CA1338818C - Immunotoxins - Google Patents
ImmunotoxinsInfo
- Publication number
- CA1338818C CA1338818C CA000589789A CA589789A CA1338818C CA 1338818 C CA1338818 C CA 1338818C CA 000589789 A CA000589789 A CA 000589789A CA 589789 A CA589789 A CA 589789A CA 1338818 C CA1338818 C CA 1338818C
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- Prior art keywords
- immunotoxin
- toxin
- mutation
- binding moiety
- cells
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- 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
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
- A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6857—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from lung cancer cell
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6865—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from skin, nerves or brain cancer cell
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- 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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Abstract
A potent and specific immunotoxin is prepared by coupling an inactivated diphtheria toxin to a binding moiety such as a monoclonal antibody or transferrin. The immunotoxins are specific for human tumors and leukemias and are indistinguishable in cell toxicity from that of the native toxin linked to the binding domain without the toxicity to other cells.
The immunotoxin is useful in treating graft versus host disease as well as selectively killing tumor cells.
The immunotoxin is useful in treating graft versus host disease as well as selectively killing tumor cells.
Description
r rJN~
Field of the Invention The present invention relates to cancer chemotherapy, and, more particularly, to a reagent which selectively kills - cancer cells and can be used to treat graft versus host disease.
Backqround of the Invention The u~e of cytotoxic products in the treatment of cancer i8 well known. The ~l;ff;~ ties associated with such 10 treatment are also well known. Of these difficulties, the lack of cancer-specific cytotoxicity has received considerable attention, ~1th~ h resolution of these difficulties has met with marginal success. Cytotoxic products kill normal cells as well as cancer cells. Such non-specificity results in a number of 15 undesirable side effects for patients undergoing cancer chemotherapy with cytotoxic products, including nausea, vomiting, diarrhea, h -Lllagic gastroenteritis, and hepatic and renal damage. Due to normal cell toxicity, the therapeutic dosage of cytotoxic products has been limited such that cancerous cells are
Field of the Invention The present invention relates to cancer chemotherapy, and, more particularly, to a reagent which selectively kills - cancer cells and can be used to treat graft versus host disease.
Backqround of the Invention The u~e of cytotoxic products in the treatment of cancer i8 well known. The ~l;ff;~ ties associated with such 10 treatment are also well known. Of these difficulties, the lack of cancer-specific cytotoxicity has received considerable attention, ~1th~ h resolution of these difficulties has met with marginal success. Cytotoxic products kill normal cells as well as cancer cells. Such non-specificity results in a number of 15 undesirable side effects for patients undergoing cancer chemotherapy with cytotoxic products, including nausea, vomiting, diarrhea, h -Lllagic gastroenteritis, and hepatic and renal damage. Due to normal cell toxicity, the therapeutic dosage of cytotoxic products has been limited such that cancerous cells are
2 0 not killed to a 8uf f icient level that subsequently prevents or delay~ new cancerous growth.
.
. - .
Current approaches to cancer chemotherapy and other immunological therapies focus on the use of cell-specific antibodies bonded to immunotoxins in order to kill specif ic 5 pop~ t; ~1n~ of cancer cells . Ideally, immunotoxins should discriminate to a high degree between target and non-target cells. The critical point, then, is the development of immunotoxins that are highly toxic for specific popul~t;ons of cells .
Monoclonal ~nt;ho~;es linked to toxic proteins (immunotoxins) can selectively kill some tumor cells ln vitro and ln v1vo. However, reagents that combine the full potency of the native toxins with the high degree of cell-type selectivity of 15 monoclonal antibodies have not previously been designed. Two heretofore inbeparable activities on one polypeptide chain of h~h~ria toxin and ricin account for the failure to construct optimal reagents. The B-chains facilitate entry of the A-chain to the cytosol, allowing immunotoxins to kill target cells 20 efficiently and bind to receptors present on mobt cells, imparting immunotoxins with a graft degree of non-target-cell toxicity .
Some toxins have been modif ied to produce a ~uitable 25 immunotoxin. The two best known are ricin and ~l;phth~ria toxin.
Antibodies which bind cell surface antigens have been linked to ~liphth~ria toxin and ricin, forming a new pharmacologic class of cell type-specific toxins. Ricin and diphtheria toxin are 60,000 to 65,000 dalton proteins with two subunits: the A-chain inhibits 30 protein synthesis when in the cytosol, and the B-chain binds cell surface receptors and facilitates passage of the A subunit into the cytosol. Two types of antibody-toxin conjugates (immunotoxins) have been shown to kill antigen-pobitive cells in vitro. Immunotoxins made by binding only the toxin A subunit to 35 an antibody have little non-target cell toxicity, but are often only ~! 1 3388 1 8 m;n;m-lly toxic to antigen-positive cells. Another type of immunotoxin is made by linking the whole toxin, A and B subunits, to the antibody and blocking the binding of the B subunit to prevent toxicity to non-target cells.
For ricin, the non-target cell binding and killing can be blocked by adding lactose to the culture media or by steric restraint imposed by linking ricin to the antibody. Intact ricin immunotoxins may have only 30-to 100-fold selectivity between antigen-positive and -negative cells, but they are highly toxic, and the best reagents can specifically kill a great many target cells .
Intact ricin and ricin A-chain immunotoxins have been found to deplete allogeneic bone marrow of T cells, which can cause graft-versus-host diseases (GVHD), or to deplete autologous marrow of tumor cells.
Diphtheria toxin is composed of two disulfide-linked aubunits: the 21, 000 dalton A-chain inhibits protein synthesis by catalyzing the ADP-ribosylation of elongation factor 2, and the 37,000-dalton B-chain binds cell surface receptors and ~;)r; 1; tat~q transport of the A-chain to the cytosol. A single molecule of either a diphtheria toxin A-chain or a ricin A-chain in the cytosol is suf f icient to kill a cell . The combination of these three activities, binding, translocation, and catalysis, produces the extreme potency of these proteins . The cell surf ace-binding domain and the phosphate-binding site are located within the carboxyl-terminal 8-kDa cyanogen bromide peptide of the B-chain.
Close to the C terminus region of the B-chain are several hydrophobic domains that can insert into membranes at low pH and appear to be important for ;rhthf~ria toxin entry.
Antibodies directed against cell surface antigens have been linked to intact ~ hthf~ria toxin or its A
subunit to selectively kill antigen-bearing target cells. Antibody toxin (immunotoxin~) or ligand toxin conjugates crmt~;n;n~ only the ~l;rhtheria A-chain have relatively low cytotoxic activity. Intact l;phth.-ria toxin conjugates can be 5 very potent, but can also have greater toxicity to normal cells.
Since the B-chain appears to facilitate entry of the A-chain to the cytosol, it is possible that its presence in whole toxin conjugates renders them more potent, although less specific.
Efforts have been made to construct more potent and specific 10 immunotoxins by separating the toxin B-chain domains involved in cell binding from the domains involved in A-chain entry.
Target celL toxicity of immunotoxins can be increased by including the toxin B-chain in the antibody-toxin complex or 15 by adding it separately. To achieve maximal 1 vitro target-cell selectivity with immunotoxins c~nt~;n;n~ intact ricin, lactose must be added to the medium to block non-target-cell binding and toxicity of the immunotoxin via the ricin B-chain. This approach is feasible in those clinical settings, such as bone marrow 20 transpl;lnt~t;~n, where the target cell population can be incubated ~L vitro in the presence of lactose. Without blockage of the B-chain binding domain, however, whole toxin conjugates have a high degree of non-target-cell toxicity, thereby limiting their usefulness in vlvo.
Construction of reagents that combine the potency of intact toxin conjugates with the cell-type selectivity of toxin A-chain conjugates may be possible if the binding site on the toxin B-chain could be irreversibly blocked. Covalent and 30 noncovalent chemical modifications that block the binding activity of ricin intracellularly also block its entry function, suggesting that the binding and translocation functions may be inseparable .
., ~_ .
~ 1 3388~ 8 Previou~ly, domain deletion was unsuccessfully used in an attempt to separate the translocation and the binding functions of ~l;rhthPria toxin B-chain. Immunotoxins made with the A-chain, intact ~i;rhthPria toxin, and a cloned fragment of 5 ~l;rhthPria toxin (MspSA) that lacks the C-tprm;n~l 17 kDa region of the B subunit were compared. The intact ~;rhthPria conjugate was 100 times more toxic that the MspSA conjugate was, which, in turn, was 100-fold more toxic than was the ~l;rhthPria toxin A-chain conjugate. The C-terminal, 17-kDa region, which C-~nt;~;nR
10 the cell surface binding site, therefore potentiates immunotoxin acidity 100-fold. It has not been possible to determine whether this C-terminal translocation activity was distinct from the binding activity.
