CA2095729A1 - Soluble mannose receptor peptides - Google Patents

Abstract

Purified soluble recombinant peptides derived from an extracellular portion of the mannose receptor protein and fragments thereof, containing one or more carbohydrate recognition domains; nucleic acid producing these fragments, and vectors and cells including such nucleic acid are disclosed. The peptides are useful for treatment of disease.

Description

~ 92/07579 2 ~ ~ ~ 7 2 9 PCT/~S91/08320 SOLUBLE
MANNOSE RECEPTOR PEPTIDES
~ac~around of the Invention This invention relates to the general field of anti-microbial and anti-viral compounds, including diagnostic compounds, as well as to methods and reagents for making and using the compounds.
It is known to use as anti-microbial agents compounds that interfere with the metabolic processes of the infective cell. For example, antibacterial agents of the sulfonamide class, as structural analogs of p-aminobenzoic acid, block purine nucleotide synthesis in susceptible microorganisms, while penicillin prevents the completion of the final stages of cell wall biosynthesis.
A number of antiviral agents such as AZT and suramin achieve their effect by targeting the uniquely retroviral enzyme reverse transcriptase. AZT has been approved for treatment of patients with the Acquired Immune Deficiency Syndrome (AIDS), caused by the Human Immunodeficiency Virus Type 1 (HIV-1). Another anti-viral agent, the polyanionic compound dextran sulfate, blocks binding of virions to target cells. The soluble mannose-binding protein prevents infection of H9 lymphoblasts by HIV-1 by binding to the high mannose glycans expressed on the envelope glycoprotein of the retrovirus (Ezekowitz et al., J. Exp. Med. 169:185-196, 1989).
Summarv In one aspect, the invention features a soluble recombinant peptide comprising at least one (and preferably two, three or more) carbohydrate recognition domains derived from an extracellular portion of mannose receptor protein (MRP). The peptide is capable of specifically targeting cells expressing mannose, N-W092/07579 ~ 09 5 7 ~ 9 PCT/~S91/083 acetylglucosamine, or fucose, by virtue of those carbohydrate recognition domain(s) (CRD). For example, the MRP-derived carbohydrate recognition domain can specifically bind eucaryotic or procaryotic pathogenic cells (e.g., bacteria, fungi, or viruses) having exposed configurations of the specified sugar moieties on their cell wall or on the envelope glycoprotein. In addition, a peptide containing the MRP-derived CRD can specifically target cancer cells which have any exposed mannose residues as a result of abberant glycosylation. Peptides according to the invention offer a probe for such cells, or a tool for delivery of specific molecules (e.g., toxins or cell specific molecules such as the T-cell antigen, CD4) to those cells, or an in vivo marker for those cells to the immune system. The domain(s) are said to be MRP-derived in that they generally contain at least 150, or preferably 300 contiguous amino acids homologous to a sequence of one or more carbohydrate recognition - domains of the mannose receptor protein, shown in Fig. 3.
The soluble peptide lacks the transmembrane and cytoplasmic regions of MRP. By peptide is meant a chain of about ten or more amino acids, including larger polypeptides and proteins, that are useful in this invention. The peptide may be glycosylated via 0- or N-linkages. By recombinant peptide is meant a peptide thatis expressed from engineered nucleic acid, defined below.
In another aspect, the invention features engineered nucleic acid (preferably cDNA) encoding such a soluble peptide. By engineered nucleic acid is meant nucleic acid removed from its natural environment ti.e., from naturally adjacent nucleic acid) by purification or recombinant DNA methodology; the term also includes synthetic nucleic acid or cDNA. This nucleic acid may be a fragment of DNA or RNA, it may be present in a vector system (e.g., a plasmid, cosmid or phage), or it may be ~'~ 92/07579 2 ~ 9 ~ 7 2 9 P ~ /~S91/08320 within the genome of an organism. In some cases, such nucleic acid is purified and includes a homogeneous preparation of desired nucleic acid.
In preferred embodiments, the peptide and nucleic acid encoding it are further characterized by at least 75% identity at the amino acid level to a sequence of at least 150, and preferably 300, contiguous amino acids of one or more carbohydrate recognition domains of mannose receptor protein most preferably the peptide includes the entire extracellular region of mannose receptor protein.
In other preferred embodiments, the nucleic acid substantially corresponds to at least 450 contiguous bases of the nucleic acid encoding the soluble extracellular fragment of mannose receptor protein, deposited in the ATCC as ATCC No. 68430 and described herein as nucleotides 1-4212 of SEQ ID N0: 1; and the nucleic acid is ligated to nucleic acid encoding the toxic part of a toxin molecule (e.g., AZT, ricin, or cholera toxin), or to nucleic acid encoding a peptide capable of fixing complement. The hybrid peptides encoded by such ligated nucleic acid are especially useful for causing an effector molecule to be targeted to an undesired cell or other organism, such as a virus.
The peptides described above, and antibodies to those peptides, may be used in therapeutic or diagnostic agents. Preferably the peptide is purified, that is, the peptide is substantially separated from contaminating peptides. Most preferably it is provided as a homogenous preparation admixed in a carrier substance suitable for therapeutic use. By therapeutic agent is meant a substance useful for the treatment of a disease or disorder; by diagnostic agent is meant a substance relating to the detection of a disease or disorder.
In yet other aspects, the invention features methods for treating an animal, e.g., a human, infected W O 92/07579 PC~r/~S91/0832~ -20957~

