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Title:  Multimeric immunotoxins

United States Patent:  6,492,498

Issued:  December 10, 2002

Inventors:  Vallera; Daniel A. (St. Louis Park, MN); Blazar; Bruce R. (Golden Valley, MN)

Assignee:  Regents of the University of Minnesota (Minneapolis, MN)

Appl. No.:  440344

Filed:  November 15, 1999

Abstract

The invention features fusion protein monomers, multimeric immunotoxic proteins, nucleic acids encoding fusion protein monomers, vectors containing the nucleic acids, and cells containing the vectors. Also encompassed by the invention are methods of killing pathogenic cells and making multimeric immunotoxic proteins.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is based upon experiments with a homodimeric immunotoxin composed of two monomer polypeptides linked by a disulfide bridge formed through cysteine residues added recombinantly to the C termini of the monomer polypeptides. The monomer polypeptide contained: (a) a toxic domain (a portion of diphtheria toxin (DT)); (b) a targeting domain which was a single chain Fv fragment (sFv) derived from antibody specific for the .epsilon. chain of murine CD3, a molecular complex associated with the antigen-specific T cell receptor (TCR) and expressed on all T cells; and (c) a non-native cysteine residue added recombinantly to the C-terminus of the monomer polypeptide. The dimeric immunotoxin was shown to specifically target T cells in that T cells exposed, both in vitro and in vivo, to it had diminished proliferative responses while exposed B cells did not. In addition, the dimeric immunotoxin ablated lethal GVHD. Toxicity studies showed that, in the GVHD model, the therapeutic dose was at least four-fold lower than the toxic dose, thereby creating a "therapeutic window." In addition, the therapeutic dose had no effect on either renal or hepatic function.

A. Fusion Protein Monomers

The fusion protein monomers of the invention contain a targeting domain linked to a toxic domain, and a moiety by which one fusion protein monomer can be joined to another fusion protein monomer. Targeting and toxic domains are discussed in the following subsections and "coupling moieties" are described in the context of multimeric immunotoxins.

A.1 Targeting Domains

A targeting domain for use in the immunotoxins of the invention can be any polypeptide (or a functional fragment thereof) that has significant binding affinity for a target molecule on the surface of a target cell (e.g., a tumor cell or an infected cell). Thus, for example, where the molecule on the surface of the target cells is a receptor, the targeting domain will be a ligand for the receptor, and where the molecule on the surface of the target cells is a ligand, the targeting domain will be a receptor for the ligand. Targeting domains can also be functional fragments of appropriate polypeptides (see below).

The invention includes, as targeting domains, antibody fragments specific for a molecule on the surface of a target cell. Antibody fragments used as targeting domains in the immunotoxins of the invention contain the antigen combining site of an antibody molecule. The antibody fragments do not contain the whole constant region of either the heavy (H) or light (L) chain of an antibody molecule. However the antibody fragments can contain segments of the constant region of either or both the H and L chain. These constant region segments can be from the N-terminal end of the constant region or from any other part of the constant regions, e.g., the hinge region of IgG or IgA heavy chains. Where the antibody fragments contain constant region amino acid residues, they will contain not more than 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) constant region amino acid residues.

An antibody fragment for use as a targeting domain contains V regions of both H and L chains of an antibody molecule. In addition, it can contain: (a) all or some of the J regions of both or either of the H and the L chain; and (b) the D region of the H chain. In general, the antibody will contain the CDR3 amino acid residues of an antibody molecule, i.e., those amino acids encoded by nucleotides at the C-termini of the V region gene segments, and/or P or N nucleotides inserted at the junctions of either the V and J, the V and D, or the D and J region gene segments during somatic B cell gene rearrangements necessary for the generation of functional genes encoding H and L chains. The antibody fragments can contain more than one (e.g., 2, 3, 4, or 5) antigen combining site, i.e., the above-described units containing components from both a H chain and a L chain.

Preferred antibody fragments are sFv fragments containing the V and, optimally, the CDR3 regions, of H and L chains joined by a flexible linker peptide. The term V region, as used in all subsequent text, unless otherwise stated, will be understood to include V regions alone and V regions and P/N nucleotides, and/or D regions, and/or J regions. They can also optionally contain up to 20 C region amino acids. Generally, but not necessarily, the heavy chain variable region (VH) will be C-terminal of the light chain variable region (VL). Linker peptides joining VH and VL regions can be 1 to about 30, even 50, amino acids long and can contain any amino acids. In general, a relatively large proportion (e.g., 20%, 40%, 60%, 80%, 90%, or 100%) of the amino acid residues in the linker will be glycine and/or serine residues.

Antibody fragments can be specific for (i.e., will have significant binding affinity for) a molecule expressed on the surface of a target cell of interest. Thus, the antibody fragments can have specific binding affinity for molecules such as T cell surface molecules (e.g., CD3 polypeptides, CD4, CD8, CD2, CD7, cytokine or growth factor receptors (see below), or TCR), B cell surface molecules a(e.g., CD19, CD20, CD22, cytokine or growth factor receptors, or Ig molecules), molecules expressed on tumor cells (e.g., those listed above for T and B cells, as well as others known in the art, e.g., melanoma, breast (e.g., her2/neu), ovarian, or colon cancer antigens), and molecules expressed on the surface of infected target cells (e.g., viral proteins and glycoproteins).