Laird and Groman, J. Virol. 19, 220 (1976) mutagenized Corynebacterium with nitrosoguanidine and ultraviolet radiation and isolated several classes of mutants within the ~l;rhthPria toxin structural gene. ~eppla and Laird further characterized several of the mutant proteins and found that three of them, CRM102, CRM103, and CRM107, retained full enzymatic activity but had defective receptor binding.
Recombinan'c DNA technology has been used to improve immunotoxin efficacy at the gene level. Greenfield et al. (1983) in Proc. ~L. ~ Sci. U.S.A. 80, 6953-6857, reported that they have cloned portions of ~i;rhthpria toxin and created a modified toxin which c r)nt~;nR the N-tPrm;n~l hydrophobic region Of ~l;rhthPria toxin but lacks the C-terminal cysteine for ease of linking to antibodies. This LL _ t, lacks the cell surface-binding site of ~irhthpria toxin but includes most of the hydrophobic region thought to f acilitate ~ e transport .
Although cleavage of ricin or ~l;rhthPria toxin into A
and B-chains had been thought to improve the specificity of the 1 3388 ~ 8 . ~
immunotoxin~ produced from the A-chain, cleavage of ricin or tl;rh~h~ria toxins into A and s-chains removes the portion of the molecule cr~nt~;n;ng residues important for transport into the cytosol of the cell. Specific cytotoxic reagents made by coupling toxin A subunits to ~nt;hs~;~5 have low systemic toxicity but also very low tumor toxicity. More potent reagents can be made by coupling intact toxins to monoclonal ~nt;ho~l;.o~, as detailed in J. T ~1l. 136: 93-98 and Proc. Natl. Acad. Sci. U.S.A. 77:
5483-5486. These reagents, however, have a high systemic toxicity due to the toxin binding to normal cells, although they can have applications n vitro in bone marrow tr~n~pl~nt~tion (cf. Science ~: 512-515).
It was found by Youle et al., as reported in ~our.
~Lm~, o~. cit., that monoclonal antibody-intact tl;rh~h~ria cell conjugates reacted ~uite differently from the intact ricin immunotoxi~s. Of the four reagents eY~m;n~tl, a monoclonal antibody against the T3 antigen linked to tl;rh~heria toxin (UCHTl-DT) had unique properties. This reagent showed greater selectivity in its toxicity to T cells as compared to stem cells than UCHTl-ricin. UCHTl-DT was found to be 10 to 100 times more selective than any previously reported immunotoxin.
Neville et al., in U.S. Patent Nos. 4,359,457 and 4,440,747, disclose that the receptor specificity of toxins can be altered by collrl ;ng the intact toxin to monoclonal antibodies directed to the cell surface antigen Thy 1.2. However, the only toxin specif ically disclosed to be treated in this manner is ricin. The same inventor~ in U.S. Patent No. 4,500,637, disclose the covalent linkage of a monoclonal antibody known as TA-l directed against human T-cells for use in treating human donor ~' 1 3388 ~ 8 bone marrow before the marrow is fused into a human recipient.
Thus, this reagent has been found to be useful in preventing graft ver~us host disease.
Another method of treating ricin to increase the rate of protein ~ynthesis inhibition is by adding excess ricin B-chain to target cells ;n~ n~nt of the amount of ricin A-chain bound to the cell surface membrane. The ricin A-chains used in this 10 procedure are conjugated to anti-Thy 1.1 monoclonal antibodie~.
This process is disclosed in Neville et al ., U. S . Patent No .
4 , 52 0 , 0 11 .
Yet another method of treating graf t versus host 15 disease i8 disclosed in Neville et al ., U. S . Patent No .
4,520,226. In this method, monoclonal antibodies specific for T-lymphocytes in human donor bone marrow are covalently linked to separate ricin toxin, combined in a mixture to form a treatment reagent, and ,~ ;n~f~ with bone marrow removed from a human 20 donor. The bone marrow-reagent mixture is then infused into an irradiated recipient, which virtually eliminates T-lymphocyte activity .
However, none of the prior art has shown effective 25 immunotoxins prepared from ~ hth.-ria toxin which have the de~ired specif icity and activity.
Summary of the Invention It is an object of the present invention to overcome the above-described deficiencies in the prior art.
Further objects are to improve anti-cancer therapy and 35 to reduce ~-~rh.oYin in cancer patients.
It is another object of the present invention to provide an immunotoxin with greater potency against cancer cells than previous immunotoxins, that at the same time is safer and 4 0 less toxic to normal cells .
.
. - .
Current approaches to cancer chemotherapy and other immunological therapies focus on the use of cell-specific antibodies bonded to immunotoxins in order to kill specif ic 5 pop~ t; ~1n~ of cancer cells . Ideally, immunotoxins should discriminate to a high degree between target and non-target cells. The critical point, then, is the development of immunotoxins that are highly toxic for specific popul~t;ons of cells .
Monoclonal ~nt;ho~;es linked to toxic proteins (immunotoxins) can selectively kill some tumor cells ln vitro and ln v1vo. However, reagents that combine the full potency of the native toxins with the high degree of cell-type selectivity of 15 monoclonal antibodies have not previously been designed. Two heretofore inbeparable activities on one polypeptide chain of h~h~ria toxin and ricin account for the failure to construct optimal reagents. The B-chains facilitate entry of the A-chain to the cytosol, allowing immunotoxins to kill target cells 20 efficiently and bind to receptors present on mobt cells, imparting immunotoxins with a graft degree of non-target-cell toxicity .
Some toxins have been modif ied to produce a ~uitable 25 immunotoxin. The two best known are ricin and ~l;phth~ria toxin.
Antibodies which bind cell surface antigens have been linked to ~liphth~ria toxin and ricin, forming a new pharmacologic class of cell type-specific toxins. Ricin and diphtheria toxin are 60,000 to 65,000 dalton proteins with two subunits: the A-chain inhibits 30 protein synthesis when in the cytosol, and the B-chain binds cell surface receptors and facilitates passage of the A subunit into the cytosol. Two types of antibody-toxin conjugates (immunotoxins) have been shown to kill antigen-pobitive cells in vitro. Immunotoxins made by binding only the toxin A subunit to 35 an antibody have little non-target cell toxicity, but are often only ~! 1 3388 1 8 m;n;m-lly toxic to antigen-positive cells. Another type of immunotoxin is made by linking the whole toxin, A and B subunits, to the antibody and blocking the binding of the B subunit to prevent toxicity to non-target cells.
For ricin, the non-target cell binding and killing can be blocked by adding lactose to the culture media or by steric restraint imposed by linking ricin to the antibody. Intact ricin immunotoxins may have only 30-to 100-fold selectivity between antigen-positive and -negative cells, but they are highly toxic, and the best reagents can specifically kill a great many target cells .
Intact ricin and ricin A-chain immunotoxins have been found to deplete allogeneic bone marrow of T cells, which can cause graft-versus-host diseases (GVHD), or to deplete autologous marrow of tumor cells.
Diphtheria toxin is composed of two disulfide-linked aubunits: the 21, 000 dalton A-chain inhibits protein synthesis by catalyzing the ADP-ribosylation of elongation factor 2, and the 37,000-dalton B-chain binds cell surface receptors and ~;)r; 1; tat~q transport of the A-chain to the cytosol. A single molecule of either a diphtheria toxin A-chain or a ricin A-chain in the cytosol is suf f icient to kill a cell . The combination of these three activities, binding, translocation, and catalysis, produces the extreme potency of these proteins . The cell surf ace-binding domain and the phosphate-binding site are located within the carboxyl-terminal 8-kDa cyanogen bromide peptide of the B-chain.
Close to the C terminus region of the B-chain are several hydrophobic domains that can insert into membranes at low pH and appear to be important for ;rhthf~ria toxin entry.
Antibodies directed against cell surface antigens have been linked to intact ~ hthf~ria toxin or its A
subunit to selectively kill antigen-bearing target cells. Antibody toxin (immunotoxin~) or ligand toxin conjugates crmt~;n;n~ only the ~l;rhtheria A-chain have relatively low cytotoxic activity. Intact l;phth.-ria toxin conjugates can be 5 very potent, but can also have greater toxicity to normal cells.
Since the B-chain appears to facilitate entry of the A-chain to the cytosol, it is possible that its presence in whole toxin conjugates renders them more potent, although less specific.
Efforts have been made to construct more potent and specific 10 immunotoxins by separating the toxin B-chain domains involved in cell binding from the domains involved in A-chain entry.
Target celL toxicity of immunotoxins can be increased by including the toxin B-chain in the antibody-toxin complex or 15 by adding it separately. To achieve maximal 1 vitro target-cell selectivity with immunotoxins c~nt~;n;n~ intact ricin, lactose must be added to the medium to block non-target-cell binding and toxicity of the immunotoxin via the ricin B-chain. This approach is feasible in those clinical settings, such as bone marrow 20 transpl;lnt~t;~n, where the target cell population can be incubated ~L vitro in the presence of lactose. Without blockage of the B-chain binding domain, however, whole toxin conjugates have a high degree of non-target-cell toxicity, thereby limiting their usefulness in vlvo.