with a bacterium, fungus, or virus. (By bacterium, fungus, or virus is meant to include any type of undesired cell or other organism that is capable of causing an infection.) One such method includes providing and administering a therapeutically effective amount of a therapeutic agent or peptide including a soluble extracellular portion of mannose receptor protein capable of specifically targeting cells expressing mannose, N-acetylglucosamine, or fucose. The therapeutic agent or peptide causes direct inhibition of growth of the infective organism, or causes host defensive cells, e.g., macrophages, to be attracted to the pathogenic organisms which are thereby inactivated. Such inactivation may be aided by the presence of complement which is fixed by the peptide. A therapeutically effective amount is that quantity which produces a significant physiological effect in the patient and is recognized by those of ordinary skill in the art to depend upon the size and weight of the animal as well as other well known factors.
In preferred embodiments, the peptide is a therapeutically effective fragment of the soluble extracellular portion of mannose receptor protein; the peptide is able to inhibit (e.g., reduce or prevent) growth of, or infection by, the bacterium, fungus, or virus, and is a peptide as described above. Most preferably, the animal is human; the infection is one that results in a bacteremia or local bacterial infection, parasitic infection, or fungal colonization, and the route of administration is either intravenous, intramuscular, oral, or local, e.g., in the form of a powder, or lotion, preferably at 5-100 ~g/ml, more preferably at 25 ~g/ml; or the virus is HIV or a related virus, and the peptide lowers the rate of infection of eucaryotic cells by the virus; the protein or peptide is W ~ 92/07579 PC~r/~S91/08320 2~95729 provided at 1-500 ~g/ml (preferably 100-150 ~g/ml) final concentration in human serum or tissue. Alternatively, lipid vesicles, or lyposomes, containing toxins or antibiotics are coated with the peptide and administered directly to the patient. Such lyposomes will be targeted to the infected area by the peptide and the content of the lyposomes released, thereby specifically retarding or preventing growth of the targeted cells or organisms in the targeted area.
In a related aspect, the invention features a coated catheter, useful for long-term administration of fluids to a patient. The catheter is coated with one of the above-described peptides, e.g., by impregnating the catheter material with the peptide. The peptide lowers the rate of bacterial, fungal or viral infection of the patient through the catheter.
In another aspect, the invention features a method for diagnosing infection by a bacterium, fungus or virus.
The method includes detecting the serum level of a pathogen that expresses one of the target glycoproteins recognized by MRP, by measuring the amount of binding of a peptide according to the invention to a sample of the serum. The detected pathogen level reflects the - infection of the patient. Preferably, the method features measuring the peptide by immunologic or fluorescent techniques.
In a related aspect, the invention features a purified antibody specifically recognizing a peptide according to the invention. The antibody is preferably provided as a homogeneous preparation of a monoclonal or polyclonal antibody. The antibody is useful for purification of the extracellular portion of mannose receptor protein or peptides thereof, according to the invention, and for diagnosis of infection as disclosed above.

W092/07579 ~ og ~ 7 ~ 9 PCT/~S91/0832~-In a final aspect, the invention features a purified soluble peptide comprising the extracellular portion of mannose receptor protein, said peptide lacking the mannose receptor protein transmembrane and cytoplasmic regions.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Brief Descri~tion of the Drawinqs Fig. la shows the nucleotide base sequence and the corresponding amino acid sequence of the extracellular portion of the mannose receptor protein, described herein as nucleotides 1-4212 of SEQ ID NO: 1.
Fig. lb shows the nucleotide base sequence and the lS corresponding amino acid sequence of the transmembrane and cytoplasmic portions of the mannose receptor protein, described herein as nucleotides 1-4212 of SEQ ID NO: 2.
Fig. 2 is a schematic diagram including the functional regions of the extracellular portion of the mannose receptor protein.
Fig. 3 shows the correspondance of the amino acid sequence of the various carbohydrate recognition domains of the mannose receptor, using the single letter amino acid code.
Descri~tion of the Preferred Embodiment Soluble recombinant peptides derived from an extracellular portion of the mannose receptor that contains one or more carbohydrate recognition domains (CRDs) are able to recognize carbohydrates with a specificity comparable to that of the native membrane-bound mannose receptor. Such soluble mannose receptor peptides can be immobilized or attached to a portion of another molecule without losing effective carbohydrate recognition capacity.
.

W ~ 92/07579 P ~ /~'S91/08320 2~9~729 Exposed sugars like mannose and N-acetylglucosamine are a feature of the cell walls of many pathogens, whereas higher organisms, including humans and animals, tend to have masked internal mannose residues that are not recognized by the mannose receptor.
Therefore, soluble mannose receptor peptides according to the invention are useful in therapeutic agents in that they specifically bind mannose-rich pathogens, including bacteria, fungi, yeasts, parasites, or the envelope glycoproteins of certain viruses. Such peptides can also specifically target cancer cells having exposed mannose residues as a result of abberant glycosylation. When such soluble peptides are attached to other entities such as macrophages or-peptide portions that fix complement, or used as a tool for the delivery of specific molecules such as toxins or cell-specific agents to mannose-rich pathogens, the peptides can direct removal of such pathogens from the patient. The soluble peptidec according to the invention are also useful as probes in diagnosis.
The amino acid sequence of the mannose receptor, from which peptides according to the invention are derived, is inferred from the cDNA seguence (Figs. la and lb) and analyzed below:
Referring to Fig. 2, the first domain is comprised of 134 amino acids at the NH2 terminus. Without being bound to any theory, it appears that this cysteine-rich region is not essential to mannose targeting according to the invention, and, therefore, it may be deleted without departing from the spirit of the invention. Preferred soluble peptides according to the invention, however, include this domain.
~ he second domain spans from residues 135-188.
Without being bound to any theory, this domain appears to be related to fibronectin type II, and it may play a role W O 92/07579 ~ 0 9 ~ 7 2 9 PC~r/~S91/0832 in interaction with the extracellular matrix and contribute to the spreading and adhesion of tissue macrophages expressing the full length receptor. As with the first domain, the second domain is not essential to the practice of the invention, but preferred peptides include it.
Carbohydrate recognition domains (CRDs) comprise the remainder of the extracellular portion of the receptor. Specifically, there are eight segments related to C-type carbohydrate recognition domains (CRDs) of animal lectins reported by Drickamer, J. Biol. Chem.
263:9557-9560 (1988). These CRDs are discussed in greater detail below, and they are central to the invention.
A transmembrane region and a COOH-terminal cytoplasmic domain are truncated from the receptor in peptides according to the invention to enhance solubility and facilitate therapeutic application of such peptides.
Surprisingly, after truncation of the transmembrane and intracellular portions of the receptor, the molecule retains carbohydrate binding capacity effective for various purposes discussed elsewhere in this application.
Preferred peptides according to the invention include at least one or more, and preferably four or more, of the eight carbohydrate recognition domains (CRDs) depicted in Fig. 2. The sequences of the individual CRDs are shown in Fig. 3 with the numbering of the starting amino acid of each CRD keyed to the amino acid sequence of the entire soluble extracellular portion of the mannose receptor as shown in Fig. la, nucleotides 1-4212 of SEQ ID NO: 1.
Those skilled in the art will recognize that it is possible to vary the specific sequence of the soluble carbohydrate-targeting peptide being used, without deviating from the concept and spirit of the invention.

