WO1988002757A1 - Hybrid proteins or polypeptides - Google Patents

Hybrid proteins or polypeptides Download PDF

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Publication number
WO1988002757A1
WO1988002757A1 PCT/AU1987/000352 AU8700352W WO8802757A1 WO 1988002757 A1 WO1988002757 A1 WO 1988002757A1 AU 8700352 W AU8700352 W AU 8700352W WO 8802757 A1 WO8802757 A1 WO 8802757A1
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hybrid
protein
epitopes
hybrid protein
polypeptide
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PCT/AU1987/000352
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French (fr)
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Christopher John Langford
Stirling John Edwards
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Saramane Pty. Ltd.;
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Publication of WO1988002757A1 publication Critical patent/WO1988002757A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/55—Vector systems having a special element relevant for transcription from bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to hybrid proteins or polypeptides which may be used as a means- of simultaneously immunising against a plurality of protein antigens, and to methods for the production thereof.
  • this invention relates to hybrid proteins or polypeptides containing multiple epitopes in the one molecule.
  • Vaccination with synthetic peptides in various forms of conjugation to carrier molecules and with a variety of adjuvant formulations have been used to elicit antibodies which are able to recognise the native protein molecule and in some cases to immunise against disease.
  • An effective vaccine against malaria may well require the simultaneous immunisation with a "cocktail" of defined antigen molecules (or peptides related to the critical epitopes). It is also likely that any large scale vaccination of human population in malaria endemic areas will also be carried out in parallel with vaccination with other defined antigen vaccines against other diseases prevalent in the region (e.g. Hepatitis B) . Accordingly, the possibility of immunising with a hybrid protein made up of a number of peptide sequences which are known to be epitopes in natural infections with malaria has been investigated. '
  • hybrid protein or polypeptide which comprises the epitopes or mimotopes of a plurality of different protein antigens.
  • hybrid means a non-naturally occurring protein or polypeptide.
  • the hybrid protein or polypeptide comprises more than one "repeat" of each epitope or mi otope in order to maximise its immunogenicity in the hybrid molecule, even if the epitope is represented only once in the native antigen molecule.
  • At least one of the epitopes or mimotopes is an epitope or mimotope of an antigen of P.falciparum.
  • the present invention also extends to the cloning of a DNA molecule that encodes the above described hybrid protein or polypeptide by incorporating the DNA molecule into a suitable vector which, in an appropriate host cell, is capable of expressing the hybrid molecule in vitro. Accordingly, in another aspect of the present invention there is provided a DNA sequence comprising a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide, said hybrid protein or polypeptide comprising the epitopes or mimotopes of a plurality of different protein antigens.
  • the present invention also extends to a recombinant DNA molecule comprising a DNA sequence as outlined above, operatively linked to an expression control sequence.
  • the invention extends further to a vector comprising a DNA sequence as outlined above, as well as to a host cell containing such a vector.
  • the hybrid polypeptide of the present invention provides a means of simultaneously immunising against a plurality of protein antigens, and accordingly the present invention also encompasses a composition for stimulating immune responses against a plurality of protein antigens which comprises a hybrid protein or polypeptide as broadly described above, togethe-r with a pharmaceutically acceptable carrier therefor, and optionally further comprising an adjuvant.
  • the invention provides a composition for stimulating immune responses against a plurality of protein antigens which comprises a virus or microorganism in association with a pharmaceutically acceptable carrier, and optionally further comprising an adjuvant, the virus or molecule having inserted therein a DNA molecule comprising a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide as broadly described above.
  • the present invention also extends to a method of stimulating immune responses which comprises administering a composition as described above.
  • the present invention provides a recombinant virus, particularly a recombinant vaccinia virus, or a composition comprising such a virus, characterised in that it includes a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide as broadly described above.
  • the virus further includes a sequence capable of being expressed as a surface or membrane-associated polypeptide segment in association with said hybrid protein or polypeptide to locate the hybrid protein or polypeptide on or at the surface of virus infected cells.
  • Figure 1 is a schematic representation of the P.falciparum malaria genes used in this study indicating the location of their tandem repeating epitopes and the construction of the hybrid S-antigen gene containing the multiple repeating elements used to express the hybrid protein using recombinant vaccinia viruses;
  • Figure 2 is a diagra matic representation of the construction of the plasmid vectors used in the cloning of the hybrid genes for expression in recombinant vaccinia viruses;
  • Figure 3 shows the sequences of the synthetic oligonucleotides used in the construction of the hybrid genes
  • Figure 4 shows the steps used in the construction of one of the hybrids containing the NANP repeat of the CS protein
  • Figure 5 shows the method of construction of hybrid genes containing multiple inserts
  • Figure 6 is a diagram showing the hybrid genes constructed and tested in recombinant vaccinia (V series) and expressed in bacteria using the glutathione S-transferase bacterial expression vector system (Sj se r ies ) ;
  • Figure 7 shows the sequence of some of the clones shown in Figure 6 to check the copy number and orientation of inserts
  • Figure 8 shows the expression of the hybrid proteins in bacteria transformed with the vaccinia virus transfection plasmids containing the hybrid genes under the control of vaccinia and bacterial promoter elements. Expression was detected using the colony immunoassay procedure with anti- ⁇ SA repeat antisera on lysates of IPTG induced bacterial colonies. Only clones V7 and VS5367 containing the MESA repeat sequence in the correct orientation gave a positive signal in this assay with the anti- ESA antiserum;
  • Figure 9 shows the expression of some of the hybrid proteins described in Figure 6 in recombinant vaccinia infected cells
  • Figure 10 shows the antibody responses of one of the rabbits immunised with recombinant vaccinia virus expressing a hybrid protein comprising five repetitive epitopes from four separate malarial antigens (pS5367.8 Figures 1 and 6) measured in an ELISA assay in which the plates were coated with synthetic peptides of the various repeating epitopes.