Construction of reagents that combine the potency of intact toxin conjugates with the cell-type selectivity of toxin A-chain conjugates may be possible if the binding site on the toxin B-chain could be irreversibly blocked. Covalent and 30 noncovalent chemical modifications that block the binding activity of ricin intracellularly also block its entry function, suggesting that the binding and translocation functions may be inseparable .
., ~_ .
~ 1 3388~ 8 Previou~ly, domain deletion was unsuccessfully used in an attempt to separate the translocation and the binding functions of ~l;rhthPria toxin B-chain. Immunotoxins made with the A-chain, intact ~i;rhthPria toxin, and a cloned fragment of 5 ~l;rhthPria toxin (MspSA) that lacks the C-tprm;n~l 17 kDa region of the B subunit were compared. The intact ~;rhthPria conjugate was 100 times more toxic that the MspSA conjugate was, which, in turn, was 100-fold more toxic than was the ~l;rhthPria toxin A-chain conjugate. The C-terminal, 17-kDa region, which C-~nt;~;nR
10 the cell surface binding site, therefore potentiates immunotoxin acidity 100-fold. It has not been possible to determine whether this C-terminal translocation activity was distinct from the binding activity.
Laird and Groman, J. Virol. 19, 220 (1976) mutagenized Corynebacterium with nitrosoguanidine and ultraviolet radiation and isolated several classes of mutants within the ~l;rhthPria toxin structural gene. ~eppla and Laird further characterized several of the mutant proteins and found that three of them, CRM102, CRM103, and CRM107, retained full enzymatic activity but had defective receptor binding.
Recombinan'c DNA technology has been used to improve immunotoxin efficacy at the gene level. Greenfield et al. (1983) in Proc. ~L. ~ Sci. U.S.A. 80, 6953-6857, reported that they have cloned portions of ~i;rhthpria toxin and created a modified toxin which c r)nt~;nR the N-tPrm;n~l hydrophobic region Of ~l;rhthPria toxin but lacks the C-terminal cysteine for ease of linking to antibodies. This LL _ t, lacks the cell surface-binding site of ~irhthpria toxin but includes most of the hydrophobic region thought to f acilitate ~ e transport .
Although cleavage of ricin or ~l;rhthPria toxin into A
and B-chains had been thought to improve the specificity of the 1 3388 ~ 8 . ~
immunotoxin~ produced from the A-chain, cleavage of ricin or tl;rh~h~ria toxins into A and s-chains removes the portion of the molecule cr~nt~;n;ng residues important for transport into the cytosol of the cell. Specific cytotoxic reagents made by coupling toxin A subunits to ~nt;hs~;~5 have low systemic toxicity but also very low tumor toxicity. More potent reagents can be made by coupling intact toxins to monoclonal ~nt;ho~l;.o~, as detailed in J. T ~1l. 136: 93-98 and Proc. Natl. Acad. Sci. U.S.A. 77:
5483-5486. These reagents, however, have a high systemic toxicity due to the toxin binding to normal cells, although they can have applications n vitro in bone marrow tr~n~pl~nt~tion (cf. Science ~: 512-515).
It was found by Youle et al., as reported in ~our.
~Lm~, o~. cit., that monoclonal antibody-intact tl;rh~h~ria cell conjugates reacted ~uite differently from the intact ricin immunotoxi~s. Of the four reagents eY~m;n~tl, a monoclonal antibody against the T3 antigen linked to tl;rh~heria toxin (UCHTl-DT) had unique properties. This reagent showed greater selectivity in its toxicity to T cells as compared to stem cells than UCHTl-ricin. UCHTl-DT was found to be 10 to 100 times more selective than any previously reported immunotoxin.
Neville et al., in U.S. Patent Nos. 4,359,457 and 4,440,747, disclose that the receptor specificity of toxins can be altered by collrl ;ng the intact toxin to monoclonal antibodies directed to the cell surface antigen Thy 1.2. However, the only toxin specif ically disclosed to be treated in this manner is ricin. The same inventor~ in U.S. Patent No. 4,500,637, disclose the covalent linkage of a monoclonal antibody known as TA-l directed against human T-cells for use in treating human donor ~' 1 3388 ~ 8 bone marrow before the marrow is fused into a human recipient.
Thus, this reagent has been found to be useful in preventing graft ver~us host disease.
Another method of treating ricin to increase the rate of protein ~ynthesis inhibition is by adding excess ricin B-chain to target cells ;n~ n~nt of the amount of ricin A-chain bound to the cell surface membrane. The ricin A-chains used in this 10 procedure are conjugated to anti-Thy 1.1 monoclonal antibodie~.
This process is disclosed in Neville et al ., U. S . Patent No .
4 , 52 0 , 0 11 .
Yet another method of treating graf t versus host 15 disease i8 disclosed in Neville et al ., U. S . Patent No .
4,520,226. In this method, monoclonal antibodies specific for T-lymphocytes in human donor bone marrow are covalently linked to separate ricin toxin, combined in a mixture to form a treatment reagent, and ,~ ;n~f~ with bone marrow removed from a human 20 donor. The bone marrow-reagent mixture is then infused into an irradiated recipient, which virtually eliminates T-lymphocyte activity .
However, none of the prior art has shown effective 25 immunotoxins prepared from ~ hth.-ria toxin which have the de~ired specif icity and activity.
Summary of the Invention It is an object of the present invention to overcome the above-described deficiencies in the prior art.
Further objects are to improve anti-cancer therapy and 35 to reduce ~-~rh.oYin in cancer patients.
It is another object of the present invention to provide an immunotoxin with greater potency against cancer cells than previous immunotoxins, that at the same time is safer and 4 0 less toxic to normal cells .
3~
13388~8 It i~ yet a further object of the present invention to provide an immunotoxin with greater selectivity between antigen positive and antigen negative cells than any previously described 5 reagent.
It is another object of the present invention to provide a reagent which aelectively kills cancer cells.
It is yet another object of this invention to provide a reagent for treating graft-versus-host-disease.
According to the present invention, a binding moiety, which can be a monoclonal antibody such as UCHT1 or transferrin 15 or epidermal growth factor or any other binding agent which binds specifically to a cell, cell type or specific receptor, is coupled to a toxin in which the native toxin binding site has been inactivated. This providea extremely potent and specif ic agents against cancer cells, and is particularly effective in 20 treatment of graft versus host disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a comparison of the toxicity of ~l;phth~ria toxin, CRM102, CRM103, and CRM107 ln vitro, as compared to native diphtheria toxin using a sixteen hour protein synthesis assay. Panel A shows the in vitro toxicity tested on Jurkat cells. Panel B shows i.IL vitro toxicity tested on Vero 3 0 cells .
Figure 2 shows the binding activity of native ~;~hth~ria toxin and the three CRM mutants to Vero cells.
Figure 3 shows the location of the CRM point mutations within the diphtheria structural gene.
Figure 4 shows a comparison of the toxicities of immunotoxins made by conjugating UCHT1 with CRM102, CRM103, CRM107, and native diphtheria toxin.
~- ~ 1 3388 1 8 DETAILED DESCRIPTION OF THE INVENTION
The present invention provides mutants of ~l;rhth~ria 5 toxin which are conjugated to a binding moiety which is a binding agent which binds specifically to a cell, cell type, or specific receptor. The binding agent may be a monoclonal antibody, transferrin or epidermal growth factor, for which receptors are found in a great variety of human tumor cells. The conjugates of 10 the present invention can be used to prepare formulations for treating a great variety of tumors without the undesirable effects of native diphtheria toxins on the patients.
Immunotoxins were made by conjugating three forms of ~1;rhthf~ria toxin, CRM102, CRM103, and CRM107 (cf. J. of Virology 220-227, 1976) that differ in only one or two amino acids from native diphtheria toxin in the C region, with UCHTI, a monoclonal antibody to the T3 antigen receptor found on human T-cells, or to transferrin, or to any one of a number of known 20 binding agents such as ~r; ~lPrr-l growth factor and polyclonal sera of certain types. The conjugation used a slight modification of previously published procedures (PNAS 77: 5483-5486, 1980).
The phenotypic designation CRM is used to designate the 25 protein product of a tox gene that is serologically identical with diphtheria toxin. The number following the CRM designation indicates the molecular weight of the protein.
The nnnt~;nf~enic mutants of coryn~ha-tF~riophage beta 30 have been classified into four major classe~. Class I consists of ten mutants, each of which produces a protein that forms a line of identity with ~l;rhth~ria toxin when tested by immunodiffusion against ~l;rhth~ria antitoxin. These mutants probably represent nonsense mutations in the structural gene for ~5 toxin. The mutant! in ~ubcl~ give a po~iti~ el~in te~t but , a negative tiE3E3ue culture test. Two po~ible explanation~ are that the mutant protein has a low level of activity or i5 produced in smaller amounts.