~ ^92/07579 PCT/~'S91/08320 g The entire extracellular portion of the mannose receptor, truncated to remove the transmembrane portion and the cytoplasmic tail of the full length receptor, is a preferred peptide, but CRD-containing fragments of the truncated receptor are also within the scope of the invention.
Not only does the invention cover CRD containing fragments of the truncated receptor, it also covers conservative mutations of the truncated receptor and its fragments. Preferably, a peptide according to the invention includes multiple (two or more and, preferably, four or more) CRDs of mannose receptor protein.
Moreover, individual CRDs can be repeated to further increase the carbohydrate binding capacity of the peptide according to the invention. Merely by way of example, and not as a limitation, the peptide can include multiple copies of one or more of the specific CRDs of the mannose receptor, shown in Fig. 3.
Isolated nucleic acid encoding a CRD of mannose rece~tor protein is useful for producing recombinant peptide fragments of the protein. In addition, the nucleic acid can be modified by standard techniques in order to express the same or modified peptides; e.g., by conservative base substitution the nucleic acid can be modified and still encode the same amino acid sequence, or the nucleic acid can be modified to encode a conservative amino acid substitution, which will preserve the tertiary structure and the distribution of charged amino acids in the peptide.
We now describe a specific cDNA clone of the extracellular portion (ectodomain) of mannose receptor protein (MRP). The clone is described not only as a specific example of the invention but also as a starting material to obtain other peptides according to the invention, using methods of producing candidate peptides W092/07579 2 0 9 ~ 7 2 9 PCT/~'S91/08~-~

and methods for screening such candidates for mannose affinity, as described below.
Example 1: Cloninq of Full Lenqth MRP and Processina of MRP cDNA to encode and express ~e~tides of the invention Sequences for probes were determined by obtaining sequence information from purified receptor. Receptor was purified from alveolar macrophages or human placenta as described (Lennartz et al., J. Biol. Chem. 262:9942, 1987).
Degenerate oligonucleotide probes were synthesized on a Dupont oligonucleotide synthesizer, purified by gel filtration, and labeled with 32p-ATP and polynucleotide kinase. Radiolabled probe was used to screen a pCDM8 placental cDNA library (gift of Dr. B. Seed, Harvard Medical School) by colony hybridization. Twenty-five positive clones were isolated by two rounds of amplification and analyzed. The longest clone (3.3kb) was found upon analysis to contain sequences encoding a number of peptides that had been derived from the placental mannose receptor (Taylor et al., J. Biol. Chem.
265:12156, 1990). This 3.3kb placental derived clone was radiolabeled and used as a probe to isolate the macrophage mannose receptor cDNAs from a 7 day macrophage cDNA library (Ezekowitz et al., J. Exp. Med., in press, December, 1990). A 750bp cDNA derived from the 5' extent - of the placental mannose receptor cDNA was utilized to isolate 5' clones from the macrophage library. A full-length cDNA was then assembled in a CDM8 expression vector.
The sequence of macrophage mannose receptor is identical to the placental form except for a C to T
polymorphism at nucleotide 2284. The initial placental clone was sequenced by double stranded sequencing using a modified T7 polymerase, Sequenase~ (U.S. Biochemical, Cleveland, Ohio) based on the Sanger chain termination ~ -92/07~79 PCT/~'S91/08320 2~9~729 method (Sanger et al., Proc. Natl. Acad. Sci., USA
74:5463, 1977). Specific oligonucleotides were synthesized and used as sequencing primers. For phage clones, 2~1 of purified stock was annealed to ~GT11 primers from each of the arms and the taq polymerase amplified product obtained after 25 cycles (94C, 30s denaturation, 55C 30s annealing, and 72C 3 minute extension) on a thermal cycler (Dorfman et al., Bio.
Techniques, 7:568, 1989), and the products were gel purified by agarose gel electrophoreisis. The purified products were digested with EcoRI, subcloned into a pUC-19 vector, and the nucleotide sequence determined as described above.
The encoded protein sequence deduced from the nucleotide sequence is shown in Figs. la and lb (SEQ ID
NO: lj. The open reading frame predicts a protein of 1438 amino acids which is consistent with the estimated molecular weight of the receptor polypeptide (150kD) after the N-linked sugars have been removed. (Lennartz et al., J. Biol. Chem. 264:2385, 1989; Taylor et al., J.
Biol. Chem. 265:12156, 1990; Ezekowitz et al., J. Exp.
Med., in press, December, 1990).
The features of the membrane bound mannose receptor protein are depicted in a schematic diagram (Fig. 2) and include (i) a typical hydrophobic signal peptide; (ii) a cys~teine rich NH2 terminal region; (iii) a fibronectin type II domain; (iv) eight carbohydrate recognition domains; (v) a hydrophobic transmembrane region; and (vi) a cytoplasmic tail. The NH2 terminal amino acid is defined by an N-terminus peptide as Leu, which is preceded by Ala-Val-Leu, a typical recognition seguence for a signal peptidase (Von Heijne, Eur. J.
Biochem. 133:17, 1983).
For encoding and expressing peptides of the invention, cDNA encoding the full length mannose receptor W092/07579 2 0 9 5 7 2 9 PCT/~S91/083?~-protein (MRP) was first derived in a CDM8 plasmid expression vector as described above. A construct of the cDNA encoding soluble mannose receptor peptide was then prepared in a CDM8 plasmid by a multiple step procedure, as follows.
- Referring to Figs. la and lb, in the first step:
an antisense primer was designed from a 3' end, at base pair 4169, to a 5' end, at base pair 4201, to contain a H~al site. The sense primer was prepared from a 5' end at base pair 3475 and encompassed the Nsil site base pair 3510. The primers were annealed to full length mannose receptor cDNA, and a 726 base pair fragment was amplified using the polymerase chain reaction technique (PCR). The full length cDNA mannose receptor in CDM8 was then digested with Nsil and Hpal which released a fragment from the unique Nsil site in the cDNA to the Hpal site in the vector, thereby removing (see Figs. la and 2) the last three amino acids of the ectodomain, the entire transmembrane region, the entire cytoplasmic domain, and some vector sequence. This fragment was replaced with the 726 bp PCR fragment, thereby creating a clone (SMR), confirmed by sequence analysis, which contained cDNA
encoding the signal peptide and the entire ectodomain of the mannose receptor (except for the last three amino acids). This clone is capable of generating a soluble mannose receptor peptide. This construct can be transfected stably or transiently into a mammalian expression system, and the soluble receptor peptide expressed is secreted into the medium.
From this plasmid, which has been deposited in the ATCC as ATCC No. 68430, a series of truncated forms of soluble mannose receptor peptide containing various numbers of carbohydrate recognition domains can be constructed by standard molecular biological techniques, either by using the polymerase chain reaction or ~'~92/07579 PCT/~S9]/08320 209~729 convenient restriction enzy~e sites to create molecules that can be secreted. Alternatively, standard molecular biological techniques may be used to isolate other nucleic acid (especially cDNA) clones encoding the extracellular portion of the mannose receptor protein by procedures analogous to those described above.
Expression vectors suitable for peptide expression also include standard bacterial, yeast, and viral expression vectors, as well as eucaryotic vectors. Those skilled in the art will realize that such vectors generally are suitable for expressing peptides of the invention.
Expression of soluble human mannose receptor peptides by these vectors and organisms can be followed using a mannan affinity column such as sepharose-~annose.
The column is first contacted with the expressed material. Peptides able to recognize and bind mannose are bound to the mannose-sepharose matrix, eluted with 50mM Tris/lOM EDTA, and identified using 8%
polyacrylamide gels (with Laemmli buffers, Nature 227:600, 1970). Those clones which produce peptides able to bind to such a column are among those useful in this invention.
USE OF THE PEPTIDES
Soluble mannose receptor peptides expressed as described above are useful for specifically targeting (or specifically recognizing) cells expressing carbohydrates such as mannose, N-acetylglucosamine, or fucose on their surface. Thus these peptides are useful in agents for diagnosing or treating infection by a wide variety of pathogenic organisms, e.g., Leishmania proamastigotes, Pneumocystis carinii, Candida albicans, Microbacteria tuberculosis (and other atypical mycobacteria), Human Immunodeficiency Virus Type 1 (HIV-1) or influenza virus.
Such agents are also useful for treating opportunistic W092/07579 2 0 9 5 7 ~ 9 PCT/~'S91/08~^~

