Title:  Artificial proteoglycans

United States Patent:  6,559,287

Issued:  May 6, 2003

Inventors:  Bennett; Kelly L. (Skillman, NJ); Wolff; Edith A. (Plainsboro, NJ); Aruffo; Alejandro A. (Belle Mead, NJ); Greenfield; Brad W. (Edmonds, WA)

Assignee:  Bristol-Myers Squibb Co. (Princeton, NJ)

Appl. No.:  235230

Filed:  January 21, 1999

Novel articifial proteoglycans containing a GAG assembly site and a control sequence required for assembly, method for enhancing the biological activity of a glycosaminoglycan binding protein using artificial proteoglycans, DNA constructs of artificial proteoglycans. The artificial proteoglycans of the present invention are useful for preparations of adjuvants for vaccination, for targeting of chemokines to non-immunogenic tumor cells to enhance cellular anti-tumor response, for preparations designed to help promote wound healing, and for treatment of immunological disorders,including rheumatoid arthritis, asthma, chronic obstructive pulmonary disorder, Lupus, inflammatory bowel disease, psoriasis, osteoarthritis, and HIV infection.

DETAILED DESCRIPTION OF THE INVENTION

The important role which proteoglycans play in regulating the function of HS-binding growth factors and chemokines has long been established. The artificial proteoglycans of the present invention can be used to target proteoglycans to a given site and thereby cause the local accumulation of GAG-binding proteins. Thus, the artifical proteoglycans of the invention are useful in preparations of adjuvants for vaccination, in the targeting of chemokines to non-immunogenic tumor cells to enhance cellular anti-tumor response, in preparations designed to help promote wound healing and for treatment of immunological disorders including rheumatoid arthritis, asthma, chronic obstructive pulmonary disorder, Lupus, inflammatory bowel disease, psoriasis, osteoarthritis, and HIV infection._In addition, the artifical proteoglycans of the invention can enhance the half life of non-GAG binding growth factors. Therefore, in the method of the invention for enhancing the biological activity of GAG binding proteins, said biological activity can be anti-tumor activity, vaccine adjuvant activity, wound healing, growth, and the like.

It is known that proteoglycans that are modified with GAGs contain the minimal assembly site sequence SG within an appropriate tertiary structure. The SG assembly site is the point at which the GAG is added to the proteoglycan. Proteoglycans that are modified with GAGs have a control sequence. We define control sequence as the sequence that defines the tertiary structure and allows for GAG assembly. In the case of HS, the control sequence includes a specific sequence that directs HS assembly. In the case of CS, the control sequence is believed to be just that which defines the tertiary structure (LindahI, U., Lidholt, K., Spillman, D., and Kjellan, L. (1994) Thrombosis Research 75, 1-32). From the data available in Zhang et. al., (1995) J. Biol. Chem. 270, 27127-27135, we have deduced that when a proteoglycan is modified with CS there is an area of 24 amino acids surrounding the assembly site in which is found the control sequence. The 24 amino acids includes the amino acids of the assembly site. We have also deduced that when a proteoglycan is modified with both CS and HS, there is an area of 24 amino acids surrounding the assembly site in which is found the control sequence, and the control sequence comprises at least three acidic amino acids and at least one hydrophobic amino acid.

It is known that CD44 isoforms containing variably spliced exon V3 are modified with HS and with CS at an assembly site. CD44 V3 contains the SG assembly site and a control sequence. The CD44 V3 control sequence defines an appropriate tertiary structure and includes a specific sequence, IDDEDFI, which we have identified. CD44 isoforms containing exon E5 are modified with CS. CD44 E5 contains the SG assembly site and a control sequence that defines an appropriate tertiary structure.

Examples of GAG binding proteins whose activity is enhanced by artificial proteoglycans of the invention include growth factors, chemokines, cytokines, enzymes, adhesion molecules, and the like. Examples of growth factors include b-FGF, a-FGF, AR, HB-EGF, TGF.beta. and the like. Examples of chemokines include RANTES, PF4, MIP-1.beta., and the like. Examples of cytokines include IL-8, GM-CSF, and the like. Examples of enzymes include Lipoprotein lipase, elastase, superoxide dismutase, and the like. Examples of adhesion molecules include laminin, thrombospondin, tenascin, and the like.