Class II mutants produce proteins that form lines of partial identity with toxin when tested against antitoxin by r-~; ffu3ion. On glab gel electrophoresis, only one of these proteins was detected, but, based on immunodiffusion tests, all 10 appear to be smaller than purified toxin. 33ither a deletion, a nonsense mutation in the structural gene for toxin, or preferential proteolysis could account for the shortened polypeptide.
Class III mutants produce two proteins serologically related to toxin, two lines being l~terted in the immunodiffusion test. One line shows full identity with purified toxin, and the other shows only partial identity.
Class IV mutants do not produce a protein serologically related to ~;rhth-~ria toxin, nor are they capable of eliciting a positive guinea pig skin test. The phenotype of these mutants has been designated CRM-. This would indicate that the intact toxin molecule is either not produced or is produced or excreted in very small amounts. This CRM phenotype likely results from such mutational events as a deletion, a very early nonsen~e mutation in the toxin structural gene leading to the production of small fragments of toxin, or a mutation in a regulatory site or gene.
The CRM102, CRM103, and CRM107 have not been classified in one of the four major classes of mutants of r~;rh~h~ria toxin, although; ~1; ffu~ion shows complete antigenic homology with diph~h~r; ~ toxin. The molecular weight of these three CRM' 8 was 35 ~lptf~rm;n~d by electrophesis to be in the range of about 62,000.
.
13388~8 Dlphtheria toxin and CRM102, CRM103, and CRM107 were conjugated to m-maleimidobenzoyl ~-hydroxysucr;n;m;rl~ ester (MBS) by incubating the toxins with a 10-fold excess of MBS for thirty minutes at room 5 temperature. The mixture was then applied to a G-25 column to remove f ree MBS f rom the toxin . UCHTl was reduced with 10 mM dithiothreitol for 30 minute8 at room temperature, and f ree DTT was separated f rom the antibody on a G-25 column. M~3S-conjugated toxin was 10 mixed with reduced antibody and incubated at room temperature f or three hours . Immunotoxins were separated from unconjugated antibody and toxin by gel filtration on a TSK-3000 HPI-C column.
Peak fractions cr~nt~;n;n~ the immunotoxins were 15 collected and tested for toxicity to an antigen positive human leukemic T-cell line. Protein synthesis was assayed by incubating 105 cells in 100 microliters of leucine-free RPMI 1640 rrntFl;n;nrJ 29~ fetal calf serum in 96 well microtiter dishes. Toxins, immunotoxins, and 20 control buffers (11 microliters) were incubated with the cells for sixteen hours at 37C. Twenty microliters of phosphate buffered saline c~nt~;n;nrJ 0.1 microCurie of l4C-leucine was then added for ~0 minutes. Cells were harvested onto glass f iber f ilters using a PHD cell 25 harvester, washed with water, dried, and counted. The results are expressed as percentage of l4C incorporation in mock-treated control cultures.
Figure 1 shows the toxiclty of CRM102, CRM103, CRM104, and native diphtheria toxin to Jurkat cells (A) 30 and Vero cells (B). Protein synthesis was a8sayed by incubating 5 x 104 Jurkat cells in 100 microliters leucine-free RPMI 1640 medium cont~;n;ng 2g6 FCS in 96-well microliter plates.
DT (-), CRM102 (X), CRM103 (O), or CRM107 (~) were 35 added in 11 microliters buffer and incubated with cells for 16 hours at 37C. The cells were then c .
pulsed with 20 microliters of PBS ~cnt~;n;n~ 0.1 microCurie of 14c-leucine, incubated for one hour at 37 C, harvested onto glass fiber filters by means of a PHD cell harvester, washed with 5 water, dried, and counted. The results are expressed as a percentage of the 14c-leucine incorporation in mock-treated control cultures.
Vero cells have a higher number of ~1;rhthpria toxin 10 receptors than do Jurkat cells, and are thus more sensitive to diphtheria toxin inhibition of protein synthesis than are Jurkat cells. CRM102 and CRM103 are 1000-fold less toxic than native r~;rhth.oria toxin ig to both Vero cells and Jurkat cells.
Figure 2 shows the binding activity of native rl;rhth~ria toxin and the three CRM mutants to Vero cells. While most cell types, including lymphoid cells such as Jurkat, have undetectable levels Of ~ h~h~ria toxin receptors, Vero cells contain 105 diphtheria toxin receptors per cell and have been used extensively to study tlirhth,oria toxin binding. At 4 C the affinity of both CRM102 and CRM103 is 100-fold less than that of native ~l;rhth~ria toxin, and the affinity of CRM107 is 800-fold less than that of native ~lirhth.oria toxin.
The reduced af f inity correlates with the reduced toxicity for CRM107 but differs by 10-fold for CRM102 and CRM103.
sinding was determined after six hours at 4 C, while toxicity was determined after 24 hours at 37 C. The discrepancy between binding and toxicity for CRM102 and CRM103 may reflect 3 0 dif f erences in temperature and time in the two assays . Binding cannot be determined at 37 C, since ener~y inhibitors commonly used to block intf~rn:ll; 7ation decrease the number of surface fl;rhth~ria toxin receptors. Alternatively, the mutations within CRM102 and CRM103 may inhibit toxin activities other than binding ~ 1 3388 1 8 that may account for the 10-fold difference between toxicity and binding .
Figure 3 shows the location of the amino acid changes 5 within the B-chain for each of the three mutations. CRM103 c-mti~; nc a gingle mutation at position 508 (Ser-Phe) . CRM102 contains a similar mutation at position 508, but has an 1;t;~n;l1 mutation at position 308 (Pro-Ser) . CRM107 C~-nt:~;n~
a single mutation at position 525 (Ser-Phe). That CRM102 has two 10 mutations while CRM103 contains only one indicates that the two mutants are independent isolate~. The presence of multiple GC-AT
transitions is consi3tent with nitrosoguanidine-induced mutagene~is .
Line 1 is the restriction map of the ~ tl~f~ria toxin structural gene, indicating the location of the sites used for sequencing. Line 2 is the expansion of the B-chain structural region, indicating the native amino acid and DNA sequence corresponding to the point mutations found within the CRM' 8 .
Mutations found within the B-chain of CRM102 (line 3), CRM103 (line 4), and CRM107 (line 5) are shown. Line 6 shows the end of the MspRT clone previou~ly described. The sequences were obtained by cloning the two MboI-ClaI fragments into M13MP and M13MP19 and sequencing by the method of Sanger et al., J. Mol. Biol. 162, 729 (1982), or by cloning the two MspI fragments into pBR322 and sequencing by the method of Gilbert and Maxam, Methods EnzYmol.
65, 499 (1980).
The 100-fold decreased binding affinity of CRM103 and CRM102 demonstrates that the serine at position 508 is important for toxin binding. The alteration at position 525 causes the 8000-fold decrease in binding activity. The mutations ~, t 338 8 1 8 at positions 508 and 525 are consistent with data which suggest that the ~;r)hthPria toxin binding domain lies within the carboxyl 17-kDa portion of the molecule.
Both mutations exchange a phenylalanine for a serine.
The relationship of binding to translocation in ~l;rhthPria toxin was P~m;nF~rl by linking each of the CRM's and native ~;~hth~ria toxin to a new binding domain, the monoclonal antibody UCHT1, which is ~3pecific for the T3 antigen on human T-cells.
Figure 4 shows that, unlike the unconjugated CRM's, all three CRM immunotoxins are highly toxic. Excess antibody blocks toxicity, demonstrating that the toxicity is antibody-mediated. The immunotoxins prepared with CRM103 and CRM107 are equally toxic as the immunotoxin prepared with native A;rhthPria toxin, whereas the immunotoxin prepared from CRM102 is approximately 10-fold less toxic. The 10-fold decrease in UCHT1-CRM102 toxicity relative to UCHT1-CRM103, despite identical binding activity of CRM102 and C~M103, suggests that the amino acid at position 303 contributes to the translocation activity of (l;rhth~ria toxin. That the conjugates prepared with CRM103 and CRM107 are as toxic as are conjugates prepared with native ~l;rhthPria toxin indicates that binding of the toxin to its receptor is not necessary for efficient translocation of the toxin-A fragment to the cytosol. Therefore, the ~l;rhth~ria toxin binding and translocation functions can be separated.