infections such as those that arise in patients with cancer, patients undergoing chemotherapy or bone marrow transplants, or patients suffering from congenital or acquired immune deficiency diseases, such as AIDS. In addition, such agents can specifically target cancer cells having exposed mannose residues as a result of abberant glycosylation.
For non-viral pathogens, removal by host defense mechanisms is achieved by directinq attachment of a soluble mannose receptor peptide, in conjunction with the cell attachment site of a receptor such as the mannose-binding protein, to the surface of phagocytic cells, thereby enhancing the clearance of the pathogens from the circulation by causing the phagocytes to recognize the pathogens. For viruses which express mannose-rich glycoproteins, direct inactivation of the virus and viral infected cells is accomplished by attaching toxins, such as ricin, cholera, diphtheria, or pertussis, or antimetabolic drugs, such as AZT, to a therapeutic soluble mannose receptor peptide. The hybrid peptide thus formed can serve to kill or inhibit growth of the target cell, such as HIV.
To form such hybrid peptides, nucleic acid encoding such toxins can be ligated by well known techniques to nucleic acid encoding a soluble mannose receptor peptide according to the invention, and the fused nucleic acid can be expressed as a single entity to form a hybrid peptide (for example, as described by Murphy, U.S. Patent No. 4,675,383, hereby incorporated by reference). (By ligated is meant linked enzymatically or chemically to form a single nucleic acid entity.) Alternatively, the two peptides can be synthesized separately and linked chemically (for example, as described by Ross, U.S. Patent No. 4,275,000, hereby incorporated by reference).

~'-92/07579 PCT/~S91/08320 209~729 Alternatively, nucleic acid encoding a complement-fixing region, e.g., the complement-fixing region of the immunoglobulin heavy chain or of the mannose-binding protein, can be engineered by standard techniques to form a hybrid molecule with nucleic acid encoding a soluble mannose receptor protein. The expression product of such nucleic acid can be used to target cells with exposed surface carbohydrate moieties and then to interact with complement components and activate complement. Activated complement will then stimulate binding of macrophages to the targeted pathogenic cells and their subsequent ingestion by the macrophages.
Example 2: Pre~aration of a fusion ~rotein A soluble mannose receptor peptide-immunoglobulin fusion protein can be prepared by digesting cDNA encoding soluble mannose receptor peptide and inserting an oligonucleotide linker te.g., a BamH1 linker). The resulting plasmid can be digested with BamH1, and the portion encoding the entire extracellular domain can be ligated to the synthetic splice donor sequence of an immunoglobulin (e.g., human IgG1) expression plasmid (Aruffo et al., Cell 61:1303-1313, 1990). Such expression vectors contain in their 3' region the immunoglobulin heavy chain constant regions two and three, which have the capacity to fix complement. A
fusion protein expressed from such a fused cDNA sequence would contain a complement-fixing region at the 3' end of a soluble mannose receptor peptide.
In another construct, cDNA encoding an immunoglobulin signal peptide fused to the NH2 terminal region, cell-attachment domain, and complement-fixing region of the mannose-binding protein (Ezekowitz, International Patent Application No. WO 89/01519, 35 February 23, 1989) can be engineered to replace the W092/07~79 2 0`9 ~ 7 2 9 PCT/~'S91/083-^