  • Figure 11 shows the purified glutathione S-transferase/hybrid protein fusion polypeptides produced by bacteria containing the Sj series of constructs shown in Figure 6.
  • the choice of the carrier protein The carrier protein used to express the multiple epitopes will vary according to the intended mode of presentation of antigen and the sequences to be expressed. In many cases the best carrier protein will need to be determined empirically as the sequence of the peptides being inserted will have differing effects on the stability of the resultant fusion protein.
  • One of the proteins chosen as the carrier protein in this example is a modified version of one of the malarial S-antigens.
  • the family of S-antigen molecules has to date not been implicated as potential malaria vaccine candidates primarily because the immunodominant repeat portion of these molecules is remarkably variant. These repeating structures vary enormously, not only in their amino acid composition which greatly affects their immunological properties, but in their length and copy number. These radical variations in protein structure do not affect their behaviour as secreted proteins.
  • the transmembrane anchor sequence from a mouse immunoglobulin gene has been added to the native S-antigen molecule of the FC27 isolate of P.falciparum and it has been shown that the hybrid protein is correctly presented as an integral membrane protein on the surface of eukaryotic cells (Langford et.al., (1986)).
  • This hybrid protein contains 13 copies of an 11 amino acid repeating epitope. Clearly this repeating sequence does not prevent the transport of the hybrid protein to the cell surface.
  • the present example replaces this simple repetitive sequence with other sequences in the hope that the new hybrid protein will be transported to the cell surface.
  • the mode of presentation of the recombinant antigen could be delivered using a live recombinant virus presentation system.
  • the hybrid protein could also be expressed in bacteria or eukaryotic cells and then purified for use as a vaccine.
  • This approach we have made use of a newly developed expression vector that vastly simplifies the purification of foreign proteins produced in bacteria (Australian Patent Application PI2195/87) .
  • Proteins are produced as fusion polypeptides with the soluble Schistosma iaponicum enzyme glutathione S-transferase (GST) and can then be easily purified by affinity purification on glutathione coupled to agarose beads. Pure fusion polypeptides can then be eluted from the beads using free glutathione prior to further purification for use as a vaccine.
  • GST glutathione S-transferase
  • Epitopes from other non-malarial antigens could also be included as well as mimotopes of conformation epitopes. Inserting these sequences as repetitive elements may also be desirable.
  • the observations on the immunodominance of the repeating peptide sequences of malarial antigens suggest that simply repeating these peptide sequences may dramatically increase their immunogenicity.
  • T-cell recognition sequences which, as well as being of importance in the development of T-cell responses, may also provide T-cell help for amanistic antibody responses when the recipient is exposed to intact pathogen.
  • the plasmid containing the hybrid anchored S-antigen gene described previously was modified to facilitate the cloning and immunological screening of the new hybrid proteins. These modifications included (a) The inclusion of a bacterial promoter upstream of the S-antigen gene and the vaccinia promoter. This was achieved by first removing the Clal site in the pBR322 region of the vector ⁇ GS62 ( Figure 2a) by partial digestion with Clal, Klenow "filling-in” and religation.
  • a Taql fragment from the strong hybrid acUV5/Trp bacterial promoter was inserted generating the new vector pGS62 tac (shown in figure 2b).
  • MCS multiple cloning site
  • the EcoRl insert from this insert was then cloned into the unique EcoRl site of the plasmid pGS62tac BamHI ⁇ to generate the plasmid ⁇ LK3 (shown in figure 2e) .
  • the plasmid pLK8 contains the S-antigen gene with less than one full copy of the 33bp repeating sequence and with a unique BamHl site located within this remaining partial repeat sequence. It is into this site that appropriately engineered sequences encoding new antigenic determinants can be cloned thus neatly replacing the S-antigen repeating epitope.
  • the incoming sequences in the situation described in- this example need to have BamHl "sticky ends" and to be engineered in such a way that the reading frame of the whole hybrid gene is maintained.
  • the epitope to be expressed should be repeated as many times as possible to maximise its immunogenicity even if this epitope is represented only once or a few times in the native antigen molecule.
  • the gene sequences encoding the repeating peptide sequences have been chemically synthesised as short oligonucleotides.
  • Chemical synthesis of the oligonucleotides also enables the codons to be "mammalianised” for optimal expression in recombinant vaccinia virus-infected mammalian cells. This may be of importance in the optimal expression of foreign proteins from species such as P.falciparum which exhibit a strong bias in their preferred codon usage away from that seen in mammalian cells.
  • the insert could also be derived from naturally occurring sequences as long as it fulfils the length and phase requirements described above.
  • oligonucleotide sequences were synthesised on an Applied Biosystems oligonucleotide synthesiser and purified by HPLC. These were then kinased prior to annealing and ligation in the standard way.
  • the oligonucleotides were designed so that the ends were complimentary in one orientation only, ensuring that only "head to tail” ligation of the double stranded monomers was possible.
  • the ligated fragments were then size fractionated on a low gelling temperature agarose gel and DNA molecules in the size range from 180 to 600b ⁇ were isolated and purified from the agarose.