Figure 4 shows the comparison of the toxicities of immunotoxins made by conjugating UCHT1 with CRM102, CRM103, CRM107, and native ~;rhthPria toxin. The antibody wa3 linked to the toxins via a thioether bond as described previously. Immunotoxins were separated from unconjugated antibody and toxin by gel filtration on a TSK-3000 HP~C column. The immunotoxin peak was collected, and toxicity was ~ 3388 1 8 evaluated with the protein synthesis assay as described in Figure 1. ~CHT1-DT (O), UCHT1-CRM102 (V), UCHT1-CRM103 (~), and UCHT1-CRM107 (C) were incubated with 5 x 10~ Jurkat cells for sixteen hours, followed by a one 5 hour pulse with l4C-leucine. Incubation with excess free UCHT1 (100 micrograms/ml) blocked toxicity.
As shown in both Figures 1 and 4, native diphtheria toxin and UCHT1-A;rhth~ria toxin inhibit Jurkat cell protein synthesis 50% at 3 x 10-llM. The selective toxicity of UCHT1-DT to T3 bearing cells is 100-fold, and exists 801ely becau~e cro~l ;nk;n~ diphtheria toxin to antibody inhibits tl;rh~h~ria toxicity 100-fold. The mutant toxins, CRM102, CRM103, and CRM107, inhibit Jurkat cell protein synthesis 5096 at 1 x 10-7M to 4 x lO~M
15 (Figure 1), whereas the IJCHT1-CRM immunotoxins act at 3 x 10-llM to 3 x 10-lM (Figure 4) . This 1000-10, ooo-fOld difference in c~1n~pntration between the CRM's and the UCHT1-CRM' 8 required to inhibit protein synthesis represents a three to four order of magnitude increase 20 in CRM immunotoxin selectivity over the native diphtheria immunotoxin.
The toxicities of the different immunotoxins were compared on non-target Vero cells, which lack antibody-binding sites but express a high number of diphtheria 25 toxin cell-surface binding sites. UCHT1-DT inhibits Vero protein synthesis 90% at 6 x 10-lM, because of toxicity via the ~ h~h~ria toxin binding site. In contrast, all three CRM immunotoxins had no effect on protein synthesis at this concentration. Thus, the 1088 30 of toxicity of the CRM's, as shown in Figure 1, is exhibited also by the CRM immunotoxins on non-target cells .
The immunotoxins as described herein can also be conjugated with human transferrin (Tfn) . Transferrin is 35 highly conserved across species, and, as a result, human transferrin exhibits species cross-reactivity that enables the comparison of the toxicity of transLerrin-toxin conjugates on cell~ derived from human (Jurkat, K562), monkey (Vero), and mou~e (Wehi, E:~-4), as shown in Table l.
t338818 .
~- ~ 11 l ~ Q' C~ R
,10 X X X X ~
U a) r~l 11i N N
c æE ~ ~ ~ r o o ~ o ~ o o o -x x O O x o X X X ~ ,,, ~a X X X ,,~ ' 'C ' ' '.C 'C ~ o ~
O - ~ ~ o :~: X,~:_ m ~ ,1 m m~ X X 'D 'D ~ ~ ,~ u I' ~i ~ N ~ o X ~ ~ o o~ ~ O O O ' O X X X X X X X X ~ ` (d ~, E~a al ~ ~ N N ~i ~) ~i ~ JJ 3 S~
3 ~
r ~q O
H ' ' ~ - ' rt U
r r r~ r~ ,, rr 3 ~ ~ ~ ~ ~ h r- o O O ~ ~ O O ~ U ~ C
~, on r-l C) O N . ~ r~ 1:` ~1 b 3 ~ æ ~ ~ Q
~338818 .
Before conjugating the toxin with transferrin, human transferrin (Tfn) was loaded with iron according to the method of .~h;n~lr~n et al., (1981), Int. J.
Cancer 27: 329 . The conjugation of Tfn was ~ hf~
5 by first generating free sulfhydryl groups on Tfn with 2-iminothiolane. The 2-iminothiolane was dissolved in 0.8M boric acid, pH 8.5, and incubated with Tfn in an 8:1 molar ratio. After a one hour ;n-llh~t;on at room temperature, the modified Tfn was separated from the 2-10 iminothiolane by gel filtration on a G-25 column. M-maleimidobenzoyl N-llydL~y~ ;n;m;de (MBS) was dissolved in dimethylformamide and added in five-fold molar exces:3 to the toxin, which was either native diphtheria toxin, CRM102, CRM103, or CRM107. This 15 mixture wa~ incubated for thirty minute~3 at room temperature, and free MBS waE3 removed from the toxin by chromatographic separation using a G-25 column. The MBS-conjugated toxin waa mixed with thiolated transferrin in 1:1:3 molar ratio and incubated for three 20 hours at room temperature. Immunotoxins were purified by gel filtration on a TSK-3000 HPLC column.
Species cross-reactivity enables one to evaluate the toxicity and the effectivene3s of the toxin conjugate in animal models as well as to examine the 25 effectiveness of the Tfn-toxin conjugate in a wide variety of human tumors~. Tfn-CRM107, when assayed on Jurkat cells, exhibited a 400, 000-fold differential in toxicity over CRM107 alone. ~ This repre3ents a con3iderable il..,~L-JV~ t in the 10, 000-fold selectivity previously observed between UCHT1-CRM107 and CRM107 on Jurkat cells.
It has also been found that K562, a human erythroleukemia cell line characterized by high levels of transferrin receptors, is also sensitive to Tfn-CRM107. Assuming that the binding and toxicity of CRM107 is reduced by 10, 000-fold relative to diphtheria ~3~
1 ~38 8 ~ 8 toxin on K562 cells, as it is on Vero and Jur~cat cells, then the differential in toxicity between CRM103 and Tfn-CRM107 for K562 cells i~ greater than one million.
Binding of the toxin conjugates was Tfn-mediated as 5 shown by the fact that free Tfn completely inhibits toxicity, as shown in Table 2.
1 3388 ~ 8 -1 . ~ ~
a~ ~ o ~ o 1- r~ o ~ ~ o ~I N ~1 ~;
O ,~
U
0~
o -- o O ~ ~ o O
h ~ E~
+ + a~
+
X X X X ~
~ o o ,,, ~ ~` ---- X X
X O O rl ~
o o N N
U
I I C) P ~
X
13388~8 Immunotoxins made with antibodies and whole toxins that are genetically altered in their binding domain possess several advantages over antibody-toxin-A-chain conjugates. First, as 5 shown with ~;rhthPria toxin, the B-chain translocation activity can be used in the absence of its binding function to increa~e reagent potency 10, 000-fold over that of A-chain conjugate~.
Reduction of the disulfide linkage leads to rapid 1088 of immunotoxin ln vivo, and the release of free antibody that can 10 bind more cells and compete with intact immunotoxins. Use of whole toxins permits construction of noncleavable thioether linkages between toxin and antibody. Intact toxins are less susceptible to proteolytic inactivation than are toxin A
fragments, and may survive longer i vivo.
The immunotoxins of the present invention have full A-chain activity and full B-chain translocation activity, but they lack the binding for native ~irhthPria toxin and possess a new binding domain, which is covalently attached. The immunotoxins 2 0 of the present invention have a greater potency than any previously ~ sed immunotoxin, and have greater selectivity between antigen po~itive and antigen negative cells.
Conjugates of the binding moiety and the inactivated 25 f1;rhthPria toxin may be made uging a variety of bif-lnrti~n~l protein coupling agents. Examples of such reagents are SPDP, iminoth;ol~nP (IT), bifunctional derivatives of imidoesters such as dimethyl adipimidate HC1, active esters such as cin;m;dyl suberate, aldehydes such as glutaraldehyde, bis-30 azido compound such as bis(~-~7;~ hPn7oyl)-ethylPnP~ m;nP~
diisocyanates such as tolylene 2, 6-diisocyanate, and bis-active fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene. The binding moiety, if it ~r~nt~; nçl a carbohydrate moiety, may be linked to the ~l;rhthPria toxin by means of a covalent bond to 1 338~ ~
an oxidized carbohydrate moiety on the antibody aa disclo~ed by U.S. Patent No. 4,671,958.
The immunotoxins of the present invention are useful in the treatment of any condition requiring bone marrow transplantation. That i8, T-cell activity from peripheral blood which r-mt~m;n~tes human bone marrow transplants can be largely eliminated by prior treatment with the immunotoxins of the present invention by preventing the reaction of T-cells in the donor marrow against the host ce}ls, causing graf t -versus-host disease. Therefore, this reagent is particularly useful in the treatment of aplastic anemia or leukemia patients who receive bone marrow tran~plants.
In treating such conditions, human bone marrow and peripheral blood r~nll~ r cells are treated with varying c~,n,--~n~rations of an immunotoxin prepared according to the present invention. The T-lymphocyte cell activity can be reduced by the immunotoxins at ~ nron~rations which have very little effect on the activity of the stem cells necessary to rPpoplll~e the patient' 8 marrow.
The protocol used for the actual treatment of human donor bone marrow is as follows: the bone marrow is removed from the human donor, treated i rl vitrQ with an immunotoxin according to the present invention, and then infused into the irradiated recipient .