The GAG binding proteins which activity is enhanced by the artificial proteoglycans of the invention can be endogenous in the treated subject, or can be administered separately. If administered separately, the GAG binding protein can be administered before, after, or concurrent with the artificial proteoglycan.

The artificial proteoglycans of the invention have at least one glycosoaminoglycan assembly site. The glycosoaminoglycan assembly site comprises the sequence SG. The glycosoaminoglycan assembly site optionally contains multiple SG sequences such as SGSG (SEQ.ID.NO.:30), SGSGSG (SEQ.ID.NO.:31), and the like.

The glycosoaminoglycan assembly site is part of the first polypeptide making up the artificial proteoglycan. The first polypeptide can be adjacent to the second polypeptide, or the first polypeptide can be contained within the second polypeptide.

The first polypeptide making up the artificial proteoglycan of the present invention comprises a control sequence and a glycosaminoglycan assembly site wherein the control sequence and the glycosaminoglycan assembly site result in modification of the polypeptide with chondroitin sulfate or both chondroitin sulfate and heparan sulfate. In one embodiment of the inventions, the control sequence is contained within 24 amino acids surrounding the glycosaminoglycan assembly site. In another embodiment of the invention, the control sequence is contained within 24 amino acids surrounding the glycosaminoglycan assembly site and comprises at least three acidic amino acids and at least one hydrophobic amino acid. In the embodiments described in which the control sequence is contained within 24 amino acids surrounding the glycosaminoglycan assembly site, the control sequence is preferably contained within 11 amino acids on either side of the glycosaminoglycan assembly site. In the embodiment described in which the control sequence comprises at least three acidic amino acids and at least one hydrophobic amino acid, the possible acidic amino acids include aspartic acid and glutamic acid, and the possible hydrophobic amino acids include phenylalanine, tyrosine, leucine, isoleucine and tryptophan. In a preferred embodiment, the control sequence comprises IDDDEDFI (SEQ.ID.NO.:29).

The first polypeptide can be a wide variety of polypeptides provided that the required control sequence is present. Thus, the first polypeptide can also contain a wide variety or other sequences depending on the source of the polypeptide and the intended biological function. It is preferred that the first polypeptide is a proteoglycan of fragment thereof. Examples of such polypeptides include receptors, antibodies, antibody fragments, receptor binding ligands, and the like. Specific examples include perlecan, fibroglycan, syndecan-3, betaglycan, syndecan-1, and the like. Preferred first polypeptides include CD44 exon V3 or CD44 exon E5, or fragments thereof containing the required control sequences.

The second targeting polypeptide making up the fusion protein artificial proteoglycan can be any polypeptide or fragment thereof which is capable of binding to the desired target, referred to herein as a "targeting polypeptide". Classes of such second binding proteins include antibodies, receptors, receptor binding ligands, and the like. Specific examples of second targeting proteins include LFA-3, VCAM-1, B7, .alpha.CD3, and the like.

Specific examples of artificial proteoglycans of the invention include the constructs LFA-3/V3_wt -Rg, LFA-3/V3_E5/8aa -Rg, VCAM-1/V3_wt -Rg, VCAM-1/V3_E5/8aa -Rg, and LFA-3/E5_wt -Rg as described in the Examples section hereof.

In the therapeutic methods of the invention, the form of administration of the artificial proteoglycan and GAG binding protein (if administered) can be any form known in the pharmaceutical art. The amount of artificial proteoglycan and GAG binding protein to be administered would depend in part on the age, weight, and general condition of the patient. Typically, a patient would be closely monitored by a physician who would determine if the dosage amount or regimen of artificial proteoglycan and GAG binding protein being administered was effective and well tolerated. Artifical proteoglycans and GAG binding proteins would be administered either alone or admixed with a pharmaceutically acceptable carrier. Administration can be parenteral or enteral depending upon the dosage form and the needs of the patient.

