, a blood level of 27 ~g/ml was detected 4 hrs after injection and 8 ~g/ml after 24 hrs (Fig. 4).
Effect of anti-TFR~LysPE40 on A431 tumors in mice Anti-TF~-LysPE40 was assayed fvr its ability to inhibit the growth of A431 cells as subcutaneous xeno-graf~s in nude mice. To produce tumors, 3 x 106 A431 cells were injected subcutaneously on day 0. In the control group treated only with diluent, the tumors grew very rapidly and the anLmals were sacrificed on day 19 with very large tumors that were penetrating the skin , ~
. .
., ~ , .
.
, . . ~, .
WO 91/Og965 PCr/~S90/074~4 '~L9 ~6 ( Fig . 5, 6 ) . In a group that received anti-TFR-LysPE40 on days 2, 4, 6, 8, no tumors were evident on day 24 whan the experiment was terminated ~ Fiy . 5A and 6 ) . In another group of animals, treatment was delayed until the tumors were about 125 mm3 in volume and gi~en on days 9, 11, 13 and 15. As soon as the treatment was initiated, the tumors stopped increasing in size (Figure 5A and 6) and then developed soft centers. His~ological examination showed that almost all of the cells in the center of ~he tumor were nonviable (Figure 7). However, at the rim of the tumor many via~le cells were observed.
To determine if the antitumor effect was dose related, treatment with various amoun~s of anti-TFR-LysPE40 was begun on day 5 when small tumors were evident (Figure 5B). Even the 5 ~g dose produced a delay in tumor growth but after the treatment was stopped on day 11, the tumors began to grow rapidly. As the dose was increased, (20 or 50 ~g), some or all of the tumors became undetect-able by day 11, but with time began to reappear and grow.
One group of animals received a single dose on day 5 of 150 ~g, which is close to the ID50. Two animals died, but in the other three animals the tumor regressed and did not reappear. When antibody alone or an immunotoxin composed of a~ antibody that did not react with the tumor (MOPC-LysPE40) was administered at 50 ~g doses, no antitumorresponse was observed.
In summary, the results indicate that LysPE40 can be efficiently coupled to antibodies yielding an immuno-toxin with high cytotoxic activity against cultured cell lines bearing the appropriate antigen, and no detectable cytotoxicity against cultured cells to which the antibody does not bind. Furthermore, an immunotoxin composed of an antibody to the human transferrin coupled to LysPE40 (anti-TFR-LysPE40~ could be administered safely in large amounts to mice and caused regression of a rapidly growing human epidermoid carcinoma implanted subcutaneously.
Nhen administered intraperi~oneally in mice, anti-TFR-LysPE40 appeared rapidly in the blood. A dose of 50 .
Wogl/09965 PCT/US90/07~64 7194~
~g gave a peak blood level 4 hrs post injection of about 30 ~g/ml, and blood levels were still 8 ~g/ml at 24 hrs ( Fig . 3) indicating that this immunotoxin has a relatively long half-life. A single dose of 100 ~g gave a peak blood level of 80 ~g/ml. since a 10 g mouse has a blood volume of about 1 ml, almost all the immunotoxin is found in the blood four hours after intraperitoneal administration.
In the treatment protocols described herein, the tumor cells were allowed to grow to form a detectable solid tumor before treatment was initiated. Under these conditions, a treatment consisting of four injections given over eight days caused obvious tumor regression (Figs. 5-8). At the lower dose levels with small tumors, or even at the high dose level with large tumors, viable tumor cells remained. By continuing the treatment for lonyer periods, a larger antitumor effect could most likely be achieved. Nevertheless, the protocol caused marked regression of a solid tumor resistant to standard chemotherapy, thereby indicating that hysPE40 when at-tached to target-specific antibodies acts as a potent immunotoxin.
A therapeutic composition in accordan~e with the present invention comprises an effective amount of the LysPE40-coupled to a target-specific ligand to kill target cells, and a pharmaceutically acceptable carrier, the target cells being those that are desired to be selective-ly killed and carry a binding site to which said ligand speciflcally binds.
Of course, ligands other than antibodies, such as receptors, growth factors and molecules which selectively recognize target cells are coupled to the LysPE40 follow-ing the methodology similar to that described herein to obtain target-specific cytotoxic entities.
It is understood that the embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggest-ed to persons skilled in the art and are to be included .
, . . -;
., wo91/09g6~ 9~6 PCr~US9~/07464 within the spirit and pur~iew of this application and scope of the appended claims.
TABLE I
ACTIVITY OF ANTI -TFR-LYSPE4 0 ON ~ARIOUS HU~N CELL LINES
CELLS ;E~soa ( ng!m~
anti-TFR-L~sPE40 L~sPE40 A431 4 . 0 >2000 KB 14 . 0 >2000 Hl!296 . 6 >2000 HUT10221. 0 >2000 CEM 22 . 5 ~2000 OVCAR2135.0 ND
OVCAR3280.O ND
OVCAR430.O ND
MOLT4 32.0 ND
aID50 is described as concentration of the immunotoxin needed for 50% inhibition of protein synthesis.