The targeting domains can also be immunoglobulin (Ig) molecules of irrelevant specificity (or immunoglobulin molecule fragments that include or contain only an Fc portion) that can bind to an Fc receptor (FcR) on the surface of a target cell (e.g., a tumor cell).

The targeting domains can be cytokines (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, the interferons (.alpha., .beta., and .gamma.), TNF-.alpha., vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) colony stimulating factors (e.g., GM-CSF), hormones (e.g., insulin, or growth hormone), ligands for signal transduction receptors (e.g., CD40 ligand, an MHC class I molecule or fragments of an MHC molecule involved in binding to CD8, an MHC class II molecule or the fragment of an MHC class II molecule involved in binding to CD4), or ligands for adhesion receptors, e.g., ICAM-1, ICAM-2, or fibronectin or a domain (e.g., one containing one or more of the "Arg-Gly-Asp" repeats) of fibronectin involved in binding to integrin molecules. In addition a targeting domain could be Fas or Fas ligand or other death domain containing polypeptides (e.g., members of the TNF receptor family) or ligands for such polypeptides (e.g., TNF-.alpha., or TWEAK)

In addition, in certain B cell lymphomas, the specificity of the cell surface Ig molecules has been defined. Thus, where such B cell lymphoma cells are the target cells, an immunotoxin of the invention could include, as the targeting domain, the antigen or a fragment containing the relevant antigenic determinant for which the surface Ig on the lymphoma cells is specific and thus has significant binding affinity. Such a strategy can also be used to kill B cells which are involved in the pathology of an autoimmune disease (e.g., systemic lupus erythematosus (SLE) or myasthenia gravis (MG)) and which express on their surface an Ig receptor specific for an autoantigen.

Similarly, malignant T cells or autoreactive T cells expressing a TCR of known specificity can be killed with an immunotoxin protein containing, as the targeting domain, a soluble MHC (class I or class II) molecule, an active (i.e., TCR-binding) fragment of such a molecule, or a multimer (e.g., a dimer, trimer, tetramer, pentamer, or hexamer) of either the MHC molecule or the active fragment. All these MHC or MHC-derived molecules can contain, within their antigenic peptide-binding clefts, an appropriate antigenic peptide. Appropriate peptide fragments could be from collagen (in the case of RA), insulin (in IDDM), or myelin basic protein (in MS). Tetramers of MHC class I molecules containing an HIV-1-derived or an influenza virus-derived peptide have been shown to bind to CD8+ T cells of the appropriate specificity [Altman et al. (1996), Science 274:94-96; Ogg et al. (1998), Science 279:2103-2106], and corresponding MHC class II multimers would be expected to be similarly useful with CD4+ T cells. Such complexes could be produced by chemical cross-linking of purified MHC class II molecules assembled in the presence of a peptide of interest or by modification of already established recombinant techniques for the production of MHC class II molecules containing a single defined peptide [Kazono et al. (1994), Nature 369:151-154; Gauthier et al. (1998), Proc. Natl. Acad. Sci. U.S.A. 95:11828-11833]. The MHC class II molecule monomers of such multimers can be native molecules composed of full-length .alpha. and .beta. chains. Alternatively, they can be molecules containing either the extracellular domains of the .alpha. and .beta. chains or the .alpha. and .beta. chain domains that form the "walls" and "floor" of the peptide-binding cleft.

In addition, the targeting domain could be a polypeptide or functional fragment that binds to a molecule produced by or whose expression is induced by a microorganism infecting a target cell. Thus, for example, where the target cell is infected by HIV, the targeting domain could be an HIV envelope glycoprotein binding molecule such as CD4, CCR4, CCR5, or a functional fragment of any of these.

The invention also includes artificial targeting domains. Thus, for example, a targeting domain can contain one or more different polypeptides, or functional fragments thereof, that bind to a target cell of interest. Thus, for example, a given targeting domain could contain whole or subregions of both IL-2 and IL-4 molecules or both CD4 and CCR4 molecules. The subregions selected would be those involved in binding to the target cell of interest. Methods of identifying such "binding" subregions are known in the art. In addition, a particular binding domain can contain one or more (e.g., 2,3, 4, 6, 8, 10, 15, or 20) repeats of one or more (e.g., 2, 3, 4, 6, 8, 15, or 20) binding subregions of one or more (e.g., 2, 3, 4, or 6) polypeptides that bind to a target cell of interest.

The targeting domains can be polypeptides of any species, e.g., a human, non-human primate (e.g., monkey), mouse, rat, guinea pig, hamster, cow, sheep, goat, horse, pig, rabbit, dog, or cat.