  • Recombinant bacterial clones containing the inserted sequence were selected by colony hybridization uussiinngg tthhee ggaammmmaa - ⁇ [[ ⁇ PP]]--ATP kinased oligonucleotides used in the initial cloning
  • Plasmid DNA was isolated from the positive clones and digested with restriction enzymes to determine the clones which contained the longest inserts. A number of these were then sequenced using the double stranded DNA sequencing procedure on alkaline denatured plasmid DNA preparations to select a clone with an insert i the correct orientation and to confirm its predicted sequence ( Figure 7) . These plasmid DNA were then transfected directly into WT vaccinia virus-infected cells to produce T recombinant virus as described previously (Langford et.al. (1986)).
  • the new insert in the hybrid gene is flanked by Sau3A sites which allow the insert to be isolated and purified from the recombinant plasmid. However only one BamHl site at the 5' of the coding strand of the insert is regenerated in this hybrid (see Figure 4) .
  • the recombinant plasmid can be linearised with BamHl, phosphatase treated and ligated with the isolated Sau3A fragment. Plasmid DNA was prepared from the transformed bacteria resulting from this cloning and digested with restriction enzymes to select clones with double (or triple) inserts. These were then sequenced to determine which contained new inserts in the correct orientation and to confirm the predicted sequence.
  • hybrid genes containing 16(V6), 32(V66) and 48(V666) copies of the 12bp repeat of the CSP gene and 10 (V3) and 20 (V33) copies of the 24bp repeat of the RESA gene have been produced (see Figure 6) .
  • the inserts from all the clones constructed in ⁇ K8 and pCL4 could be isolated as Sau3A fragments and cloned into the unique BamHl site of any other construct. This allows a polymer of inserts in order to be built in as a new 3amHl site will be created following the insertion of each new insert. As the Sau3A fragment can ligate in either orientation into the BamHl site the orientation needs to be checked in each case by DNA sequencing. A number of these hybrid genes including one construct (V55367) containing inserts encoding the S-antigen, CS, RESA 3', RESA 5' and MESA repeats were cloned into recombinant vaccinia viruses. These constructs are shown in Figure 6.
  • BamHl/EcoRl fragments of the genes described in the V-series of constructs shown in Figure 6 were subcloned into the BamHl/EcoRl cloning sites at the 3' end of the E-, iaponicum glutathione S-transferase (GST) gene in the expression vector ⁇ GEX-1 (or an earlier version of the vector; pSjlO Ba ⁇ lO) as described' in Australian Patent Application No.PI 2195/87.
  • GST E-, iaponicum glutathione S-transferase
  • the GST gene is under the control of the strong, IPTG inducible trp/lac (tac) promoter (Arnann et al., 1983).
  • This expression system has been used to purify a wide range of parasite and non-parasite polypeptides, the majority of which are produced as abundant, soluble and stable fusion proteins that can be purified on glu a hione-agarose.
  • Plasmid DNAs were sequenced directly following alkaline denaturation using the double stranded sequencing procedures adapted f om those described by Chen and Seeburg (1985). Synthetic oligonucleotide primers complimentary to S-antigen sequences located 3 ' (5*-AATGGATTAATAGAAGG-3 • ) and
  • the strong hybrid bacterial promoter located upstream of the S-antigen gene in these hybrid constructs enables the expression of the hybrid protein to be checked in bacteria transformed with the plasmid DNA.
  • a number of bacterial clones containing hybrid genes with RESA 24bp repeat inserts were picked into arrays on nitrocellulose, replicated and grown in the presence of IPTG. Colonies were then lysed with SDS and SDS/chloro orm. Filters were then blocked and then probed with antibodies raised in rabbits to chemically synthesised polypeptides encoding the repeat epitopes conjugated to KLH. This was followed by I 125 protein A binding, washing and autoradiograph . The expression of the specific insert sequences could be demonstrated in bacteria for most hybrids prior to their insertion into vaccinia virus.
  • Rabbits were immunised with 10 PFU of each recombinant virus by intrader al injection on the middle of the back. Sera were collected at weekly intervals and assayed in an ELISA in which synthetic peptides of the repeating epitopes, conjugated to BSA or ⁇ -galactosidase fusion polypeptides of malarial antigens purified from bacteria, were used to coat the plates. Antibody responses specific to the sequences encoded by the inserts could be detected in all cases.
  • Protein purified from cloned S S53 was used to immunize rabbits and mice producing antibodies which were capable of recognizing each of the repeating epitopes contained in the hybrid molecule (data not shown) . These purified hybrid proteins also proved to be excellent substitutes for synthetic peptides as diagnostic reagents in ELISAs and cell proliferation assays.

Abstract

A hybrid protein or polypeptide which comprises the epitopes or mimotopes of a plurality of different protein antigens may be used to stimulate an immune response against the different antigens. A recombinant virus such as vaccinia virus, may include a nucleotide sequence capable of being expressed as such a hybrid protein or polypeptide.

Description

"HYBRID PROTEINS OR POLYPEPTIDES"
■ This invention relates to hybrid proteins or polypeptides which may be used as a means- of simultaneously immunising against a plurality of protein antigens, and to methods for the production thereof. In particular, this invention relates to hybrid proteins or polypeptides containing multiple epitopes in the one molecule.
In recent years there has been rapid progress in the characterisation of defined protein antigens which are crucial in the development of host-protective immune responses to foreign pathogens. The vast majority of B-cell epitopes which are recognised appear to be non-linear or conformational epitopes. Only in the case of a few antigens (e.g. the foot and mouth disease virus VPl 140-152 peptide and the polio virus VPl, 1-14 peptide) has it be demonstrated that a linear array of amino acid sequences constitute a functional portion of the protein which can be blocked with antibodies.
Vaccination with synthetic peptides in various forms of conjugation to carrier molecules and with a variety of adjuvant formulations have been used to elicit antibodies which are able to recognise the native protein molecule and in some cases to immunise against disease.