The effective amount of the artifical proteoglycan depends upon many factors such as the intended biological effect as well as the age, weight, sex, health, etc. of the subject. For wound healing, a typical effective amount is about 1 .mu.g to about 50 mg per kg of body weight per day. For anti-tumor activity, a typical effective amount is about 1 .mu.g to about 50 mg per kg of body weight per day. When used as a vaccine adjuvant, the artificial proteoglycan typically comprises about 0.00001 to about 1 weight percent of the total vaccine composition. For treatment of immunological disorders, a typical effective amount is about 1 .mu.g to about 50 mg per kg of body weight per day. Therefore, the present invention is also directed to a method for enhancing wound healing comprising administering to a subject about 1 .mu.g to about 50 mg per kg of body weight per day of the artificial proteoglycan of the invention. Also, the present invention is directed to a method for enhancing the cellular anti-tumor response comprising administering to a subject about 1 .mu.g to about 50 mg per kg of body weight per day the artificial proteoglycan of the invention. Also, the present invention is directed to a vaccine composition comprising about 0.00001 to about 1 weight percent of the artificial proteoglycan of the invention, based on total composition weight. Also, the present invention is directed to a method for treating immunological disorders including rheumatoid arthritis, asthma, chronic obstructive pulmonary disorder, Lupus, inflammatory bowel disease, psoriasis, osteoarthritis, and HIV infection comprising administering to a subject about 1 .mu.g to about 50 mg per kg of body weight per day the artificial proteoglycan of the invention.

If GAG binding protein is to be administered, the same factors considered above for administering the artificial proteoglycan should be taken into account. In general, the dosages and amounts of GAG binding protein are the same or similar to the dosages and amounts of artificial proteoglycans recited above.

The artificial proteoglycan of the invention can be prepared using standard recombinant nucleic acid technology known in the art to prepare nucleic acid encoding the proteoglycan and expressing the proteoglycan in a suitable host cell. Preferably, the nucleic acid molecule is a DNA molecule and the nucleic acid sequence is a DNA sequence. All DNA sequences are represented herein by formulas whose left to right orientation is in the conventional direction of 5' to 3'.

It is also contemplated that the present invention encompasses modified sequences. As used in the present application, the term "modified", when referring to a nucleotide or polypeptide sequence, means a nucleotide or polypeptide sequence which differs from the wild-type sequence found in nature.

The DNA sequences of the present invention can be obtained using various methods well-known to those of ordinary skill in the art. At least three alternative principal methods may be employed:

(i) the isolation of a double-stranded DNA sequence from genomic DNA or complementary DNA (cDNA) which contains the sequence;

(2) the chemical synthesis of the DNA sequence; and

(3) the synthesis of the DNA sequence by polymerase chain reaction (PCR).

In the first approach, a genomic or cDNA library can be screened in order to identify a DNA sequence coding for all or part of the desired peptide. Various techniques can be used to screen the genomic DNA or cDNA libraries.

For example, labeled single stranded DNA probe sequences duplicating a sequence present in the target genomic DNA or cDNA coding for all or part of the desired peptide can be employed in DNA/DNA hybridization procedures carried out on cloned copies of the genomic DNA or cDNA which have been denatured to single stranded form.

A genomic DNA or cDNA library can also be screened for a genomic DNA or cDNA coding for all or part of the desired peptide using immunoblofting techniques.

In one typical screening method suitable for either immunoblotting or hybridization techniques, the genomic DNA library, which is usually contained in a vector, or cDNA library is first spread out on agar plates, and then the clones are transferred to filter membranes, for example, nitrocellulose membranes. A DNA probe can then be hybridized or an antibody can then be bound to the clones to identify those clones containing the genomic DNA or cDNA coding for all or part of the desired peptide.

In the second approach, the DNA sequences of the present invention coding for all or part of the desired peptide can be chemically synthesized. For example, the DNA sequence coding for the artificial proteoglycan can be synthesized as a series of 100 base oligonucleotides that can be sequentially ligated (via appropriate terminal restriction sites or complementary terminal sequences) so as to form the correct linear sequence of nucleotides.

In the third approach, the DNA sequences of the present invention coding for all or part of the desired peptide can be synthesized using PCR. Briefly, pairs of synthetic DNA oligonucleotides at least 15 bases in length (PCR primers) that hybridize to opposite strands of the target DNA sequence are used to enzymatically amplify the intervening region of DNA on the target sequence. Repeated cycles of heat denaturation of the template, annealing of the primers and extension of the 3'-termini of the annealed primers with a DNA polymerase results in amplification of the segment defined by the 5' ends of the PCR primers. See, White et al., Trends Genet. 5, 185-189 (1989).