The amino acid sequence of the targeting domains of the invention can be identical to the wild-type sequence of appropriate polypeptide. Alternatively, the targeting domain can contain deletions, additions, or substitutions. All that is required is that the targeting domain have at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the wild-type polypeptide to bind to the target molecule. Substitutions will preferably be conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

Particularly useful as targeting domains are those whose nucleotide sequences have been defined and made public. Indeed, the nucleotide sequences encoding the H and L chains of many appropriate antibodies have been defined and are available to the public in, for example, scientific publications or data bases accessible to the public by mail or the internet. For example, the nucleic acid sequences (and references disclosing them) encoding the following polypeptides were obtained from GenBank at the National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.: VH and VL of an antibody specific for human CD4 [Weissenhorn et al. (1992) Gene 121(2):271-278]; VH and VL of an antibody specific for human CD3 [GenBank Accession Nos. AF078547 and AF078546]; VH and VL of an antibody specific for human CD7 [Heinrich et al. (1989) J. Immunol. 143:3589-3597]; human IL-1.alpha. [Gubler et al. (1986) J. Immunol. 136(7):2492-2497]; human IL-3 [Yang et al. (1986) Cell 47(1):3-10]; human IL-4 (genomic DNA sequence) [Arai et al. (1989) J. Immunol. 142(1):274-282]; human IL-4 (cDNA sequence) [Yokota et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83(16):5894-5898]; human GM-CSF [Wong et al. (1985) Science 228(4701):81-815]; human VEGF [Weindel et al. (1992) Biochem. Biophys. Res. Comm. 183(3):1167-1174]; human EGF [Bell et al. (1986) Nucleic Acids Res. 14(21):8427-8446]; and human CD40 ligand [Graf et al. (1992) Eur. J. Immunol. 22(12):3191-3194].

However, the invention is not limited to the use of targeting domains whose nucleotide sequences are currently available. Methods of cloning nucleic acid molecules encoding polypeptides and establishing their nucleotide sequences are known in the art [e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1989) and Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N.Y., 1989)]

A.2 Toxic Domains

Toxic domains useful in the invention can be any toxic polypeptide that mediates a cytotoxic effect on a cell. Preferred toxic polypeptides include ribosome inactivating proteins, e.g., plant toxins such as an A chain toxin (e.g., ricin A chain), saporin, bryodin, gelonin, abrin, or pokeweed antiviral protein (PAP), fungal toxins such as .alpha.-sarcin, aspergillin, or restrictocin, bacterial toxins such as DT or Pseudomonas exotoxin A, or a ribonuclease such as placental ribonuclease or angiogenin. As with the targeting domains, the invention includes the use of functional fragments of any of the polypeptides. Furthermore, a particular toxic domain can include one or more (e.g., 2, 3, 4, or 6) of the toxins or functional fragments of the toxins. In addition, more than one functional fragment (e.g. 2, 3, 4, 6, 8, 10, 15, or 20) of one or more (e.g., 2, 3, 4, or 6) toxins can be included in the toxic domain. Where repeats are included, they can be immediately adjacent to each other, separated by one or more targeting fragments, or separated by a linker peptide as described above.

The amino acid sequence of the toxic domains of the invention can be identical to the wild-type sequence of appropriate polypeptide. Alternatively, the toxic domain can contain deletions, additions, or substitutions. All that is required is that the targeting domain have at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the wild-type polypeptide to kill relevant target cells. It could be desirable, for example, to delete a region in a toxic polypeptide that mediates non-specific binding to cell surfaces. Substitutions will preferably be conservative substitutions (see above).

Particularly useful as toxic domains are those toxic polypeptides whose nucleotide sequences have been defined and made public. Indeed, the nucleotide sequences encoding many of the toxic polypeptides listed above have been defined and are available to the public. For example, the nucleic acid sequences (and references disclosing them) encoding the following toxic polypeptides were obtained from GenBank at the National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.: gelonin [Nolan et al. (1993) Gene 134(2):223-227]; saporin [Fordham-Skelton et al. (1991) Mol. Gen. Genet. 229(3);460-466]; ricin A-chain [Shire et al. (1990) Gene 93:183-188]; .alpha.-sarcin [Oka et al. (1990) Nucleic Acids Res. 18(7):1897; restrictocin [Lamy et al. (1991) Mol. Microbiol. 5(7):1811-1815]; and angiogenin [Kurachi et al. (1985) Biochemistry 24(20) :5494-5499].

However, the invention is not limited to the use of toxic domains whose nucleotide sequences are currently available. Methods of cloning nucleic sequences encoding known polypeptides and establishing their nucleotide sequences are known in the art. [Maniatis et al., supra, Ausubel et al., supra].

Toxic and targeting domains can be disposed in any convenient orientation with respect to each other in the fusion proteins of the invention. Thus, the toxic domain can be N-terminal of the targeting domain or vice versa. The two domains can be immediately adjacent to each or they can be separated by a linker (see above).

Smaller fusion proteins (less than 100 amino acids long) can be conveniently synthesized by standard chemical means. In addition, fusion proteins can be produced by standard in vitro recombinant DNA techniques and in vivo recombination/genetic recombination (e.g., transgenesis), using the nucleotide sequences encoding the appropriate polypeptides or peptides. The fusion proteins can also be made by a combination of chemical and recombinant methods.

Methods well known to those skilled in the art can be used to construct expression vectors containing relevant coding sequences and appropriate transcriptional/translational control signals. See, for example, the techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y., 1989], and Ausubel et al., Current Protocols in Molecular Biology, [Green Publishing Associates and Wiley Interscience, N.Y., 1989].

Expression systems that may be used for small or large scale production of the fusion proteins of the invention include, but are not limited to, microorganisms such as bacteria (for example, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the invention; yeast (for example, Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the nucleic acid molecules of the invention (see below); insect cell systems infected with recombinant virus expression vectors (for example, baculovirus) containing the nucleic acid molecules of the invention; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (for example, Ti plasmid) containing fusion protein nucleotide sequences; or mammalian cell systems (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the adenovirus late promoter and the vaccinia virus 7.5K promoter). Also useful as host cells are primary or secondary cells obtained directly from a mammal, transfected with a plasmid vector or infected with a viral vector.