Recent experiments have suggested that non-linear conformational epitopes might be mimicked by synthetic linear sequences (termed "mimotopes") which might also be used as immunogens,
However there is an accumulating body of data which suggests that higher order structural features or additional sequence elements may be required in addition to the basic epitope to elicit the appropriate antibody and T-cell responses relevant to an effective immune response. This may mean that chemically synthesised peptides suitable for effective immunisation may need to be larger than first envisaged and peptide vaccines may therefore not turn out to be as economical to manufacture as was first envisaged. This may mean that although the linear peptide sequence is the target of host protective immune responses, it may need to be present in the context of a larger protein molecule produced in bacteria, yeast, recombinant viruses or other commercially viable expression system.
One of the striking features of studies to date on the protein antigens of Plasmodium falciparum has been the preponderance of tandem arrays of repetitive amino acid sequences. In the majority of cases these sequences are the immunodominant epitopes of the antigen and there are early indications that some may be the targets of host protective immune responses. These epitopes appear to be recognised as linear determinants.
An effective vaccine against malaria may well require the simultaneous immunisation with a "cocktail" of defined antigen molecules (or peptides related to the critical epitopes). It is also likely that any large scale vaccination of human population in malaria endemic areas will also be carried out in parallel with vaccination with other defined antigen vaccines against other diseases prevalent in the region (e.g. Hepatitis B) . Accordingly, the possibility of immunising with a hybrid protein made up of a number of peptide sequences which are known to be epitopes in natural infections with malaria has been investigated.'
The prevalence of repetitive peptide sequences as dominant antigenic determinants in many of the antigens of P.falciparum has provided a unique opportunity to investigate vaccination procedures involving the presentation of a plurality of short peptide sequences in the one hybrid protein molecule.
According to the present invention, there is provided a hybrid protein or polypeptide which comprises the epitopes or mimotopes of a plurality of different protein antigens.
The term "hybrid" as used herein means a non-naturally occurring protein or polypeptide.
Preferably, the hybrid protein or polypeptide comprises more than one "repeat" of each epitope or mi otope in order to maximise its immunogenicity in the hybrid molecule, even if the epitope is represented only once in the native antigen molecule.
In one particularly preferred aspect of the invention, in the hybrid protein or polypeptide broadly described above at least one of the epitopes or mimotopes is an epitope or mimotope of an antigen of P.falciparum.
The present invention also extends to the cloning of a DNA molecule that encodes the above described hybrid protein or polypeptide by incorporating the DNA molecule into a suitable vector which, in an appropriate host cell, is capable of expressing the hybrid molecule in vitro. Accordingly, in another aspect of the present invention there is provided a DNA sequence comprising a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide, said hybrid protein or polypeptide comprising the epitopes or mimotopes of a plurality of different protein antigens.
The present invention also extends to a recombinant DNA molecule comprising a DNA sequence as outlined above, operatively linked to an expression control sequence. The invention extends further to a vector comprising a DNA sequence as outlined above, as well as to a host cell containing such a vector.
As broadly described above, the hybrid polypeptide of the present invention provides a means of simultaneously immunising against a plurality of protein antigens, and accordingly the present invention also encompasses a composition for stimulating immune responses against a plurality of protein antigens which comprises a hybrid protein or polypeptide as broadly described above, togethe-r with a pharmaceutically acceptable carrier therefor, and optionally further comprising an adjuvant. In an alternative embodiment, the invention provides a composition for stimulating immune responses against a plurality of protein antigens which comprises a virus or microorganism in association with a pharmaceutically acceptable carrier, and optionally further comprising an adjuvant, the virus or molecule having inserted therein a DNA molecule comprising a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide as broadly described above.
The present invention also extends to a method of stimulating immune responses which comprises administering a composition as described above.
In one particularly preferred embodiment, the present invention provides a recombinant virus, particularly a recombinant vaccinia virus, or a composition comprising such a virus, characterised in that it includes a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide as broadly described above. Preferably also, the virus further includes a sequence capable of being expressed as a surface or membrane-associated polypeptide segment in association with said hybrid protein or polypeptide to locate the hybrid protein or polypeptide on or at the surface of virus infected cells.
The following detailed description of the simultaneous delivery of a number of potentially important epitopes of P.falciparum in one hybrid protein, is given by way of illustration of the present invention. It will be readily appreciated by those skilled in the art that the techniques described can be readily adapted to other malarial antigens or epitopes or to non-malarial linear epitopes from other protein antigens.
In the drawings:
Figure 1 is a schematic representation of the P.falciparum malaria genes used in this study indicating the location of their tandem repeating epitopes and the construction of the hybrid S-antigen gene containing the multiple repeating elements used to express the hybrid protein using recombinant vaccinia viruses;
Figure 2 is a diagra matic representation of the construction of the plasmid vectors used in the cloning of the hybrid genes for expression in recombinant vaccinia viruses;
Figure 3 shows the sequences of the synthetic oligonucleotides used in the construction of the hybrid genes;
Figure 4 shows the steps used in the construction of one of the hybrids containing the NANP repeat of the CS protein;
Figure 5 shows the method of construction of hybrid genes containing multiple inserts;
Figure 6 is a diagram showing the hybrid genes constructed and tested in recombinant vaccinia (V series) and expressed in bacteria using the glutathione S-transferase bacterial expression vector system (Sj se r ies ) ;
Figure 7 shows the sequence of some of the clones shown in Figure 6 to check the copy number and orientation of inserts;
Figure 8 shows the expression of the hybrid proteins in bacteria transformed with the vaccinia virus transfection plasmids containing the hybrid genes under the control of vaccinia and bacterial promoter elements. Expression was detected using the colony immunoassay procedure with anti- ΞSA repeat antisera on lysates of IPTG induced bacterial colonies. Only clones V7 and VS5367 containing the MESA repeat sequence in the correct orientation gave a positive signal in this assay with the anti- ESA antiserum;
Figure 9 shows the expression of some of the hybrid proteins described in Figure 6 in recombinant vaccinia infected cells;
Figure 10 shows the antibody responses of one of the rabbits immunised with recombinant vaccinia virus expressing a hybrid protein comprising five repetitive epitopes from four separate malarial antigens (pS5367.8 Figures 1 and 6) measured in an ELISA assay in which the plates were coated with synthetic peptides of the various repeating epitopes.