The DNA sequences of the present invention coding for all or part of the desired peptides can also be modified (i.e., mutated) to prepare various mutations. Such mutations may be either degenerate, i.e., the mutation changes the amino acid sequence encoded by the mutated codon, or non-degenerate, i.e., the mutation does not change the amino acid sequence encoded by the mutated codon. These modified DNA sequences may be prepared, for example, by mutating the desired DNA sequence so that the mutation results in the deletion, substitution, insertion, inversion or addition of one or more amino acids in the encoded polypeptide using various methods known in the art. For example, the methods of site-directed mutagenesis described in Morinaga et al., Bio/Technol. 2, 636-639 (1984), Taylor et al., Nucl. Acids Res. 13, 8749-8764 (1985) and Kunkel, Proc. Natl. Acad. Sci. USA 82, 482-492 (1985) may be employed. In addition, kits for site-directed mutagenesis may be purchased from commercial vendors. For example, a kit for performing site-directed mutagenesis may be purchased from Amersham Corp. (Arlington Heights, Ill.). In addition, disruption, deletion and truncation methods as described in Sayers et al., Nucl. Acids Res. 16, 791-802 (1988) may also be employed. Both degenerate and non-degenerate mutations may be advantageous in producing or using the polypeptides of the present invention. For example, these mutations may permit higher levels of production, easier purification, or provide additional restriction endonuclease recognition sites. All such modified DNA and polypeptide molecules are included within the scope of the present invention.

The present invention further concerns expression vectors comprising a DNA sequence coding for the artificial proteoglycan. The expression vectors preferably contain all or part of one of the DNA sequences having the nucleotide sequences encoding CD44 exon V3 or CD44 exon E5. Further preferred are expression vectors comprising one or more regulatory DNA sequences operatively linked to the DNA sequence coding for the proteoglycan. As used in this context, the term "operatively linked" means that the regulatory DNA sequences are capable of directing the replication and/or the expression of the DNA sequence coding for the proteoglycan.

Expression vectors of utility in the present invention are often in the form of "plasmids", which refer to circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

Expression vectors useful in the present invention typically contain an origin of replication, a promoter located in front (i.e., upstream of) the DNA sequence encoding the proteoglycan and followed by the DNA sequence encoding the proteoglycan. The DNA sequence coding for the proteoglycan is followed by transcription termination sequences and the remaining vector. The expression vectors may also include other DNA sequences known in the art; for example, stability leader sequences which provide for stability of the expression product, secretory leader sequences which provide for secretion of the expression product, sequences which allow expression of the proteoglycan to modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium), marking sequences which are capable of providing phenotypic selection in transformed host cells, stability elements such as centromeres which provide mitotic stability to the plasmid, and sequences which provide sites for cleavage by restriction endonucleases. The characteristics of the actual expression vector used must be compatible with the host cell which is to be employed. Suitable promoters include, for example, the SV-40 promoter. It is also preferred that the expression vector include a sequence coding for a selectable marker. The selectable marker is preferably Amp^R or Tet^R. All of these materials are known in the art and are commercially available.

Suitable expression vectors containing the desired coding and control sequences may be constructed using standard recombinant DNA techniques known in the art, many of which are described in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).

The present invention additionally concerns host cells containing an expression vector which comprises a DNA sequence coding for the artificial proteoglycan of the invention. The host cells preferably contain an expression vector which comprises all or part of one of the DNA sequence having the nucleotide sequences substantially. Further preferred are host cells containing an expression vector comprising one or more regulatory DNA sequences capable of directing the replication and/or the expression of and operatively linked to a DNA sequence coding for the proteoglycan. Suitable host cells include COS cells and CHO cells (DG44).

Expression vectors may be introduced into host cells by various methods known in the art. For example, transfection of host cells with expression vectors can be carried out by the polyethylene glycol mediated protoplast transformation method. However, other methods for introducing expression vectors into host cells, for example, electroporation, biolistic injection, or protoplast fusion, can also be employed.

Once an expression vector has been introduced into an appropriate host cell, the host cell may be cultured under conditions permitting expression of the desired polypeptide.