Fusion proteins of the invention also include those described above, but modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.

B. Multimeric Immunotoxins

The multimeric immunotoxins of the invention will contain two or more (e.g., three, four, five, six, or eight) of the monomeric fusion proteins described above. In a preferred embodiment, they will be dimeric. Each monomer can be identical, i.e., contain the same targeting and toxic domains and have the same amino acid sequence. Alternatively, they can be different. Thus, they can contain, for example, the same targeting domains but different toxic domains, different targeting domains but the same toxic domains, or different targeting domains and different toxic domains. Where different targeting domains are used, they will generally have significant binding affinity for either the same cell-surface molecule or for different molecules on the surface of the same cell.

The monomer fusion proteins of the invention can be linked to each by methods known in the art. For example, a terminal or internal cysteine residue on one monomer can be utilized to form a disulfide bond with a terminal or internal cysteine residue on another monomer.

Monomers can also be cross-linked using any of a number of known chemical cross linkers. Examples of such reagents are those which link two amino acid residues via a linkage that includes a "hindered" disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. In FIG. 1 is shown how one such reagent, 4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.(2-pyridyldithio)toluene (SMPT), would form such a linkage between two monomers (designated "DTsFv") utilizing a terminal lysine on one of the monomers and a terminal cysteine on the other. Heterobifunctional reagents which cross-link by a different coupling moiety on each monomer polypeptide can be particularly useful in generating, for example, dimeric immunotoxins involving two different monomers. Thus, the coupling moiety on one monomer could be a cysteine residue and on the other a lysine residue. In this way, the resulting dimers will be heterodimers rather than either homodimers or a mixture of homodimers and heterodimers. Other useful cross-linkers, which are listed in the Pierce Products catalog (1999/2000), include, without limitation, reagents which link two amino groups (e.g., N-5-Azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-Bis-maleimidobutane) an amino group and a sulfhydryl group (e.g., m-Maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-Azidosalicylamido]butylamine), and an amino group and a guanadium group that is present in the side chain of arginine (e.g., p-Azidophenyl glyoxal monohydrate).

While these cross-linking methods can involve residues ("coupling moieties") that are native to either of the domains of the monomers, they can also be used to cross-link non-native ("heterologous") residues incorporated into the polypeptide chains. While not necessarily the case, such residues will generally be amino acids (e.g., cysteine, lysine, arginine, or any N-terminal amino acid). Non-amino acid moieties include, without limitation, carbohydrates (e.g., on glycoproteins) in which, for example, vicinal diols are employed [Chamow et al. (1992) J. Biol. Chem. 267, 15916-15922]. The cross-linking agent 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), for example, can be used to cross-link a carbohydrate residue on one monomer and a sulfhydryl group on another. They can be added during, for example, chemical synthesis of a monomer or a part of the monomer. Alternatively, they can be added by standard recombinant nucleic acid techniques known in the art.

The coupling moieties can be positioned anywhere in the monomer fusion proteins, provided that the activity of the resulting immunotoxin multimer is not compromised. Thus, the linkage must not result in disruption of the structure of a targeting domain such that it can no longer bind to the cell-surface molecule for which it is specific. Furthermore, the linkage must not result in the disruption of the structure of the toxic domain such that it ablates the ability of the immunotoxin to kill its respective target cell. Using standard binding and toxicity assays known to those in the art, candidate multimeric immunotoxins employing linkages involving different residues on the monomers can be tested for their ability to bind and kill target cells of interest. Using molecular modeling techniques, it will frequently be possible to predict regions on a targeting domain or toxic domain that would be appropriate for the insertion of moieties by which inter-monomer linkages could be formed. Thus, for example, regions predicted to be on the exterior surface of a targeting domain, but unlikely to be involved in binding to a target molecule, could be useful regions in which to an insert an appropriate moiety in the targeting domain. Similarly, regions predicted to be on exterior surface of a toxic domain, but unlikely to be involved in the toxic activity, could be useful regions in which to an insert an appropriate moiety in the toxic domain.

The coupling moieties will preferably be at the termini (C or N) of the monomers. They can be, as indicated above, a cysteine residue on each monomer, or a cysteine on one and a lysine on the other. Where they are two cysteine residues, cross-linking can be effected by, for example, exposing the monomers to oxidizing conditions.

It can be desirable in some cases to eliminate, for example, one or more native cysteine residues in a monomer in order to restrict cross-linking to only non-native moieties inserted into the monomers. A potentially troublesome cysteine could, for example, be replaced by an alanine or a tyrosine residue. This can be done by, for example, standard recombinant techniques. Naturally, these replacements should not, compromize the activity of the resulting multimeric immunotoxin (see above).

It is understood that in immunotoxins containing more than two monomers, at least one of the monomers will have more than one cross-linking moiety. Such multimers can be constructed "sequentially", such that each monomer is joined to the next such that the terminal two monomers in the chain only have one residue involved in an inter-monomer bond while the "internal" monomers each have two moieties involved in inter-monomer bonds. Alternatively, one monomer could be linked to multiple (e.g., 2, 3, 4, or 5) other monomers. In these cases the first monomer would be required to contain multiple native and/or non-native cross-linkable moieties. A multimeric immunotoxin could also be formed by a combination of these two types of structure.