Figure 11 shows the purified glutathione S-transferase/hybrid protein fusion polypeptides produced by bacteria containing the Sj series of constructs shown in Figure 6.
The choice of the carrier protein The carrier protein used to express the multiple epitopes will vary according to the intended mode of presentation of antigen and the sequences to be expressed. In many cases the best carrier protein will need to be determined empirically as the sequence of the peptides being inserted will have differing effects on the stability of the resultant fusion protein.
One of the proteins chosen as the carrier protein in this example is a modified version of one of the malarial S-antigens. The family of S-antigen molecules has to date not been implicated as potential malaria vaccine candidates primarily because the immunodominant repeat portion of these molecules is remarkably variant. These repeating structures vary enormously, not only in their amino acid composition which greatly affects their immunological properties, but in their length and copy number. These radical variations in protein structure do not affect their behaviour as secreted proteins.
The transmembrane anchor sequence from a mouse immunoglobulin gene has been added to the native S-antigen molecule of the FC27 isolate of P.falciparum and it has been shown that the hybrid protein is correctly presented as an integral membrane protein on the surface of eukaryotic cells (Langford et.al., (1986)). This hybrid protein contains 13 copies of an 11 amino acid repeating epitope. Clearly this repeating sequence does not prevent the transport of the hybrid protein to the cell surface. The present example replaces this simple repetitive sequence with other sequences in the hope that the new hybrid protein will be transported to the cell surface.
The mode of presentation of the recombinant antigen The hybrid antigens could be delivered using a live recombinant virus presentation system. We have used recombinant vaccinia viruses as an example of this approach.
The hybrid protein could also be expressed in bacteria or eukaryotic cells and then purified for use as a vaccine. As an example of this approach we have made use of a newly developed expression vector that vastly simplifies the purification of foreign proteins produced in bacteria (Australian Patent Application PI2195/87) . Proteins are produced as fusion polypeptides with the soluble Schistosma iaponicum enzyme glutathione S-transferase (GST) and can then be easily purified by affinity purification on glutathione coupled to agarose beads. Pure fusion polypeptides can then be eluted from the beads using free glutathione prior to further purification for use as a vaccine.
The choice of linear sequences for insertion in the hybrid protein.
The sequences used in the construction of the hybrid proteins described herein were selected from a large number of repetitive malarial antigens with some regard to the potential of these antigens as vaccine molecules. Thus the dominant immunogenic epitope of the P. falciparum sporozoite (NANP) was an obvious choice as ceptide and recombinant protein preparations of this basic sequence are presently being used in human vaccine trials (Zavala et.al. (1985)).. Similarly recent results on the protection of Aotus monkeys with fragments of the RESA molecule (Collins et.al. (1986)) have focused attention on two regions of repeating amino acids with sequences DDEHVΞΞPTVA and EΞNVEHDA. There are many other candidate vaccine antigens of P.falciparum many of which contain i munodominant repetitive amino acid sequences. From these the GESKΞT and SGASA repetitive sequences of the MESA and Pfl95 major merozoi e surface antigens, respectively, have been chosen as further examples.
Epitopes from other non-malarial antigens could also be included as well as mimotopes of conformation epitopes. Inserting these sequences as repetitive elements may also be desirable. The observations on the immunodominance of the repeating peptide sequences of malarial antigens suggest that simply repeating these peptide sequences may dramatically increase their immunogenicity.
As well as incorporating the relevant epitopes involved in antibody responses it may also be advantageous to incorporate T-cell recognition sequences which, as well as being of importance in the development of T-cell responses, may also provide T-cell help for amanistic antibody responses when the recipient is exposed to intact pathogen.
General methods for the construction of the hybrid proteins
Prior to the construction of the hybrid genes containing the new repeating sequences the plasmid containing the hybrid anchored S-antigen gene described previously (Langford et.al. (1986)) was modified to facilitate the cloning and immunological screening of the new hybrid proteins. These modifications included (a) The inclusion of a bacterial promoter upstream of the S-antigen gene and the vaccinia promoter. This was achieved by first removing the Clal site in the pBR322 region of the vector ρGS62 (Figure 2a) by partial digestion with Clal, Klenow "filling-in" and religation. Into the now unique Clal site a Taql fragment from the strong hybrid acUV5/Trp bacterial promoter was inserted generating the new vector pGS62 tac (shown in figure 2b). Using this vector the expression of genes inserted into the multiple cloning site (MCS) of the vector can be checked in bacteria as well as in virus-infected cells.
(b) The creation of a unique BamHl cloning site in the body of the S-antigen gene. This was achieved by first deleting the BamHl site from the MCS of the vector ρGS62tac by BamHl digestion, Klenow "filling-in" and religation to generate the new plasmid pGS62-tac [BamΔ] (figure 2c). The 1088 bp EcoRl fragment from pVA20 which also contains a BamHl site was cloned into the EcoRl site of the MCS of ρGS62tac BamHlΔ to generate the plasmid ρCL4 (figure 2d) .