cysteine-rich and fibronectin binding domains of a soluble mannose receptor peptide. The fusion protein expressed from such a cDNA sequence would contain a complement-fixing region in the amino terminal portion of the molecule, preceding the carbohydrate recognition domains.
Soluble mannose receptor peptides according to the invention may be administered by routine methods in pharmaceutically acceptable carrier substances, i.e., inert substances suitable for pharmaceutical use such as the dispensing of drugs or medicine. For example, they can be administered in an aerosol form to treat, e.g., Pneumocystis carinii. Alternatively, they may be administered orally or parenterally, e.g., they can be lS injected directly into the blood stream of an animal, especially humans, to a level of between 1-500 ~g/ml serum (most preferably, 100-150 ~g/ml) final concentration, and this dose repeated to maintain this level. The peptides can be administered prophylactically or after infection.
In a specific prophylactic use, soluble mannose receptor peptides may be used to coat intravenous or urethral catheters (e.g., by chemical impregnation of the catheter material with the peptide) to prevent infection in immunocompromised patients (e.g., cancer patients subjected to long term intravenous chemotherapy). Such catheters will bind infective organisms and prevent their entry into the patient.
In a specific therapeutic use, the soluble mannose receptor peptide may be applied topically in powder or lotion form (at a concentration of between 56-100 ~g/ml), for example, to treat local infections, such as bacterial infection, yeast infection, or infection with Trichophyton, which causes athlete's foot.

~ ~92/07579 2 0 9 5 7 2 9 PCT/~S91/08320 Soluble mannose receptor peptides can also be used as a diagnostic tool, e.g., for the diagnosis of fungal diseases. Fungi infecting an animal will shed a mannose-rich polysaccharide into the serum. A sample of serum S from a patient (e.g., l00~l) can be analyzed with fluorescently-labelled soluble mannose receptor peptide to observe binding to the fungal polysaccharide coat, and the degree of binding can be related to the degree of fugal infection remaining following a course of treatment. In an alternative diagnostic method, the degree of binding of the soluble mannose receptor peptide can be detected by using a labelled antibody that specifically recognizes the peptide. Appropriate subsequent treatment can be planned accordingly.
Other Embodiments As described above, the invention generally features peptides that include a MRP-derived carbohydrate recognition domain. The genetic material encoding soluble mannose receptor peptide (deposited as described above as ATCC No. 68430) can be used to generate a large number of recombinant peptides by fragmenting the full-length nucleic acid and expressing candidate fragments.
Alternatively, as described above, standard molecular biological techniques may be used to isolate other nucleic acid (especially cDNA) clones encoding soluble mannose receptor peptides. These clones can also be used to express candidate fragment peptides. As described, preferred fragments are those containing multiple CRDs.
Various assays may be used to determine whether a particular candidate peptide has carbohydrate recognition ability.
In addition to the affinity column chromatographic assay described above, another assay invokes binding and uptake of Il25-labeled mannose-BSA. Specifically,~
mannose-BSA (EY Labs, CA) is radiolabeled as described ~'092/07579 PCT/~S91/083'^
2 0 9 ~ ~ 2 9 (Ezekowitz et al., J. Exp. Med. 154:60, 1981), and binding and uptake of radiolabled ligand is performed on COS-I cells transfected with cloned cDNA encoding the candidate peptide. COS-I cells transfected with CD64 S serve as controls, and thioglycollate eliclted mouse peritoneal macrophages serve as a positive control.
Another assay utilizes antibody that specifically recognizes a soluble mannose receptor peptide. The antibody may be linked with a fluorescent tag and antibody-peptide binding identified flow cytometrically.
Alternatively, the antibody may be immobilized for 2ssay use or employed in an enzyme linked immunosorbent assay or ELIZA test.
Deposits Plasmid SMR, in CDM8 in E-coli strain MC1061/P3, was deposited on Oct. 2, 1990, with the American Type Culture Collection (ATCC) as ATCC No. 68430.
Applicant's assignee, Children's Medical Center Corporation, represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited microorganism, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant's assignee acknowledges its duty to ~'^92/07579 PCT/~iS91/08320 2~9~729 replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.

209~ 29 WO 92/07~79 PCT/~'S91/08321~

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tA~) LYCTR: 51~5 bose polr~
tS) TlPE: nuKlelc cid 4 0 tC) ST~A~DED~E# : 51r~le tD) Tt~OLOGr: l inerr tii) SEC~OE DESCRIPTIOII: 50 ID ~O. 1:

~- 92/07579 2 0 9 5 7 2 9 PCI/~S91/08320 Het Arg Leu Pro Leu Leu Leu Val Phe Ala Ser Val lle Pro Gly Ala Val Leu Leu Leu Asp Thr Arg Gln Phe Leu I le Tyr Asn Glu Asp His Lys Arg Cy5 Val Asp Ala Val Ser Pro Ser Ala Val Gln Thr Ala Ala Cys Asn Gln Asp Ala Glu Ser Gln Lys Phe Arg Trp Val Ser Glu Ser Gln lle Met Ser Val Ala Phe Lys Leu Cys Leu Gly Val Pro Ser Lys Thr Asp Trp Val Ala l le Thr Leu Tyr Ala Cys Asp Ser Lys Ser Glu Phe Gln Lys Trp Glu Cys Lys Asn Asp Thr Leu Leu Gly l le Lys Gly Glu Asp Leu Phe Phe Asn Tyr Gly Asn Arg Gln Glu Lys Asn lle Met 95 lûû 105 110 Leu Tyr Lys Gly ser Gly Leu Trp Ser Arg Trp Lys lle Tyr Gly Thr Thr Asp Asn Leu Cys Ser Arg Gly Tyr Glu Ala Het Tyr Thr Leu Leu Gly Asn Ala Asn Gly Als Thr Cys Ala Phe Pro Phe Lys Phe Glu Asn Lys Trp Tyr Ala ~sp Cys Thr Ser Ala Gly ~rg Ser ~sp Gly Trp Leu Trp Cys Gly Thr Thr Thr Asp Tyr Asp Thr Asp Lys Leu Phe Gly Tyr 4 O 175 18û 185 190 Cys Pro Leu Lys Phe Glu Gly Ser Glu Ser Leu Trp Asn Lys Asp Pro Leu Thr Ser Val Ser Tyr Gln lle Asn Ser Lys Ser Ala Leu Thr Trp WO 92/07579 ~ ~) 9 ~ 7 2 9 PCI /I,'S91/083?'`

His Gln Ala Arg Lys Ser Cys Gln Gln Gln Asn Ala Glu Leu Leu Ser I le Thr Glu l le His Glu Gln Thr Tyr Leu Thr Gly Leu Thr Ser Ser Leu Thr Ser Gly Leu Trp lle Gly Leu Asn Ser Leu Ser Phe Asn Ser 0 Gly Trp Gln Trp Ser Asp Arg Ser Pro Phe Arg Tyr Leu Asn Trp Leu Pro Gly Ser Pro Ser Ala Glu Pro Gly Lys Ser Cys Val Ser Leu Asn Pro Gly Lys Asn Ala Lys Trp Glu Asn Leu Glu Cys Val Gln Lys Leu Gly Tyr ILe Cys Lys Lys Gly Asn Thr Thr Leu Asn Ser Phe Val lle Pro Ser Glu Ser Asp Val Pro Thr llis Cys Pro Ser Gln Trp Trp Pro Tyr Ala Gly His Cys Tyr Lys ILe His Arg Asp Glu Lys Lys lle Gln Arg Asp Ala Leu Thr Thr Cys Arg Lys Glu Gly Gly Asp Leu Thr Ser lle His Thr lle Glu Glu Leu Asp Phe lle lle Ser Gln Leu Gly Tyr Glu Pro Asn Asp Glu Leu Trp lle Gly Leu Asn Asp lle Lys lle Gln liet Tyr Phe Glu Trp Ser Asp Gly Thr Pro Val Thr Phe Thr Lys Trp Leu Arg Gly Glu Pro Ser His Glu Asn Asn Arg Gln Glu Asp Cys Val Vsl ~let Lys Gly Lys Asp Gly Tyr Trp Ala Asp Arg Gly Cys Glu Trp Pro Leu Gly Tyr lle Cys Lys Met Lys Ser Arg Ser Gln Gly Pro Glu ~' 92/07579 2 0 9 ~ 7 2 9 P ~ /~'S91/08320 lle Val Glu Val Glu Lys Gly Cys Arg Lys Gly Trp Lys Lys His His Phe Tyr Cys Tyr Met lle Gly His Thr Leu Ser Thr Phe Ala Glu Ala Asn Gln Thr Cys Asn Asn Glu Asn Ala Tyr Leu Thr Thr lle Glu Asp Arg Tyr Glu Gln Ala Phe Leu Thr Ser Phe Val Gly Leu Arg Pro Glu Lys Tyr Phe Trp Thr Gly Leu Ser Asp ile Gln Thr Lys Gly Thr Phe CAG TGG ACC ATC GAG GAA GAG GTT CGG TTC ACC CAC TGG AAT TCA GAT t839 Gln Trp Thr lle Glu Glu Glu Val Arg Phe Thr His Trp Asn Ser.Asp Met Pro Gly Arg Lys Pro Gly Cys Val Ala Het Arg Thr Gly lle Ala Gly Gly Leu Trp Asp Val Leu Lys Cys Asp Glu Lys Ala Lys Phe Val Cys Lys Nis Trp Ala Glu Gly Val Thr His Pro Pro Lys Pro Thr Thr Thr Pro Glu Pro Lys Cys Pro Glu Asp Trp Gly Ala Ser Ser Arg Thr Ser Leu Cys Phe Lys Leu Tyr Ala Lys G~y Lys His Glu Lys Lys Thr Trp Phe Glu Ser Arg Asp Phe Cys Arg ALa Leu Gly Gly Asp Leu Ala Ser lle Asn Asn Lys Glu Glu Gln Gln Thr lle Trp Arg Leu lle Thr AIA Ser Gly Ser Tyr His Lys Leu Phe Trp Leu Gly Leu Thr Tyr Gly Ser Pro Ser Glu Gly Phe Thr Trp Ser Asp Gly Ser Pro Val Ser Tyr Glu Asn Trp Ala Tyr Gly Glu Pro Asn Asn Tyr Gln Asn Val Glu Tyr WO 92~07~;79 2 0 9 5 7 2 9 pcr/~s9l/o832r Cys Gly Glu Leu Lys Gly Asp Pro Thr Met Ser Trp Asn Asp lle Asn cys Glu His Leu Asn Asn Trp lle Cys Gln lle Gln Lys Gly Gln Thr 750 755 760 ~65 Pro Lys Pro Glu Pro Thr Pro Ala Pro Gln Asp Asn Pro Pro Val Thr Glu Asp Gly Trp Val I le Tyr Lys Asp Tyr Gln Tyr Tyr Phe Ser Lys Glu Lys Glu Thr Het Asp Asn Ala Arg Ala Phe Cys Lys Arg Asn Phe - 15 800 ôOS 870 Gly Asp Leu Val Ser lle Gln Ser Glu Ser Glu Lys Lys Phe Leu Trp ôlS 820 825 Lys Tyr Val Asn Arg Asn Asp Ala Gln Ser Ala Tyr Phe lle Gly Leu Leu lle Ser Leu Asp Lys Lys Phe Ala Trp Met Asp Gly Ser Lys Val Asp Tyr Val Ser Trp Ala Thr Gly Glu Pro Asn Phe Ala Asn Glu Asp Glu Asn Cys Val Thr Met Tyr Ser Asn Ser Gly Phe Trp Asn Asp lle Asn Cys Gly Tyr Pro Asn Ala Phe lle Cys Gln Arg His Asn Ser Ser lle Asn Ala Thr Thr Val Met Pro Thr Met Pro Ser Val Pro Ser Gly Cys Lys Glu Gly Trp Asn Phe Tyr Ser Asn Lys Cys Phe Lys lle Phe 93û 935 940 Gly Phe ~let Glu Glu Glu ~rg Lys Asn Trp Gln Glu Ala Arg Lys Ala Cys lle Gly Phe Gly Gly Asn Leu Val Ser lle Gln Asn Glu Lys Glu Gln Aln Phe Leu Thr Tyr His Het Lys Asp Ser Thr Phe Ser Ala Trp W ~)2/07579 2 0 9 5 72 9 PCTIUS91/08320 Thr &Iy Leu Asn Asp Val Asn Ser Glu His Thr Phe Leu Trp Thr Asp Gly Arg Gly Val His Tyr Thr Asn Trp Gly Lys Gly Tyr Pro Gly Gly Arg Arg Ser Ser Leu Ser Tyr &lu Asp Ala Asp Cys Val Val l le l le Gly Gly Ala Ser Asn Glu Ala Gly Lys Trp Met Asp Asp Thr Cys Asp Ser Lys Arg Gly Tyr lle Cys Gln Thr Arg Ser Asp Pro Ser Leu Thr Asn Pro Pro Ala Thr lle Gln Thr Asp Gly Phe Val Lys Tyr Gly Lys Ser Ser Tyr Ser Leu Met Arg Gln Lys Phe Gln Trp His Glu Ala Glu Thr Tyr Cys Lys Leu His Asn Ser Leu lle Ala Ser lle Leu Asp Pro Tyr Ser Asn Ala Phe Ala Trp Leu Gln Met Glu Thr Ser Asn Glu Arg Val Trp lle Alo Leu Asn Ser Asn Leu Thr Asp Asn Gln Tyr Thr Trp Thr Asp Lys Trp Arg Val Arg Tyr Thr Asn Trp Ala Ala Asp Glu Pro Lys Leu Lys Ser Ala Cys Val Tyr Leu Asp Leu Asp Gly Tyr Trp Lys Thr Ala His Cys Asn Glu Ser Phe Tyr Phe Leu Cys Lys Arg Ser Asp Glu lle Pro Ala Thr Glu Pro Pro Gln Leu Pro Gly Arg Cys Pro Glu Ser Asp His Thr Ala Trp lle Pro Phe His Gly His Cys Tyr Tyr lle Glu Ser Ser Tyr Thr Arg Asn Trp Gly Gln Ala Ser Leu Glu Cys Leu wo 92/o7j79 2 0 9 ~ 7 2 9 2 6 - PCI /~'S91 /0832(\