Host cells containing an expression vector which contains a DNA sequence coding for the proteoglycan may be identified by one or more of the following six general approaches: (a) DNA-DNA hybridization; (b) the presence or absence of marker gene functions; (d) assessing the level of transcription as measured by the production of proteoglycan mRNA transcripts in the host cell; (d) detection of the gene product immunologically; (e) colorimetric detection; and (f) enzyme assay, (d) being the preferred method of identification.

In the first approach, the presence of a DNA sequence coding for the proteoglycan can be detected by DNA-DNA or RNA-DNA hybridization using probes complementary to the DNA sequence.

In the second approach, the recombinant expression vector host system can be identified and selected based upon the presence or absence of certain marker gene functions (e.g., dihydorfolate reductase (mehotrexate is the selection component), etc.). A marker gene can be placed in the same plasmid as the DNA sequence coding for the proteoglycan under the regulation of the same or a different promoter used to regulate the proteoglycan coding sequence. Expression of the marker gene in response to induction or selection indicates the presence of the entire recombinant expression vector which carries the DNA sequence coding for the proteoglycan.

In the third approach, the production of proteoglycan mRNA transcripts can be assessed by hybridization assays. For example, polyadenylated RNA can be isolated and analyzed by Northern blotting or nuclease protection assay using a probe complementary to the RNA sequence. Alternatively, the total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the proteoglycan can be assessed immunologically, for example, by Western blotting.

In the fifth approach, the expression of the proteoglycan can be assessed by complementation analysis.

In the sixth approach, expression of the proteoglycan can be measured by assaying for proteoglycan activity using known methods.

The DNA sequences of expression vectors, plasmids or DNA molecules of the present invention may be determined by various methods known in the art. For example, the dideoxy chain termination method as described in Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977), or the Maxam-Gilbert method as described in Proc. Natl. Acad. Sci. USA 74, 560-564 (1977) may be employed.

It should, of course, be understood that not all expression vectors and DNA regulatory sequences will function equally well to express the DNA sequences of the present invention. Neither will all host cells function equally well with the same expression system. However, one of ordinary skill in the art may make a selection among expression vectors, DNA regulatory sequences, and host cells using the guidance provided herein without undue experimentation and without departing from the scope of the present invention.

The present invention further concerns a method for producing the artificial proteoglycan comprising culturing a host cell containing an expression vector capable of expressing the proteoglycan. Preferably the expression vector is pD18.

The present invention is also directed to the artificial proteoglycan. All amino acid residues identified herein are in the natural L-configuration. In keeping with standard polypeptide nomenclature, J. Biol. Chem. 243, 3557-3559 (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence:

All amino acid sequences are represented herein by formulas whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus.

It is preferred that the artificial proteoglycan of the invention be obtained by production in eukaryotic host cells expressing a DNA sequence coding for the proteoglycan. For example, the DNA sequence of CD44 exon V3 may be synthesized using PCR as described above and inserted into a suitable expression vector, which in turn may be used to transform a suitable host cell. The recombinant host cell may then be cultured to produce the proteoglycan modified with, in the case of CD44 V3, HS and CS. Techniques for the production of polypeptides by these means are known in the art, and are described herein.

The polypeptides produced in this manner may then be isolated and purified to some degree using various protein purification techniques. For example, chromatographic procedures such as ion exchange chromatography, gel filtration chromatography and immunoaffinity chromatography may be employed.

The polypeptides of the present invention have been defined by means of determined DNA and deduced amino acid sequencing. Due to the degeneracy nature of the genetic code, which results from there being more than one codon for most of the amino acid residues and stop signals, other DNA sequences which encode the same amino acid may be used for the production of the polypeptides of the present invention. In addition, it will be understood that allelic variations of these DNA and amino acid sequences naturally exist, or may be intentionally introduced using methods known in the art. These variations may be demonstrated by one or more amino acid differences in the overall sequence, or by deletions, substitutions, insertions, inversions or additions of one or more amino acids in said sequence. Such amino acid substitutions may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphiphatic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, tyrosine. Other contemplated variations include salts and esters of the aforementioned polypeptides, as well as precursors of the aforementioned polypeptides, for example, precursors having N-terminal substituents such as methionine, N-formylmethionine used and leader sequences. All such variations are included within the scope of the present invention.

Claim 1 of 1 Claim

What we claim is:

1. An artificial proteoglycan which is LFA-3/V3_wt -Rg.

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