(c) The deletion of the repeating sequences of the S-antigen gene. To achieve this the isolated EcoRl fragment of pV8 (Langford et.al., (1986)) was digested with Sau3A. This enzyme cuts once in each of the S-antigen repeats. The non-repeat fragments 5* and 3' to the repeat portions were isolated and ligated into the EcoRl site of the vector ρUC8 to generate the new construct pSAD AhalO. The Sph 1 linkered mouse membrane IgGl cDNA fragment encoding the transmembrane anchor sequence was then cloned into the unique Sph 1 site of this plasmid to generate the new plasmid pAlϋ. The EcoRl insert from this insert was then cloned into the unique EcoRl site of the plasmid pGS62tac BamHIΔ to generate the plasmid ρLK3 (shown in figure 2e) . The plasmid pLK8 contains the S-antigen gene with less than one full copy of the 33bp repeating sequence and with a unique BamHl site located within this remaining partial repeat sequence. It is into this site that appropriately engineered sequences encoding new antigenic determinants can be cloned thus neatly replacing the S-antigen repeating epitope.
Replacement of the S-antigen repetitive sequences with new repeating epitopes.
The incoming sequences in the situation described in- this example need to have BamHl "sticky ends" and to be engineered in such a way that the reading frame of the whole hybrid gene is maintained. This requires that the total length of the insert DNA should be an equal multiple of 3 base pairs and that the reading frame be in phase with the GAT codon of the GGATCC BamHl site at the 5' end of the (coding strand of the) insert.
It is also believed that the epitope to be expressed should be repeated as many times as possible to maximise its immunogenicity even if this epitope is represented only once or a few times in the native antigen molecule.
To achieve this, in this example the gene sequences encoding the repeating peptide sequences have been chemically synthesised as short oligonucleotides. Chemical synthesis of the oligonucleotides also enables the codons to be "mammalianised" for optimal expression in recombinant vaccinia virus-infected mammalian cells. This may be of importance in the optimal expression of foreign proteins from species such as P.falciparum which exhibit a strong bias in their preferred codon usage away from that seen in mammalian cells. However, the insert could also be derived from naturally occurring sequences as long as it fulfils the length and phase requirements described above.
Complementary oligonucleotide sequences (Figure 3) were synthesised on an Applied Biosystems oligonucleotide synthesiser and purified by HPLC. These were then kinased prior to annealing and ligation in the standard way. The oligonucleotides were designed so that the ends were complimentary in one orientation only, ensuring that only "head to tail" ligation of the double stranded monomers was possible. The ligated fragments were then size fractionated on a low gelling temperature agarose gel and DNA molecules in the size range from 180 to 600bρ were isolated and purified from the agarose. These size fractionated molecules were then ligated with BamHl cut calf intestinal phosphatase treated, p K8 plasmid DNA in the presence of two kinased synthetic oligonucleotides with the sequence 5'-GATCCC-3' and 5 ' -GATCGG-3" . These adaptors were designed to allow the ligation of the 3* overhanging CC and GG ends of the repeating oligonucleotide fragment to the 5' overhanging GATC sticky ends of the BamHl cut ρLK8 and pCL4 and to ensure that the insert sequence was "in frame" with the S-antigen sequence (see example shown in Figure 4).
Recombinant bacterial clones containing the inserted sequence were selected by colony hybridization uussiinngg tthhee ggaammmmaa -~[[ Δ PP]]--ATP kinased oligonucleotides used in the initial cloning
Plasmid DNA was isolated from the positive clones and digested with restriction enzymes to determine the clones which contained the longest inserts. A number of these were then sequenced using the double stranded DNA sequencing procedure on alkaline denatured plasmid DNA preparations to select a clone with an insert i the correct orientation and to confirm its predicted sequence (Figure 7) . These plasmid DNA were then transfected directly into WT vaccinia virus-infected cells to produce T recombinant virus as described previously (Langford et.al. (1986)).
Increasing the length of the repeating epitope by further subcloning.
The new insert in the hybrid gene is flanked by Sau3A sites which allow the insert to be isolated and purified from the recombinant plasmid. However only one BamHl site at the 5' of the coding strand of the insert is regenerated in this hybrid (see Figure 4) . Thus the recombinant plasmid can be linearised with BamHl, phosphatase treated and ligated with the isolated Sau3A fragment. Plasmid DNA was prepared from the transformed bacteria resulting from this cloning and digested with restriction enzymes to select clones with double (or triple) inserts. These were then sequenced to determine which contained new inserts in the correct orientation and to confirm the predicted sequence. These new hybrids again have a unique BamHl site at the 5' end of the insert, and this process can be repeated many times over thereby increasing the size of the insert to any desired length. In this example hybrid genes containing 16(V6), 32(V66) and 48(V666) copies of the 12bp repeat of the CSP gene and 10 (V3) and 20 (V33) copies of the 24bp repeat of the RESA gene have been produced (see Figure 6) .
Construction of hybrid genes encoding multiple repeating epitopes.
The inserts from all the clones constructed in ρ K8 and pCL4 could be isolated as Sau3A fragments and cloned into the unique BamHl site of any other construct. This allows a polymer of inserts in order to be built in as a new 3amHl site will be created following the insertion of each new insert. As the Sau3A fragment can ligate in either orientation into the BamHl site the orientation needs to be checked in each case by DNA sequencing. A number of these hybrid genes including one construct (V55367) containing inserts encoding the S-antigen, CS, RESA 3', RESA 5' and MESA repeats were cloned into recombinant vaccinia viruses. These constructs are shown in Figure 6.