C. Nucleic Acids Encoding Fusion Proteins

The invention includes nucleic acids (e.g., cDNA, genomic DNA, synthetic DNA, or RNA) encoding any of the above fusion proteins of the invention. The nucleic acids can be double-stranded or single-stranded (i.e., a sense or an antisense strand). A RNA molecule can be produced by in vitro transcription. The nucleic acid molecules are not limited to coding sequences and can include some or all of the non-coding sequences that lie upstream or downstream of a particular coding sequence. The nucleic acids can have nucleotide sequences that are identical to those of nucleic acids encoding the wild-type targeting and toxic domains. Alternatively, they can contain codons other than wild-type codons but which, due to the degeneracy of the genetic code, encode toxic or targeting domains with amino acid sequences identical to relevant wild-type polypeptides. Furthermore, the nucleic acids can encode targeting or toxic domains with any of the above described deletions, additions, or substitutions.

Generally, the nucleic acids will include "hybrid genes," containing a first portion and a second portion. The first portion will encode a targeting domain and second portion will encode a toxic domain. Between the first and second portions can be codons encoding a linker (see above). Furthermore, where required, the nucleic acids can contain one or more codons encoding a heterologous (i.e., not existing in the wild-type polypeptide) coupling moiety, e.g., cysteine or lysine.

The coding sequences contain a leader sequence that encodes a hydrophobic signal peptide. The leader sequence is at the 5' end of the sequence encoding the fusion protein. The signal peptide is generally immediately N-terminal of the mature polypeptide (fusion protein) but can be separated from it by one or more (e.g., 2, 3, 4, 6, 8, 10, 15 or 20) amino acids, provided that the leader sequence is in frame with the nucleic acid sequence encoding the fusion protein. The signal peptide, which is generally cleaved from the fusion protein prior to secretion, directs fusion proteins into the lumen of an appropriate cell's endoplasmic reticulum (ER) during translation and the fusion proteins are then secreted, via secretory vesicles, into the environment of the cell. In this way, the producing cells remain viable since interaction of the toxin with the protein synthetic machinery in the cytosol of the cell is prevented by the membrane bilayers of the ER and secretory vesicles.

Useful leader peptides can be the native leader peptide of the relevant targeting domain (e.g., VH or VL) or a functional fragment of the native leader. Alternatively, the leader can be that of another exported polypeptide. For example, the signal peptide can have the amino acid sequence MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:1). In addition, the peptide sequence KDEL (SEQ ID NO:2) has been shown to act as a retention signal for the ER.

The invention also includes vectors containing the above nucleic acids. The vectors are preferably expression vectors. In the expression vectors of the invention, the nucleic acid sequence encoding a fusion protein of interest with an initiator methionine and, preferably, a signal sequence is "operably linked" to one or more transcriptional regulatory elements (TRE), e.g., a promoter or enhancer-promoter combination.

A promoter is a TRE composed of a region of a DNA molecule, typically within 100 nucleotide pairs upstream of the point at which transcription starts. Promoters are clustered around the initiation site for RNA polymerase II. Enhancers provide expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site. The coding sequence in the expression vector is operatively linked to a transcription terminating region. To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the peptide or polypeptide between one and about fifty nucleotides downstream (3') of the promoter. A list of promoters is provided in Table 1.