CGA ATC GGT CC TCT CTG GTT CC ATT CAA AGt CCT CCA CM TCC AtT 3903 Arg ~et Cly Scr Ser Lcu Val Ser lle Glu Ser Ala Ale Glu Ser Ser 1255 ~260 1265 TTT CTG TCA TAT CCC CTT GAG CCA CTT ~ AGT MA ACC MT TTT TCG 395' Phe Leu Ser Tyr Arg Vrl Glu Pro Leu Lys Ser Lys Ihr Asn Phe Trp ATA GGA TTG TTC.ACA AAT CTT GAA GGG ACG TGG CTG TGG ATA AAT AAC 3999 lle Gly Leu Phe lrg Asn Val Glu Gly Thr Trp Leu Trp 11~ Asn Asn 1205 1290 ~295 Ser Pro Val Ser Phe V~l Asn Trp Asn Tht Gly Asp Pro Ser Gly Clu Arg Asn Asp Cys Val Thr Leu His Ala Ser Ser Gly Phe Trp Ser Asn ATT CAC TGT TCT TCC TAC AAA GGA TAT ATT TGT AAA AGA CCl AAA ATT 4143 lle His Cys Ser Ser Tyr Lys Gly Tyr lle Cys Lys Arg Pro Lys lle 1335 13~0 1345 lle ~sp Als Lys Pro Thr His Clu Leu Leu Thr Thr Lys Al~ Asp Thr 1350 1355 13~0 Arg Ly~ het Asp Pro Ser Lrs Pro Ser Ser Asn Val Ala Gly Val V-l ATC ATT CTC ATC CTC CTC ATT TTA ACC CGT UT CGC CTT GCC GCC TAT $287 lle ll~ V~l lle Leu Leu lle Leu Thr Gly Al- Cly Leu Als Al~ Tyr TTC TTT TAT AAG AAA AGA CCT GTG CAC CTA CCT tAA CAG GU GtC TTT '335 Phe Phe Tyr Lys Lrs Arg Jlrg Val Nis Leu Pro Gln Glu Gly A(s Phe 13ff 1400 1'.~5 1~10 GAA AAC ACT CTC TAT TTT AAC AGT CAC TCA AGt CCA GCA ACT ACT CAT 43U
Clu Asn Thr Leu Tyr Phe Asn Ser Cln Ser Ser Pro Cly Thr S-r Asp 1415 1-.20 1~25 ATC AAA GAT tTC GTC GCt AAT ATT GAA CAC AAT GAA CAC TCC CTC ATC U31 l~et Lys Asp Leu Val Glr Asn lle Glu Gln Asn Glu Nis Ser V-l lle 1'.30 1~35 lU0 TAC TACCTCAATG CGATTCTGAC ATATTTCAAT TTtATAA~AT TCTMCTGAA U84 Erd ATTTAAMTT TTTAGTTCA~ ICTCATTCTT TTCTTTMAA TCAGTACTCA ATTGTACTCC 4544 TCTCTCCTIT TTTCCTTTGC CTMTTCMC AAATMTTU TTClTTTCTA CCCTCGCAAC 4~44 ATATTTTCAT MMCACGGA TMCMTCCT CATTACTACC TTTTMMTA TTTTAGATM 46~4 ATCCACAGCA CCACACCACC ACATCTAACC ATTACTCATG CGTAGCTGAT CTCAGCTTCA ~.n-.
TCTGGATTTT MGCACTCTA CAAACAATGA ACCTTCTTGG CATATTTTM CCACCTCCCA '78 AAATGTGTTA CCTATTMAT TGTMCTCAG CAAGTAGAAG ACCATTTCAA MGTCAGCTA U~
CMATTTCCT CAAGTCGCAT AMAATGTAG TCAGTTTTCT CTTTTACCAG TTTTTATTTC