Expression and purification of hybrid protein vaccines in bacteria
BamHl/EcoRl fragments of the genes described in the V-series of constructs shown in Figure 6 were subcloned into the BamHl/EcoRl cloning sites at the 3' end of the E-, iaponicum glutathione S-transferase (GST) gene in the expression vector ρGEX-1 (or an earlier version of the vector; pSjlO Ba ΔlO) as described' in Australian Patent Application No.PI 2195/87. In this vector system the GST gene is under the control of the strong, IPTG inducible trp/lac (tac) promoter (Arnann et al., 1983).
This expression system has been used to purify a wide range of parasite and non-parasite polypeptides, the majority of which are produced as abundant, soluble and stable fusion proteins that can be purified on glu a hione-agarose.
RESULTS Checking the clones by sequencing.
All clones shown in Figure 6 have been sequenced at various stages in their construction and again immediately prior to transformation into virus infected cells to produce the viral recombinants. Instability of the repetitive elements of Plas odial antigen genes has been a frequent problem in their manipulation in recombinant bacteria. However, in this example, there was no evidence that the copy-number of the repeating sequences of any of the hybrids was altered during the cloning procedures. Plasmid DNAs were sequenced directly following alkaline denaturation using the double stranded sequencing procedures adapted f om those described by Chen and Seeburg (1985). Synthetic oligonucleotide primers complimentary to S-antigen sequences located 3 ' (5*-AATGGATTAATAGAAGG-3• ) and
5' (5r-GCTTTCCATGTCCTTCAGC-3') to the BamHl cloning site in pLKS allowed the sequence and orientation of the insert to be checked from each side. As a rapid check for orientation of the insert normally only one dideoxy sequencing reaction was necessary. For example in the sequencing gel shown in Figure 7B only "A" reactions were performed. In this example, clones with inserts with the sequence 5'-AACGCCAACCCC-3 ' in the correct orientation would be expected to produce a sequence pattern made up of uniformly spaced doublets interrupted by gaps of 4 nucleotides in length. This pattern is interrupted every 192bp in the clones containing multiple inserts of the CS repeating sequence because of the 5'-GATCCC-3 ' adaptor sequences at the end of each single insert sequence.
Checking for the expression of the inserted sequences in bacteria.
The strong hybrid bacterial promoter located upstream of the S-antigen gene in these hybrid constructs enables the expression of the hybrid protein to be checked in bacteria transformed with the plasmid DNA. In the example shown in Figure 8, a number of bacterial clones containing hybrid genes with RESA 24bp repeat inserts were picked into arrays on nitrocellulose, replicated and grown in the presence of IPTG. Colonies were then lysed with SDS and SDS/chloro orm. Filters were then blocked and then probed with antibodies raised in rabbits to chemically synthesised polypeptides encoding the repeat epitopes conjugated to KLH. This was followed by I 125 protein A binding, washing and autoradiograph . The expression of the specific insert sequences could be demonstrated in bacteria for most hybrids prior to their insertion into vaccinia virus.
Checking for the expression of the hybrid proteins in recombinant vaccinia infected cells.
Once recombinant vaccinia clones had been identified, monolayers of BSC-1 cells were infected with the viruses and incubated for 18hr. Cells were then harvested and precipitated by centrifugation. The pellets were then suspended in SDS sample buffer, boiled and centrifuged to remove insoluble material. This sample was then subjected to SDS/PAGE and immunoblotting using the rabbit anti-peptide antisera. Figure 9 shows the expression of a number of these hybrid proteins. In most cases a stable antigenic protein is produced.
Immunisation of animals.
Rabbits were immunised with 10 PFU of each recombinant virus by intrader al injection on the middle of the back. Sera were collected at weekly intervals and assayed in an ELISA in which synthetic peptides of the repeating epitopes, conjugated to BSA or β-galactosidase fusion polypeptides of malarial antigens purified from bacteria, were used to coat the plates. Antibody responses specific to the sequences encoded by the inserts could be detected in all cases.
In Figure 10, the antibody responses of one of many rabbits immunised with the virus expressing the V55367 hybrid protein are shown. Antibodies recognising the CSP, MESA, RESA 8-amino acid, RESA 11-amino acid and S-antigen repeating epitopes could be detected in all animals following immunisation with this single hybrid protein.
Hybrid proteins produced in bacteria
Constructs shown in Figure 6 (Sj series) expressed abundant, stable fusion proteins once the hydrophobic mouse IgG transmembrane region sequence was removed (by cleaving the hybrid genes with Sphl followed by religation thus releasing the transmembrane gene region) . The fusion proteins could be purified on glutathione agarose beads and eluted using free glutathione, yielding up to lOmg of purified protein per liter of bacterial culture. Commassie stained polyacrylamide gel analysis of these purified proteins are shown in Figure 11.
Protein purified from cloned S S53 was used to immunize rabbits and mice producing antibodies which were capable of recognizing each of the repeating epitopes contained in the hybrid molecule (data not shown) . These purified hybrid proteins also proved to be excellent substitutes for synthetic peptides as diagnostic reagents in ELISAs and cell proliferation assays. REFERENCES
1. A ann et al (1983) Gene, 7__, 167-178.
2. Chen and Seeburg (1935) DNA, 4, p.165.
3. Collins et.al. (1986) Nature, 122, p.259.
4. Langford et.al. (1986), Mol.Cellular Biol. 6_, p.3191,
5. Zavala et.al. (1985), Science 228. p.1436.

Claims

1. A hybrid protein or polypeptide which comprises the epitopes or mimotopes of a plurality of different protein antigens.