                                                TABLE 1
                                               PROMOTERS
    PROMOTER TYPE        PROMOTER ELEMENT     REFERENCES
    CONSTITUTIVE         .beta.-actin         Liu et al., Mol. Cell Biol.
     10:3432-40 (1990)
                         tubulin              Angelichio et al., Nucleic Acids
     Res. 19:5037-43 (1991)
                         CMV                  see Invitrogen
                         SV40 enhancer        see Pharmacia
                         RSV-LTR              see Invitrogen
                         Adenovirus enhancer  Inoue et al., Biochem Biophys Res
     Commun 173:1311-6 (1990)
    TISSUE-SPECIFIC
    LIVER                serum amyloid A      Li et al., Nucleic Acids Res
     20:4765-72 (1992)
                         phenylalanine        Wang et al., J Biol Chem
     269:9137-46 (1994)
                         hydroxylase
                         IGFBP-1              Babajko et al., PNAS 90:272-6
     (1993)
                         apolipoprotein B     Brooks et al., Mol Cell Biol
     14:2243-56 (1994)
                         albumin              Pinkert et al., Genes Dev
     1:268-76 (1987)
                         vitellogenin         Corthesy et al., Mol Endocrinol
     5:159-69 (1991)
                         angiotensinogen      Brasier et al., Embo J 9:3933-44
     (1990)
                         haptoglobin          Yang et al., Genomics 18:374-80
     (1993)
                         PEPCK                Short et al., Mol Cell Biol
     12:1007-20 (1992)
                         factor IX            Jallat et al., Embo J 9:3295-301
     (1990)
                         transferrin          Idzerda et al., Mol Cell Biol
     9:5154-62 (1989)
                         .beta.-fibrinogen    Dalmon et al., Mol Cell Biol
     13:1183-93 (1993)
                         kininogen            Chen et al., Mol Cell Biol
     13:6766-77 (1993)
                         CRP                  Toniatti et al., Mol Biol Med
     7:199-212 (1990)
    KIDNEY               renin                Fukamizu et al., Biochem Biophys
     Res Commun 199:183-90 (1994)
    HEART                cardiac myosin       Lee et al., J Biol Chem
     267:15875-85 (1992)
                         light
                         chain
                         cardiac troponin C   Parmacek et al., Mol Cell Biol
     12:1967-76 (1992)
                         .alpha.-cardiac myosin Gulick et al., J Biol Chem
     266:9180-5 (1991)
                         heavy chain
                         MCK                  Johnson et al., Mol Cell Biol
     9:3393-9 (1989)
                         troponin I
                         atrial natriuretic   Rockman et al., PNAS 88:8277-81
     (1991) erratum 88(21):9907
                         factor
    LUNG                 pulmonary            Glasser et al., Am J Physiol
     L349-56 (1991)
                         surfactant
                         protein SP-C
    PANCREAS/ISLET       insulin              Dandoy et al., Nucleic Acids Res
     19:4925-30 (1991); and
                                              Selden et al., Nature 321:525-8
     (1986)
                         pancreatic amylase   Osborn et al., Mol Cell Biol
     7:326-34 (1987)
    BRAIN/GLIA           GFAP                 Brenner et al., J Neurosci 1030-7
     (1994)
                         JCV                  Henson et al., J Biol Chem
     269:1046-50 (1994)
                         MBP                  Miskimins et al., Brain Res Dev
     Brain Res 65:217-21 (1992)
                         serotonin 2          Ding et al., Brain Res Mol Brain
     Res 20:181-91 (1993)
                         receptor
                         myelin PO            Monuki et al., Mech Dev 42:15-32
     (1993)
                         myelin proteolipid   Berndt et al. J Biol Chem
     267:14730-7 (1992)
                         protein
    INDUCIBLE
    A) IMMUNE            IL-2                 Thompson et al., Mol Cell Biol
     12:1043-53 (1992)
    SYSTEM/NATURAL       IL-4                 Todd et al., J Exp Med
     177:1663-74 (1993)
                         IL-6                 Libermann et al., Mol Cell Biol
     10:2327-34 (1990); and
                                              Matsusaka et al., PNAS 90:10193-7
     (1993)
                         IL-8                 Matsusaka et al., PNAS 90:10193-7
     (1993)
                         IL-10                Kim et al., J Immunol 148:3618-23
     (1992)
                         TNF-.alpha.          Drouet et al., J Immunol
     147:1694-700 (1991)
                         IL-1                 Shirakawa et al., Mol Cell Biol
     13:1332-44 (1993)
                         MIP-1                Grove et al., Mol Cell Biol
     13:5276-89 (1993)
                         IFN-.gamma.          Penix et al., J Exp Med
     178:1483-96 (1993)
                         VCAM-1               Iademarco et al., J Biol Chem
     267:16323-9 (1992)
                         ICAM-1               Voraberger et al., J Immunol
     147:2777-86 (1991)
                         ELAM-1               Whelan et al., Nucleic Acids Res
     19:2645-53 (1991)
                         tissue factor        Mackman et al., J Exp Med
     174:1517-26 (1991)
                         IFN-.beta.           Visvanathan et al., Embo J
     8:1129-38 (1989)
                         c-jun                Muegge et al., PNAS 90:7054-8
     (1993)
                         junB                 Nakajima et al., Mol Cell Biol
     13:3017-41 (1993)
                         c-fos                Morgan et al., Cell Prolif
     25:205-15 (1992)
                         iNOS                 Xie et al., J Exp Med 177:1779-84
     (1993)
                         G-CSF                Shannon et al., Growth Factors
     7:181-93 (1992)
                         GM-CSF               Miyatake et al., Mol Cell Biol
     11:5894-901 (1991)
    B) IMMMNE            NF-KB                Lenardo et al., Cell 58:227-9
     (1989)
    SYSTEM/SYNTHETIC     NF-IL6               Akira et al., Embo J 9:1897-906
     (1990)
    multiple copies of   IL6-response         Wegenka et al., Mol Cell Biol
     13:276-88 (1993)
    binding sites        element
                         CRE                  Brindle et al., Curr Opin Genet
     Dev 2:199-204 (1992)
                         AP-1                 Auwerx et al., Oncogene 7:2271-80
     (1992)
                         p91/stat             Larner et al., Science 261:1730-3
     (1993)
                         combinations of
                         multiple NF-KB and
                         NF-ILG or
                         combinations with
                         the other elements
    C) EXOGENOUS/NON-    IPTG inducible/lac   see Stratagene LacSwitch .TM., La
     Jolla, CA
    MAMMALIAN            repressor/operon
                         system
                         ecdysone-inducible   Burtis et al., Cell 61:85-99
     (1990)
                         promoter/ecdysone
                         receptor
                         Na-salicylate-       Yen, J Bacteriol 173:5328-35
     (1991)
                         inducible promoter
                         PG/regulator nahR
                         nalidixic acid       Rangwala et al., Biotechnology
     9:477-9 (1993)
                         inducible recA
                         promoter

Suitable expression vectors include, without limitation, plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses, adeno-associated viruses, lentiviruses and herpes viruses, among others.