N.D. - ~ot done.
TABLE II
INCIDENCE OF TUMOR IN NUDE NICE TREATED WITH
Athymic mice were injected subcutaneously with 3 x 106 A431 cells. On days 5, 7, 9 and 11, the animals were injected I.P. with anti-TFR-LysPE40 at the indicated dose.
Number of animals with tumors/total animals is shown on different days after tumor transplantation.
Treatment _ _DAYS POST TUMOR IMPhANTATION _ None 5/5 5/5 5/5 - - ~
5 ~g 5/6 6/6 6/6 6/6 6/6 20 ~ 1/5 1/5 3/5 3/5 3/5 4/5 50 ~ 0/5 0/5 lJ5 1/5 1/5 2/5 lS0 uq~ 0/3_ 0/3 0/3 _ 0/3 0/3 0/3 ~Single dose on day 5 . ~
Claims (7)
1. An improved Pseudomonas exotoxin (PE) of the type including a deletion in the receptor binding domain Ia of the native toxin, wherein the improvement comprises a recombinant PE molecule possessing at least one lysine residue in domain Ia of the PE molecule which otherwise will be devoid of a lysine residue when having a deletion in the receptor binding domain Ia, said lysine residue providing an essential linkage for effective coupling of the recombinant PE to other molecules.
2. A cytotoxic molecule comprising the recombinant PE of claim 1 coupled to a target-specific ligand substantially at said domain Ia lysine.
3. The cytotoxic molecule of claim 2 wherein said ligand is an antibody of a growth factor.
4. A composition comprising an acceptable amount of the cytotoxic molecule of claim 2 to kill target cells and a pharmaceutically acceptable carrier.
5. A method for achieving targeted cytotoxicity, comprising contacting cells targeted to be killed with cytotoxic amount of the cytotoxic molecule of claim 3, said target cells being those having binding sites to which the target-specific ligand binds, but the composition being without detectable cytotoxicity to cells which lack said binding sites.
6. The use of exotoxin as set out in claim 1, for the preparation of a medicament for achieving targeted cytotoxicity in connection with cells targeted to be killed.
7. The use of the cytotoxin molecule of claim 2 for achieving targeted cytotoxicity in connection with cells targeted to be killed.
Applications Claiming Priority (2)
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US45416289A | 1989-12-21 | 1989-12-21 | |
US454,162 | 1989-12-21 |
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CA2071946A1 true CA2071946A1 (en) | 1991-06-22 |
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EP (1) | EP0506854A4 (en) |
JP (1) | JPH04506976A (en) |
AU (1) | AU636453B2 (en) |
CA (1) | CA2071946A1 (en) |
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US5977307A (en) * | 1989-09-07 | 1999-11-02 | Alkermes, Inc. | Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins |
US5527527A (en) * | 1989-09-07 | 1996-06-18 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical agent conjugates |
US5672683A (en) * | 1989-09-07 | 1997-09-30 | Alkermes, Inc. | Transferrin neuropharmaceutical agent fusion protein |
US6329508B1 (en) | 1989-09-07 | 2001-12-11 | Alkermes, Inc. | Transferrin receptor reactive chimeric antibodies |
US5571894A (en) * | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US5939531A (en) * | 1991-07-15 | 1999-08-17 | Novartis Corp. | Recombinant antibodies specific for a growth factor receptor |
JPH07509444A (en) * | 1991-11-26 | 1995-10-19 | アルカーメス・インコーポレーテツド | Method for producing transferrin receptor-specific antibody-nerve drug or diagnostic drug complex |
US6066718A (en) * | 1992-09-25 | 2000-05-23 | Novartis Corporation | Reshaped monoclonal antibodies against an immunoglobulin isotype |
US6015555A (en) * | 1995-05-19 | 2000-01-18 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
US8932586B2 (en) | 2011-09-06 | 2015-01-13 | Intrexon Corporation | Modified forms of Pseudomonas exotoxin A |
WO2016146833A1 (en) | 2015-03-19 | 2016-09-22 | F. Hoffmann-La Roche Ag | Biomarkers for nad(+)-diphthamide adp ribosyltransferase resistance |
EP3184547A1 (en) | 2015-10-29 | 2017-06-28 | F. Hoffmann-La Roche AG | Anti-tpbg antibodies and methods of use |
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US4545985A (en) * | 1984-01-26 | 1985-10-08 | The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services | Pseudomonas exotoxin conjugate immunotoxins |
US4892827A (en) * | 1986-09-24 | 1990-01-09 | The United States Of America As Represented By The Department Of Health And Human Services | Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects |
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- 1990-12-21 JP JP91502841A patent/JPH04506976A/en active Pending
- 1990-12-21 CA CA002071946A patent/CA2071946A1/en not_active Abandoned
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