2. A hybrid protein or polypeptide according to claim 1, which comprises more than one repeated sequence of each of said epitopes or mimotopes.
3. A hybrid protein or polypeptide according to claim 1, wherein at least one of said epitopes or mimotopes is an epitope or mimotope of an antigen of P. falciparum.
4. A hybrid protein or polypeptide according to claim 3, wherein at least one of said epitopes or mimotopes is selected from the group of epitopes of P. falciparum antigens consisting of NANP, DDEHVEEPTVA, EENVEHDA, GESET and QSGASA.
5. A hybrid protein or polypeptide according to claim 1, further comprising a surface or membrane-associated polypeptide segment.
6. A hybrid protein or polypeptide according to claim I, further comprising a T-cell recognition sequence
7. A DNA molecule comprising a nucleotide sequence capable of being expressed as a hybrid protein or polypeptide according to any one of claims 1 to 6.
8. A recombinant DNA molecule comprising a nucleotide sequence according to claim 7, operatively linked to an expression control sequence.
9. A cloning vector comprising a nucleotide sequence according to claim 7.
10. A host cell comprising a cloning vector according to claim 9.
11. A composition for stimulating an immune response against a plurality of different protein antigens which comprises a hybrid protein or polypeptide according to any one of claims 1 to 6, together with a pharmaceutically acceptable carrier or diluent.
12. A composition according to claim 11, further comprising an adjuvant.
13. A method of stimulating an immune response which comprises administering a composition according to claim 11 or claim 12.
14. A recombinant virus comprising a nucleotide sequence capable of' being expressed as a hybrid protein or polypeptide according to any one of claims 1 to 6.
15. A recombinant virus according to claim 14, wherein said virus is vaccinia virus.
16. A composition for stimulating an immune response against a plurality of different protein antigens, which comprises a recombinant virus according to claim 14 or claim 15, together with a pharmaceutically acceptable carrier or diluent.
17. A composition according to claim 16, further comprising an adjuvant.
18. A method of stimulating an immune response which comprises administering a composition according to claim 16 or claim 17.
PCT/AU1987/000352 1986-10-17 1987-10-16 Hybrid proteins or polypeptides WO1988002757A1 (en)

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EP0730606A1 (en) * 1994-09-14 1996-09-11 Progenitor, Inc. Hu-B1.219, A NOVEL HUMAN HEMATOPOIETIN RECEPTOR
US5763211A (en) * 1994-09-14 1998-06-09 Progenitor, Inc. Isolated nucleic acid encoding Hu-B1.219, a novel human hematopoietin
WO1998023731A3 (en) * 1996-11-27 1998-11-12 Univ Catholique Louvain Chimeric target molecules having a regulatable activity
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EP0492500A2 (en) * 1990-12-24 1992-07-01 BEHRINGWERKE Aktiengesellschaft Protecting plasmodium falciparum hybrid proteins, containing partial sequences of malaria antigens HRPII and SERP
EP0492500A3 (en) * 1990-12-24 1993-02-10 Behringwerke Aktiengesellschaft Protecting plasmodium falciparum hybrid proteins, containing partial sequences of malaria antigens hrpii and serp
US5395614A (en) * 1990-12-24 1995-03-07 Behringwerke Aktiengesellschaft Protective Plasmodium falciparum hybrid proteins which contain part-sequences of the malaria antigens HRPII and SERP, the preparation and use thereof
US5869610A (en) * 1994-09-14 1999-02-09 Progenitor, Inc. Hu-B1.219, a novel human hematopoietin receptor
US5912123A (en) * 1994-09-14 1999-06-15 Progenitor, Inc. Detection of the leptin receptor in reproductive organs and methods for regulating reproductive biology
US5763211A (en) * 1994-09-14 1998-06-09 Progenitor, Inc. Isolated nucleic acid encoding Hu-B1.219, a novel human hematopoietin
US6953670B2 (en) 1994-09-14 2005-10-11 Indevus Pharmaceuticals, Inc. Methods for identifying compounds capable of supplementing the biological activity of leptin
US5856098A (en) * 1994-09-14 1999-01-05 Progenitor, Inc. Detection of a leptin receptor variant
EP0730606A1 (en) * 1994-09-14 1996-09-11 Progenitor, Inc. Hu-B1.219, A NOVEL HUMAN HEMATOPOIETIN RECEPTOR
US5882860A (en) * 1994-09-14 1999-03-16 Progenitor, Inc. Detection of a leptin receptor variant and methods for regulating obesity
EP0730606A4 (en) * 1994-09-14 1997-03-12 Progenitor Inc Hu-B1.219, A NOVEL HUMAN HEMATOPOIETIN RECEPTOR
US6005080A (en) * 1994-09-14 1999-12-21 Progenitor, Inc. Antibodies specific for Hu-B1.219, a novel hematopoietin receptor
US6355237B1 (en) 1994-09-14 2002-03-12 Progenitor, Inc. Methods for using the obese gene and its gene product to stimulate hematopoietic development
US6451523B1 (en) 1994-09-14 2002-09-17 Interneuron Pharmaceuticals, Inc. Detection of a leptin receptor variant and methods for regulating obesity
US6524806B1 (en) 1994-09-14 2003-02-25 Indevus Pharmaceuticals, Inc. Methods of screening for ligands of human hemetopoietin receptor HU-B1.219
US6838079B2 (en) 1994-09-14 2005-01-04 Indevus Pharmaceuticals, Inc. Methods for using the obese product to stimulate hematopoietic development
WO1998023731A3 (en) * 1996-11-27 1998-11-12 Univ Catholique Louvain Chimeric target molecules having a regulatable activity

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