The expression vectors of the invention containing the above described coding sequences have a variety of uses. They can be used, for example, to transfect or transduce either prokaryotic (e.g., bacteria) cells or eukaryotic cells (e.g., yeast, insect, or mammalian) cells. Such cells can then be used, for example, for large or small scale in vitro production of the relevant fusion protein by methods known in the art (see above). In essence, such methods involve culturing the cells under conditions which maximize production of the fusion proteins and isolating the fusion proteins from the cells or from the culture medium. The transduced/transfected cells can be used as targeting cells for delivery of the immunotoxic protein to a target cell by administration of the transduced/transfected cells to a subject harboring the target cell. Alternatively, the vector itself can be delivered to the subject.

The cells of the invention can, for example, be transduced with: (a) a single expression vector containing a nucleic acid sequence (e.g., a genomic DNA sequence, a cDNA sequence, or an RNA sequence) encoding one of the above fusion proteins of the invention; (b) two (or more) vectors, each containing a coding sequence encoding a different fusion protein; or (c) a single vector containing (two more) coding sequence, each encoding a different fusion protein, and each coding sequence being separately transcribed and/or translated. In cases (b) and (c), the fusion proteins encoded by the two (or more) coding regions are designed so that they associate post-translationally within the target cell by either covalent (e.g., disulfide) bonds or non-covalent (e.g., hydrophobic or ionic) interactions to form multimeric proteins of the invention.

D. Administration of a Multimeric Immunotoxic Protein

The multimeric immunotoxic proteins of the invention can be delivered to a cell population in vitro in order, for example, to deplete the population of cells expressing a cell surface molecule to which the targeting domain of an appropriate fusion protein binds. For example, the population of cells can be bone marrow cells from which it desired to remove T cells prior to use of the bone marrow cells for allogeneic or xenogeneic bone marrow transplantation. Alternatively, it may be desirable to deplete bone marrow cells of contaminating tumor cells prior to use of the bone marrow cells for bone marrow transplantation (autologous, allogeneic, or syngeneic) in a cancer patient. In such in vitro administrations, the cells to be depleted can be cultured with: (a) the isolated Age multimeric immunotoxic protein itself; (b) one or more expression vectors encoding one or more fusion proteins capable of associating to form a multimeric immunotoxic protein; or (c) cells transduced or transfected with one or more expression vectors encoding one or more fusion proteins which associate to form a multimeric immunotoxic protein. The mixture is cultured to allow for production of the immunotoxin (where the vector or genetically manipulated cells are added), binding of the immunotoxin to the target cells, and killing of the target cells.

Alternatively, a multimeric immunotoxic protein can be administered as a therapeutic agent to a subject in which it is desired to eliminate a cell population expressing a cell surface molecule to which the targeting domain of the fusion protein binds. Appropriate subjects include, without limitation, transplant (e.g., bone marrow, heart, kidney, liver, pancreas, lung) recipients, those with any of a variety of tumors (e.g., hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors, genitourinary tumors, and ovarian tumors, bone tumors, vascular tissue tumors), those with any of a variety of autoimmune diseases (e.g., RA, IDDM, MS, MG, or SLE), or those with an infectious disease involving an intracellular microorganism (e.g., Mycobacterium tuberculosis, Salmonella, influenza virus, measles virus, hepatitis C virus, human immunodeficiency virus, and Plasmodium falciparum). In transplant recipients, the multimeric immunotoxic protein is delivered, for example, to T cells, thereby resulting in the death of a substantial number, if not all, of the T cells. In the case of a hematopoietic (e.g., bone marrow) cell transplant, the treatment can diminish or abrogate both host-versus-graft rejection and GVHD. Delivery of an appropriate multimeric immunotoxic protein to tumor cells can result in the death of a substantial number, if not all, of the tumor cells. In the case of infection, the multimeric immunotoxic protein is delivered to the infected cells, thereby resulting in the death of a substantial number of, in not all, the cells and thus a substantial decrease in the number of, if not total elimination of, the microorganisms. In autoimmune diseases, the multimeric immunotoxic protein can contain a targeting domain directed at the T cells (CD4+ and/or CD8+) and/or B cells capable of producing antibodies that are involved in the tissue destructive immune responses of the diseases.

Subjects receiving such treatment can be any mammal, e.g., a human (e.g., a human cancer patient), a non-human primate (e.g., a chimpanzee, a baboon, or a rhesus monkey), a horse, a pig, a sheep, a goat, a bovine animal (e.g., a cow or a bull), a dog, a cat, a rabbit, a rat, a hamster, a guinea pig, or a mouse.

The therapeutic methods of the invention fall into 2 basic classes, i.e., those using in vivo approaches and those using ex vivo approaches.

D. 1 In Vivo Approaches

In one in vivo approach, the multimeric immunotoxic protein itself is administered to the subject. Generally, the immunotoxins of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. They are preferably delivered directly to an appropriate tissue, e.g., lymphoid tissue such as spleen, lymph nodes, or gut-associated lymphoid tissue in which an immune response (as, for example, in GVHD or an autoimmune disease) is occurring. The dosage required depends on the choice of the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 .mu.g/kg. Wide variations in the needed dosage are to be expected in view of the variety of multimeric immunotoxic proteins available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of the polypeptide in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.