The foregoing description of the specific ~ l;r- ~
will 80 fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are ;nt-on-lP~ to be comprehended within the meaning and range of equivalents of the disclosed ,t.
133~8 1 ~
embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.
~`
13388~8 It i~ yet a further object of the present invention to provide an immunotoxin with greater selectivity between antigen positive and antigen negative cells than any previously described 5 reagent.
It is another object of the present invention to provide a reagent which aelectively kills cancer cells.
It is yet another object of this invention to provide a reagent for treating graft-versus-host-disease.
According to the present invention, a binding moiety, which can be a monoclonal antibody such as UCHT1 or transferrin 15 or epidermal growth factor or any other binding agent which binds specifically to a cell, cell type or specific receptor, is coupled to a toxin in which the native toxin binding site has been inactivated. This providea extremely potent and specif ic agents against cancer cells, and is particularly effective in 20 treatment of graft versus host disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a comparison of the toxicity of ~l;phth~ria toxin, CRM102, CRM103, and CRM107 ln vitro, as compared to native diphtheria toxin using a sixteen hour protein synthesis assay. Panel A shows the in vitro toxicity tested on Jurkat cells. Panel B shows i.IL vitro toxicity tested on Vero 3 0 cells .
Figure 2 shows the binding activity of native ~;~hth~ria toxin and the three CRM mutants to Vero cells.
Figure 3 shows the location of the CRM point mutations within the diphtheria structural gene.
Figure 4 shows a comparison of the toxicities of immunotoxins made by conjugating UCHT1 with CRM102, CRM103, CRM107, and native diphtheria toxin.
~- ~ 1 3388 1 8 DETAILED DESCRIPTION OF THE INVENTION
The present invention provides mutants of ~l;rhth~ria 5 toxin which are conjugated to a binding moiety which is a binding agent which binds specifically to a cell, cell type, or specific receptor. The binding agent may be a monoclonal antibody, transferrin or epidermal growth factor, for which receptors are found in a great variety of human tumor cells. The conjugates of 10 the present invention can be used to prepare formulations for treating a great variety of tumors without the undesirable effects of native diphtheria toxins on the patients.
Immunotoxins were made by conjugating three forms of ~1;rhthf~ria toxin, CRM102, CRM103, and CRM107 (cf. J. of Virology 220-227, 1976) that differ in only one or two amino acids from native diphtheria toxin in the C region, with UCHTI, a monoclonal antibody to the T3 antigen receptor found on human T-cells, or to transferrin, or to any one of a number of known 20 binding agents such as ~r; ~lPrr-l growth factor and polyclonal sera of certain types. The conjugation used a slight modification of previously published procedures (PNAS 77: 5483-5486, 1980).
The phenotypic designation CRM is used to designate the 25 protein product of a tox gene that is serologically identical with diphtheria toxin. The number following the CRM designation indicates the molecular weight of the protein.
The nnnt~;nf~enic mutants of coryn~ha-tF~riophage beta 30 have been classified into four major classe~. Class I consists of ten mutants, each of which produces a protein that forms a line of identity with ~l;rhth~ria toxin when tested by immunodiffusion against ~l;rhth~ria antitoxin. These mutants probably represent nonsense mutations in the structural gene for ~5 toxin. The mutant! in ~ubcl~ give a po~iti~ el~in te~t but , a negative tiE3E3ue culture test. Two po~ible explanation~ are that the mutant protein has a low level of activity or i5 produced in smaller amounts.
Class II mutants produce proteins that form lines of partial identity with toxin when tested against antitoxin by r-~; ffu3ion. On glab gel electrophoresis, only one of these proteins was detected, but, based on immunodiffusion tests, all 10 appear to be smaller than purified toxin. 33ither a deletion, a nonsense mutation in the structural gene for toxin, or preferential proteolysis could account for the shortened polypeptide.
Class III mutants produce two proteins serologically related to toxin, two lines being l~terted in the immunodiffusion test. One line shows full identity with purified toxin, and the other shows only partial identity.
Class IV mutants do not produce a protein serologically related to ~;rhth-~ria toxin, nor are they capable of eliciting a positive guinea pig skin test. The phenotype of these mutants has been designated CRM-. This would indicate that the intact toxin molecule is either not produced or is produced or excreted in very small amounts. This CRM phenotype likely results from such mutational events as a deletion, a very early nonsen~e mutation in the toxin structural gene leading to the production of small fragments of toxin, or a mutation in a regulatory site or gene.
The CRM102, CRM103, and CRM107 have not been classified in one of the four major classes of mutants of r~;rh~h~ria toxin, although; ~1; ffu~ion shows complete antigenic homology with diph~h~r; ~ toxin. The molecular weight of these three CRM' 8 was 35 ~lptf~rm;n~d by electrophesis to be in the range of about 62,000.
.
13388~8 Dlphtheria toxin and CRM102, CRM103, and CRM107 were conjugated to m-maleimidobenzoyl ~-hydroxysucr;n;m;rl~ ester (MBS) by incubating the toxins with a 10-fold excess of MBS for thirty minutes at room 5 temperature. The mixture was then applied to a G-25 column to remove f ree MBS f rom the toxin . UCHTl was reduced with 10 mM dithiothreitol for 30 minute8 at room temperature, and f ree DTT was separated f rom the antibody on a G-25 column. M~3S-conjugated toxin was 10 mixed with reduced antibody and incubated at room temperature f or three hours . Immunotoxins were separated from unconjugated antibody and toxin by gel filtration on a TSK-3000 HPI-C column.
Peak fractions cr~nt~;n;n~ the immunotoxins were 15 collected and tested for toxicity to an antigen positive human leukemic T-cell line. Protein synthesis was assayed by incubating 105 cells in 100 microliters of leucine-free RPMI 1640 rrntFl;n;nrJ 29~ fetal calf serum in 96 well microtiter dishes. Toxins, immunotoxins, and 20 control buffers (11 microliters) were incubated with the cells for sixteen hours at 37C. Twenty microliters of phosphate buffered saline c~nt~;n;nrJ 0.1 microCurie of l4C-leucine was then added for ~0 minutes. Cells were harvested onto glass f iber f ilters using a PHD cell 25 harvester, washed with water, dried, and counted. The results are expressed as percentage of l4C incorporation in mock-treated control cultures.
Figure 1 shows the toxiclty of CRM102, CRM103, CRM104, and native diphtheria toxin to Jurkat cells (A) 30 and Vero cells (B). Protein synthesis was a8sayed by incubating 5 x 104 Jurkat cells in 100 microliters leucine-free RPMI 1640 medium cont~;n;ng 2g6 FCS in 96-well microliter plates.
DT (-), CRM102 (X), CRM103 (O), or CRM107 (~) were 35 added in 11 microliters buffer and incubated with cells for 16 hours at 37C. The cells were then c .
pulsed with 20 microliters of PBS ~cnt~;n;n~ 0.1 microCurie of 14c-leucine, incubated for one hour at 37 C, harvested onto glass fiber filters by means of a PHD cell harvester, washed with 5 water, dried, and counted. The results are expressed as a percentage of the 14c-leucine incorporation in mock-treated control cultures.
Vero cells have a higher number of ~1;rhthpria toxin 10 receptors than do Jurkat cells, and are thus more sensitive to diphtheria toxin inhibition of protein synthesis than are Jurkat cells. CRM102 and CRM103 are 1000-fold less toxic than native r~;rhth.oria toxin ig to both Vero cells and Jurkat cells.
Figure 2 shows the binding activity of native rl;rhth~ria toxin and the three CRM mutants to Vero cells. While most cell types, including lymphoid cells such as Jurkat, have undetectable levels Of ~ h~h~ria toxin receptors, Vero cells contain 105 diphtheria toxin receptors per cell and have been used extensively to study tlirhth,oria toxin binding. At 4 C the affinity of both CRM102 and CRM103 is 100-fold less than that of native ~l;rhth~ria toxin, and the affinity of CRM107 is 800-fold less than that of native ~lirhth.oria toxin.
The reduced af f inity correlates with the reduced toxicity for CRM107 but differs by 10-fold for CRM102 and CRM103.
sinding was determined after six hours at 4 C, while toxicity was determined after 24 hours at 37 C. The discrepancy between binding and toxicity for CRM102 and CRM103 may reflect 3 0 dif f erences in temperature and time in the two assays . Binding cannot be determined at 37 C, since ener~y inhibitors commonly used to block intf~rn:ll; 7ation decrease the number of surface fl;rhth~ria toxin receptors. Alternatively, the mutations within CRM102 and CRM103 may inhibit toxin activities other than binding ~ 1 3388 1 8 that may account for the 10-fold difference between toxicity and binding .