W~` ~2t07579 2 0 9 5 7 2 9 PCI /~:S91 /0832() CACTCCAATT ATITAL~CT TTATTTGTAC ATGTGCAGM GAATAAGGCA GCTG.~GA~TC 49~4 TTGTT7CCCC CAAGAG~`GTT TTACAGGCTG AGTGTIGCM ATGIGTTCTT TGTCCIGTTA 5024 TATtTATATC AGGAATAtAA GGAIGIGAAA IAAAACIGTA AATTTGCAIA ACIGGAIGTA 50~4 CTIAGAIAAl GTGAAATAM CATTAAAGAC AAGGTCTATT TTT~ATA~M AAAAAAAAAA 5144

Claims (30)

WO 92/07579 PCT/US91/083?

What is claimed is:

1. A soluble recombinant peptide comprising at least one carbohydrate recognition domain, derived from an extracellular portion of mannose receptor protein, said peptide lacking the mannose receptor protein transmembrane and cytoplasmic regions, said peptide being capable of specifically targeting cells expressing mannose, N-acetylglucosamine, or fucose.

2. The peptide of claim 1 comprising a sequence with greater than 75% homology to a fragment of at least 150 contiguous amino acids of mannose receptor protein.

3. The peptide of claim 1 comprising at least 150 contiguous amino acids of mannose receptor protein.

4. The peptide of claim 1 comprising a sequence with greater than 75% homology to a fragment of at least 300 contiguous amino acids of mannose receptor protein.

5. The peptide of claim 1 comprising at least 300 contiguous amino acids of mannose receptor protein.

6. The peptide of claim 1 comprising at least one complete carbohydrate recognition domain from mannose receptor protein, shown in Fig. 3.

7. The peptide of claim 1 comprising at least two complete carbohydrate recognition domains from mannose receptor protein, shown in Fig. 3.

? 92/07579 PCT/US91/08320

8. The peptide of claim 1 comprising at least two copies of a carbohydrate recognition domain from mannose reception protein, shown in Fig. 3.

9. The peptide of claim 1 further comprising a segment capable of fixing complement.

10. The peptide of claim 9 wherein said peptide comprises a complement-fixing region of immunoglobulin heavy chain.

11. The peptide of claim 1 further comprising a cytotoxin.

12. The peptide of claim 11 wherein said cytotoxin is AZT, ricin, pertussis, or cholera toxin.

13. Engineered nucleic acid encoding the peptide of claim 1.

14. The engineered nucleic acid of claim 13 wherein said nucleic acid is cDNA.

15. A nucleic acid fragment substantially corresponding to at least 450 contiguous bases of the nucleic acid encoding the soluble extracellular fragment of mannose receptor protein (SEQ ID NO: 1), deposited in the ATCC as ATCC No. 68430, said fragment encoding a soluble peptide capable of specifically targeting cells expressing mannose, N-acetylglucosamine, or fucose.

16. Engineered nucleic acid substantially corresponding to the nucleic acid encoding the soluble WO 92/07579 PCT/US91/0832?

extracellular fragment of mannose receptor protein (SEQ ID
NO: 1), deposited in the ATCC as ATCC No. 68430.

17. An expression vector comprising the engineered nucleic acid of claim 13.

18. A recombinant cell comprising the engineered nucleic acid of claim 13.

19. A therapeutic agent comprising a therapeutically effective amount of the peptide of claim 1 administered in a pharmaceutically acceptable carrier substance.

20. The therapeutic agent of claim 19 comprising a lyposome coated with the soluble peptide.

21. A method for treating an animal infected with a bacterium, a fungus, or a virus, said method comprising the steps of providing the therapeutic agent of claim 19, and administering to the animal a therapeutically effective amount of said agent.

22. The method of claim 21 wherein said animal is human.

23. The method of claim 22 wherein said peptide is administered by application of a powder or a lotion comprising said peptide to the foot.

24. The method of claim 21 wherein said agent lowers the level of infection of eucaryotic cells by said bacterium, fungus, or virus.

25. A purified antibody specifically recognizing the peptide of claim 1.

26. A method for diagnosing, in an animal, an infection by a bacterium, a fungus, or a virus, said method comprising the steps of providing a sample of serum from the animal, contacting said serum with an effective amount of the peptide of claim 1, and measuring the amount of binding of said peptide to said serum sample.

27. The method of claim 26 wherein said animal is human and said infection is by a fungus.

28. The method of claim 26 wherein said measuring step comprises determining the amount of binding of an antibody specific for said peptide to a serum sample containing said peptide.

29. The method of claim 26 wherein said measuring step comprises fluorescence detection.

30. A purified soluble peptide comprising the extracellular portion of mannose receptor protein, said peptide lacking the mannose receptor protein transmembrane and cytoplasmic regions.