In another in vivo approach, an expression vector containing one or more coding sequences encoding one or more fusion proteins of the invention, each coding sequence being separately transcribed, can be delivered to an appropriate cell of the subject. Alternatively, more than one expression vector, each containing a coding sequence encoding a different fusion protein, can be delivered to the appropriate cell. As the latter process would require that each expression vector be incorporated into a cell of interest, the approach using a single vector containing one or more coding sequences will be more efficient. The fusion proteins are designed such that, after translation, the fusion proteins will be multimerized by normal physiological mechanisms within the cell. Thus, for example, the fusion proteins can be linked by the formation of inter-fusion protein disulfide bonds or by non-covalent hydrophobic interactions between two or more fusion proteins.

Expression vectors and genetic constructs can be any of those described above. Expression vectors can be administered systemically to a subject. However, expression of the coding sequence will preferably be directed to a tissue or organ of the subject containing the target cells. For example, expression can be directed to a transplanted tissue or cell (e.g., a hematopoietic cell). An appropriate expression vector can, for example, be delivered directly to a tumor or, at the time of surgery, to tissues in the region of the body of the subject from which the tumor was surgically removed. Similarly, expression vectors can be delivered directly to the site of an infection or an autoimmune attack, e.g., joints in RA or the pancreas in IDDM.

It is not required that expression of the fusion protein be directed to the target cell itself. Indeed, expression will preferably not be by the target cell alone since, in this case, killing of the target cells by the multimeric immunotoxic proteins would result in the depletion of the source of the multimeric immunotoxic protein.

Delivery of the expression vectors can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles approximately 1-10 .mu.m in diameter can be used. The expression vector is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the expression vector. Once released, the expression vector is expressed within the cell. A second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of expression vector that is taken up by cells only upon release from the micro-particle through biodegradation. These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 .mu.m and preferably larger than 20 .mu.m). Microparticles useful for nucleic acid delivery, methods for making them, and methods of use are described in greater detail in U.S. Pat. No. 5,783,567, incorporated herein by reference in its entirety.

Another way to achieve uptake of vectors is through the use of liposomes, prepared by standard methods. The vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies. Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells [Cristiano et al. (1995), J. Mol. Med. 73:479]. Alternatively, tissue specific targeting can be achieved by the use of tissue-specific TRE. A variety of tissue specific TRE and relevant references are listed in Table 1.

Expression vectors can be administered in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles suitable for administration to a mammalian subject such as, for example, a human patient, e.g., physiological saline. A therapeutically effective amount is an amount of the expression vector which is capable of producing a medically desirable result in a treated mammal, e.g., a human patient. As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for administration of an expression vector is from approximately 106to 1012 copies of the expression vector. This dose can be repeatedly administered, as needed. Routes of administration include, without limitation, intramuscular, intravenous, subcutaneous, intraperitoneal, intrarectal, intravaginal, intranasal, intragastric, intratracheal, or intrapulmonary routes. In addition, administration can be oral or transdermal, employing a penetrant such as a bile salt, a fusidic acid or another detergent. The injections can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, or 10-fold).

D.2 Ex Vivo Approaches

An ex vivo strategy can involve transfecting or transducing targeting cells obtained from the subject with an expression vector containing one or more coding sequence encoding one or more fusion proteins. For the reasons given above, where the multimeric immunotoxic protein contains more than one species of fusion protein, it is preferred to use a single vector encoding each fusion protein. The transfected or transduced targeting cells are then returned to the subject, either at the site of the disease or systemically. Cells for use in these ex vivo methods can be any of a wide range of types including, without limitation, fibroblasts, bone marrow cells, macrophages, monocytes, dendritic cells, epithelial cells, endothelial cells, keratinocytes, or muscle cells which act as a source of the fusion protein for as long as they survive in the subject.

The ex vivo methods include the steps of harvesting cells from a subject, culturing the cells, transfecting or transducing them with one or more expression vectors, and maintaining the cells under conditions suitable for expression of the fusion protein(s). Expression vectors and genetic constructs can be any of those described above. These methods are known in the art of molecular biology. The transfection or transduction step is accomplished by any standard means used for ex vivo gene therapy, including calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced are optionally selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells may then be lethally irradiated (if desired) and injected or implanted into the patient.

While it is preferred that the cells to be used for the ex vivo methods be autologous (i.e., obtained from the subject to which they are being administered following genetic manipulation), it is understood that they need not be autologous.

These methods of the invention can be applied to any of the diseases and species listed here. Methods to test whether a multimeric immunotoxic protein is therapeutic for a particular disease can be by methods known in the art. Where a therapeutic effect is being tested, a test population of subjects displaying signs or symptoms of the disease (e.g., cancer or RA patients or experimental animals) is treated with a test multimeric immunotoxic protein, using any of the above described strategies. A control population, also displaying signs or symptoms of the disease, is treated, using the same methodology, with a placebo. Disappearance or a decrease of the disease signs or symptoms in the test subject indicates that the multimeric immunotoxic protein is an effective therapeutic agent.

Claim 1 of 28 Claims

What is claim is:

1. A fusion protein molecule comprising a toxic domain, a targeting domain, and at least one heterologous coupling moiety, wherein cysteine residues forming disulfide bonds within said fusion protein are:

(i) cysteine residues native to the toxic domain and form disulfide bonds within the toxic domain; or

(ii) cysteine residues native to the targeting domain and form disulfide bonds within the targeting domain, and

wherein the at least one heterologous coupling moiety is a moiety through which a second fusion protein molecule can be bound to the fusion protein molecule.
 


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