Figure 3 shows the location of the amino acid changes 5 within the B-chain for each of the three mutations. CRM103 c-mti~; nc a gingle mutation at position 508 (Ser-Phe) . CRM102 contains a similar mutation at position 508, but has an 1;t;~n;l1 mutation at position 308 (Pro-Ser) . CRM107 C~-nt:~;n~
a single mutation at position 525 (Ser-Phe). That CRM102 has two 10 mutations while CRM103 contains only one indicates that the two mutants are independent isolate~. The presence of multiple GC-AT
transitions is consi3tent with nitrosoguanidine-induced mutagene~is .
Line 1 is the restriction map of the ~ tl~f~ria toxin structural gene, indicating the location of the sites used for sequencing. Line 2 is the expansion of the B-chain structural region, indicating the native amino acid and DNA sequence corresponding to the point mutations found within the CRM' 8 .
Mutations found within the B-chain of CRM102 (line 3), CRM103 (line 4), and CRM107 (line 5) are shown. Line 6 shows the end of the MspRT clone previou~ly described. The sequences were obtained by cloning the two MboI-ClaI fragments into M13MP and M13MP19 and sequencing by the method of Sanger et al., J. Mol. Biol. 162, 729 (1982), or by cloning the two MspI fragments into pBR322 and sequencing by the method of Gilbert and Maxam, Methods EnzYmol.
65, 499 (1980).
The 100-fold decreased binding affinity of CRM103 and CRM102 demonstrates that the serine at position 508 is important for toxin binding. The alteration at position 525 causes the 8000-fold decrease in binding activity. The mutations ~, t 338 8 1 8 at positions 508 and 525 are consistent with data which suggest that the ~;r)hthPria toxin binding domain lies within the carboxyl 17-kDa portion of the molecule.
Both mutations exchange a phenylalanine for a serine.
The relationship of binding to translocation in ~l;rhthPria toxin was P~m;nF~rl by linking each of the CRM's and native ~;~hth~ria toxin to a new binding domain, the monoclonal antibody UCHT1, which is ~3pecific for the T3 antigen on human T-cells.
Figure 4 shows that, unlike the unconjugated CRM's, all three CRM immunotoxins are highly toxic. Excess antibody blocks toxicity, demonstrating that the toxicity is antibody-mediated. The immunotoxins prepared with CRM103 and CRM107 are equally toxic as the immunotoxin prepared with native A;rhthPria toxin, whereas the immunotoxin prepared from CRM102 is approximately 10-fold less toxic. The 10-fold decrease in UCHT1-CRM102 toxicity relative to UCHT1-CRM103, despite identical binding activity of CRM102 and C~M103, suggests that the amino acid at position 303 contributes to the translocation activity of (l;rhth~ria toxin. That the conjugates prepared with CRM103 and CRM107 are as toxic as are conjugates prepared with native ~l;rhthPria toxin indicates that binding of the toxin to its receptor is not necessary for efficient translocation of the toxin-A fragment to the cytosol. Therefore, the ~l;rhth~ria toxin binding and translocation functions can be separated.
Figure 4 shows the comparison of the toxicities of immunotoxins made by conjugating UCHT1 with CRM102, CRM103, CRM107, and native ~;rhthPria toxin. The antibody wa3 linked to the toxins via a thioether bond as described previously. Immunotoxins were separated from unconjugated antibody and toxin by gel filtration on a TSK-3000 HP~C column. The immunotoxin peak was collected, and toxicity was ~ 3388 1 8 evaluated with the protein synthesis assay as described in Figure 1. ~CHT1-DT (O), UCHT1-CRM102 (V), UCHT1-CRM103 (~), and UCHT1-CRM107 (C) were incubated with 5 x 10~ Jurkat cells for sixteen hours, followed by a one 5 hour pulse with l4C-leucine. Incubation with excess free UCHT1 (100 micrograms/ml) blocked toxicity.
As shown in both Figures 1 and 4, native diphtheria toxin and UCHT1-A;rhth~ria toxin inhibit Jurkat cell protein synthesis 50% at 3 x 10-llM. The selective toxicity of UCHT1-DT to T3 bearing cells is 100-fold, and exists 801ely becau~e cro~l ;nk;n~ diphtheria toxin to antibody inhibits tl;rh~h~ria toxicity 100-fold. The mutant toxins, CRM102, CRM103, and CRM107, inhibit Jurkat cell protein synthesis 5096 at 1 x 10-7M to 4 x lO~M
15 (Figure 1), whereas the IJCHT1-CRM immunotoxins act at 3 x 10-llM to 3 x 10-lM (Figure 4) . This 1000-10, ooo-fOld difference in c~1n~pntration between the CRM's and the UCHT1-CRM' 8 required to inhibit protein synthesis represents a three to four order of magnitude increase 20 in CRM immunotoxin selectivity over the native diphtheria immunotoxin.
The toxicities of the different immunotoxins were compared on non-target Vero cells, which lack antibody-binding sites but express a high number of diphtheria 25 toxin cell-surface binding sites. UCHT1-DT inhibits Vero protein synthesis 90% at 6 x 10-lM, because of toxicity via the ~ h~h~ria toxin binding site. In contrast, all three CRM immunotoxins had no effect on protein synthesis at this concentration. Thus, the 1088 30 of toxicity of the CRM's, as shown in Figure 1, is exhibited also by the CRM immunotoxins on non-target cells .
The immunotoxins as described herein can also be conjugated with human transferrin (Tfn) . Transferrin is 35 highly conserved across species, and, as a result, human transferrin exhibits species cross-reactivity that enables the comparison of the toxicity of transLerrin-toxin conjugates on cell~ derived from human (Jurkat, K562), monkey (Vero), and mou~e (Wehi, E:~-4), as shown in Table l.
t338818 .
~- ~ 11 l ~ Q' C~ R
,10 X X X X ~
U a) r~l 11i N N
c æE ~ ~ ~ r o o ~ o ~ o o o -x x O O x o X X X ~ ,,, ~a X X X ,,~ ' 'C ' ' '.C 'C ~ o ~
O - ~ ~ o :~: X,~:_ m ~ ,1 m m~ X X 'D 'D ~ ~ ,~ u I' ~i ~ N ~ o X ~ ~ o o~ ~ O O O ' O X X X X X X X X ~ ` (d ~, E~a al ~ ~ N N ~i ~) ~i ~ JJ 3 S~
3 ~
r ~q O
H ' ' ~ - ' rt U
r r r~ r~ ,, rr 3 ~ ~ ~ ~ ~ h r- o O O ~ ~ O O ~ U ~ C
~, on r-l C) O N . ~ r~ 1:` ~1 b 3 ~ æ ~ ~ Q
~338818 .
Before conjugating the toxin with transferrin, human transferrin (Tfn) was loaded with iron according to the method of .~h;n~lr~n et al., (1981), Int. J.
Cancer 27: 329 . The conjugation of Tfn was ~ hf~
5 by first generating free sulfhydryl groups on Tfn with 2-iminothiolane. The 2-iminothiolane was dissolved in 0.8M boric acid, pH 8.5, and incubated with Tfn in an 8:1 molar ratio. After a one hour ;n-llh~t;on at room temperature, the modified Tfn was separated from the 2-10 iminothiolane by gel filtration on a G-25 column. M-maleimidobenzoyl N-llydL~y~ ;n;m;de (MBS) was dissolved in dimethylformamide and added in five-fold molar exces:3 to the toxin, which was either native diphtheria toxin, CRM102, CRM103, or CRM107. This 15 mixture wa~ incubated for thirty minute~3 at room temperature, and free MBS waE3 removed from the toxin by chromatographic separation using a G-25 column. The MBS-conjugated toxin waa mixed with thiolated transferrin in 1:1:3 molar ratio and incubated for three 20 hours at room temperature. Immunotoxins were purified by gel filtration on a TSK-3000 HPLC column.
Species cross-reactivity enables one to evaluate the toxicity and the effectivene3s of the toxin conjugate in animal models as well as to examine the 25 effectiveness of the Tfn-toxin conjugate in a wide variety of human tumors~. Tfn-CRM107, when assayed on Jurkat cells, exhibited a 400, 000-fold differential in toxicity over CRM107 alone. ~ This repre3ents a con3iderable il..,~L-JV~ t in the 10, 000-fold selectivity previously observed between UCHT1-CRM107 and CRM107 on Jurkat cells.
It has also been found that K562, a human erythroleukemia cell line characterized by high levels of transferrin receptors, is also sensitive to Tfn-CRM107. Assuming that the binding and toxicity of CRM107 is reduced by 10, 000-fold relative to diphtheria ~3~
1 ~38 8 ~ 8 toxin on K562 cells, as it is on Vero and Jur~cat cells, then the differential in toxicity between CRM103 and Tfn-CRM107 for K562 cells i~ greater than one million.
Binding of the toxin conjugates was Tfn-mediated as 5 shown by the fact that free Tfn completely inhibits toxicity, as shown in Table 2.
1 3388 ~ 8 -1 . ~ ~
a~ ~ o ~ o 1- r~ o ~ ~ o ~I N ~1 ~;
O ,~
U
0~
o -- o O ~ ~ o O
h ~ E~
+ + a~
+
X X X X ~
~ o o ,,, ~ ~` ---- X X
X O O rl ~
o o N N
U
I I C) P ~
X
13388~8 Immunotoxins made with antibodies and whole toxins that are genetically altered in their binding domain possess several advantages over antibody-toxin-A-chain conjugates. First, as 5 shown with ~;rhthPria toxin, the B-chain translocation activity can be used in the absence of its binding function to increa~e reagent potency 10, 000-fold over that of A-chain conjugate~.
Reduction of the disulfide linkage leads to rapid 1088 of immunotoxin ln vivo, and the release of free antibody that can 10 bind more cells and compete with intact immunotoxins. Use of whole toxins permits construction of noncleavable thioether linkages between toxin and antibody. Intact toxins are less susceptible to proteolytic inactivation than are toxin A
fragments, and may survive longer i vivo.
The immunotoxins of the present invention have full A-chain activity and full B-chain translocation activity, but they lack the binding for native ~irhthPria toxin and possess a new binding domain, which is covalently attached. The immunotoxins 2 0 of the present invention have a greater potency than any previously ~ sed immunotoxin, and have greater selectivity between antigen po~itive and antigen negative cells.
Conjugates of the binding moiety and the inactivated 25 f1;rhthPria toxin may be made uging a variety of bif-lnrti~n~l protein coupling agents. Examples of such reagents are SPDP, iminoth;ol~nP (IT), bifunctional derivatives of imidoesters such as dimethyl adipimidate HC1, active esters such as cin;m;dyl suberate, aldehydes such as glutaraldehyde, bis-30 azido compound such as bis(~-~7;~ hPn7oyl)-ethylPnP~ m;nP~
diisocyanates such as tolylene 2, 6-diisocyanate, and bis-active fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene. The binding moiety, if it ~r~nt~; nçl a carbohydrate moiety, may be linked to the ~l;rhthPria toxin by means of a covalent bond to 1 338~ ~
an oxidized carbohydrate moiety on the antibody aa disclo~ed by U.S. Patent No. 4,671,958.
The immunotoxins of the present invention are useful in the treatment of any condition requiring bone marrow transplantation. That i8, T-cell activity from peripheral blood which r-mt~m;n~tes human bone marrow transplants can be largely eliminated by prior treatment with the immunotoxins of the present invention by preventing the reaction of T-cells in the donor marrow against the host ce}ls, causing graf t -versus-host disease. Therefore, this reagent is particularly useful in the treatment of aplastic anemia or leukemia patients who receive bone marrow tran~plants.
In treating such conditions, human bone marrow and peripheral blood r~nll~ r cells are treated with varying c~,n,--~n~rations of an immunotoxin prepared according to the present invention. The T-lymphocyte cell activity can be reduced by the immunotoxins at ~ nron~rations which have very little effect on the activity of the stem cells necessary to rPpoplll~e the patient' 8 marrow.
The protocol used for the actual treatment of human donor bone marrow is as follows: the bone marrow is removed from the human donor, treated i rl vitrQ with an immunotoxin according to the present invention, and then infused into the irradiated recipient .
The foregoing description of the specific ~ l;r- ~
will 80 fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are ;nt-on-lP~ to be comprehended within the meaning and range of equivalents of the disclosed ,t.
133~8 1 ~
embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.
~`
Claims (21)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An immunotoxin comprising a moiety which binds to a specific receptor covalently linked to a mutant diphtheria toxin, wherein said mutant diphtheria toxin consists of an A chain polypeptide and a B chain polypeptide, wherein said B chain polypeptide is inactivated as to membrane binding but retains full A-chain translocating activity.
2. An immunotoxin of claim 1, wherein said mutant diphtheria toxin contains at least one point mutation selected from the group consisting of mutation of serine residue 508 to another amino acid, mutation of serine residue 525 to another amino acid, and mutation of proline residue 308 to another amino acid.
3. An immunotoxin of claim 1, wherein said mutant diphtheria toxin contains a point mutation selected from the group consisting of mutation of serine residue 508 to phenylalanine, mutation of serine residue 525 to phenylalanine, and mutation of proline residue 308 to serine, together with mutation of serine residue 525 to phenylalanine.
4. An immunotoxin of any one of claims 1, 2 or 3 wherein said binding moiety is selected from the group consisting of a monoclonal antibody, epidermal growth factor and transferrin.
5. An immunotoxin of claim 4, wherein said binding moiety is a monoclonal antibody UCHT1.
6. An immunotoxin of claim 4, wherein said binding moiety is epidermal growth factor.
7. A method for preparing an immunotoxin comprising:
i) linking a mutant diphtheria toxin, wherein said mutant diphtheria toxin consists of an A chain polypeptide and a B chain polypeptide, wherein said B chain polypeptide is inactivated as to membrane binding but retains full A-chain translocating activity, to an ester, to form a linked toxin;
ii) Reducing a binding moiety wherein said binding moiety is one which specifically binds to a receptor;
iii) mixing the linked toxin with the reduced binding moiety and incubating the mixture to conjugate the binding moiety to the mutant toxin to form an immunotoxin; and iv) separating the immunotoxin from unconjugated binding moiety and linked toxin.
i) linking a mutant diphtheria toxin, wherein said mutant diphtheria toxin consists of an A chain polypeptide and a B chain polypeptide, wherein said B chain polypeptide is inactivated as to membrane binding but retains full A-chain translocating activity, to an ester, to form a linked toxin;
ii) Reducing a binding moiety wherein said binding moiety is one which specifically binds to a receptor;
iii) mixing the linked toxin with the reduced binding moiety and incubating the mixture to conjugate the binding moiety to the mutant toxin to form an immunotoxin; and iv) separating the immunotoxin from unconjugated binding moiety and linked toxin.
8. The method of claim 7, wherein said mutant diphtheria toxin contains at least one point mutation selected from the group consisting of mutation of cerine residue 508 to another amino acid, mutation of serine residue 525 to another amino acid, and mutation of proline residue 308 to another amino acid.
9. The method of claim 7, wherein said mutant diphtheria toxin contains a point mutation selected from the group consisting of mutation of serine residue 508 to phenylalanine, mutation of serine residue 525 to phenylalanine, and mutation of proline residue 308 to serine, together with mutation of serine residue 525 to phenylalanine.
10. The method of claim 7, wherein said binding moiety is selected from the group consisting of a monoclonal antibody, epidermal growth factor and transferrin.
11. The method of claim 7, wherein said binding moiety is a monoclonal antibody UCHT1.
12. The method of claim 7, wherein said binding moiety is epidermal growth factor.
13. Use of an immunotoxin according to any one of claims 1, 2, 3, 5 or 6 in preparing a composition for treatment of graft versus host disease.
14. Use of an immunotoxin according to claim 4 in preparing a composition for treatment of graft versus host disease.
15. Use of an immunotoxin according to any one of claims 1, 2, 3, 5 or 6 in preparing a composition for the selective killing of tumor cells in a mammalian host.
16. Use of an immunotoxin according to claim 4 in preparing a composition for the selective killing of tumor cells in a mammalian host.
17. The use of claim 15, wherein said tumor cells are leukemia cells.
18. The use of claim 16, wherein said tumor cells are leukemia cells.
19. A composition for treatment of graft versus host disease comprising an immunotoxin of any one of claims 1, 2, 3, 5 or 6 and a pharmaceutically acceptable diluent or excipient.
20. A composition for treatment of a tumor in a mammalian host comprising an immunotoxin of any one of claims 1, 2, 3, 5 or 6 and a pharmaceutically acceptable diluent or excipient.
21. A composition for treatment of leukemia in a mammalian host comprising an immunotoxin of any one of claims 1, 2, 3, 5 or 6 and a pharmaceutically acceptable diluent or excipient.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/301,376 US5208021A (en) | 1987-10-05 | 1989-01-25 | Method of preparing diphtheria immunotoxins |
| CA000589789A CA1338818C (en) | 1987-10-05 | 1989-02-01 | Immunotoxins |
| US07/925,417 US5352447A (en) | 1987-10-05 | 1992-08-10 | Immunotoxins for treatment of intracranial lesions and as adjunct to chemotherapy |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10517287A | 1987-10-05 | 1987-10-05 | |
| CA000589789A CA1338818C (en) | 1987-10-05 | 1989-02-01 | Immunotoxins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1338818C true CA1338818C (en) | 1996-12-31 |
Family
ID=25672419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000589789A Expired - Fee Related CA1338818C (en) | 1987-10-05 | 1989-02-01 | Immunotoxins |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1338818C (en) |
-
1989
- 1989-02-01 CA CA000589789A patent/CA1338818C/en not_active Expired - Fee Related
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