WO2001023430A9 - Produits de fusion chitinase-immunoglobuline - Google Patents

Produits de fusion chitinase-immunoglobuline

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Publication number
WO2001023430A9
WO2001023430A9 PCT/US2000/026960 US0026960W WO0123430A9 WO 2001023430 A9 WO2001023430 A9 WO 2001023430A9 US 0026960 W US0026960 W US 0026960W WO 0123430 A9 WO0123430 A9 WO 0123430A9
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WO
WIPO (PCT)
Prior art keywords
chitinase
human
product
immunoglobulin
fusion product
Prior art date
Application number
PCT/US2000/026960
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English (en)
Other versions
WO2001023430A2 (fr
WO2001023430A3 (fr
Inventor
Daniel S Allison
Gregory N Dietsch
Patrick W Gray
Kevin D Shaw
Bart H Steiner
Original Assignee
Icos Corp
Daniel S Allison
Gregory N Dietsch
Patrick W Gray
Kevin D Shaw
Bart H Steiner
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Icos Corp, Daniel S Allison, Gregory N Dietsch, Patrick W Gray, Kevin D Shaw, Bart H Steiner filed Critical Icos Corp
Priority to AU77410/00A priority Critical patent/AU7741000A/en
Publication of WO2001023430A2 publication Critical patent/WO2001023430A2/fr
Publication of WO2001023430A3 publication Critical patent/WO2001023430A3/fr
Publication of WO2001023430A9 publication Critical patent/WO2001023430A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates generally to chitinase immunoglobulin fusion products comprising chitinase or fragments or variants thereof, and uses of such products.
  • Chitin is a linear homopolymer of ⁇ -(l,4)-linked N-acetylglucosamine residues. This polysaccharide is second only to cellulose as the most abundant organic substance.
  • the exoskeleton of arthropods is composed of chitin.
  • fungi and other parasites contain chitin in their outer cell wall, where it serves important structural and protective roles. Disruption of the fungal cell wall and membrane has been a useful therapeutic strategy against fungi and parasites. For example, Amphotericin B and fluconazole exert their antifungal activity by affecting membrane steroids. Despite the existence of antifungal therapeutics, fungal infections of humans have increasingly become responsible for life-threatening disorders. See,
  • the fungal species and parasites responsible for these diseases include Candida, Aspergillus, Cryptococcms, Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, Fusarium and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.
  • Chitin can be degraded by the enzyme chitinase.
  • Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi. Chitinases have been cloned from numerous bacterial [e.g., Serratia marcescens, Jones et al., EMBOJ., 5:467-473 (1986)], plant [e.g., tobacco, Heitz et al., Mol. Gen.
  • WO 96/40940 which reported two distinct human chitotriosidase cDNAs encoding a 50 kD and a 39kD product, both of which were fully enzymatically active. Renkema et al, Eur. J. Biochem., 244:219-2 ⁇ 5 (1997) reported that human chitinase is initially produced in macrophages as a 50 kD protein that is in part processed into a 39 kD form that accumulates in lysozymes, and also reported that alternative splicing generates a distinct human chitinase mRNA species encoding a 40 kD chitinase. Cloning of human chitinase cDNA was also reported in International Patent Publication No. WO 97/47752 (Gray).
  • immunoadhesins which are chimeric molecules that combine the functional portion of a physiologically active protein with an immunoglobulin sequence, have been reported in the art, and are also known as "Ig-chimeras,” “Ig- fusion proteins,” “Fc-fusion proteins,” and “receptor-globulins.” See, e.g., Harvill et al, Immunotechnology 1 :95-105 (1995) regarding IL-2 fusions and Zheng et al, J. Immunol, 154:5590-5600 (1995) regarding IL-10 fusions; Fell, U.S. Patent No. 5,204,244.
  • the present invention relates to chitinase immunoglobulin fusion products comprising chitinase or fragments or variants thereof fused to at least a portion of an immunoglobulin chain.
  • the invention provides novel purified and isolated polynucleotides (i.e., DNA and RNA, both sense and antisense strands) encoding such fusion products; methods for the recombinant production of such fusion products; purified and isolated chitinase immunoglobulin fusion products; pharmaceutical and diagnostic compositions comprising such fusion products; and corresponding diagnostic or therapeutic uses, e.g., for detecting chitin, binding chitin, and treating fungal infections.
  • polynucleotides i.e., DNA and RNA, both sense and antisense strands
  • a presently preferred chitinase immunoglobulin fusion product comprises human chitinase fused at its C-terminus to the N-terminus of an IgG4 heavy chain constant region.
  • a chitinase immunoglobulin fusion product for treatment of fungal infections may be accompanied by the concurrent administration of other non-chitinase antifungal therapeutic agents, including non-enzymatic antifungal agents.
  • Figure 1 shows residual spleen fungal infection in mice challenged with sublethal doses of C. albicans and treated with varying amounts of rCH/Fc, amphotericin B or a combination of the two drugs.
  • Figure 2 shows residual kidney fungal infection in mice challenged with sublethal doses of C. albicans and treated with varying amounts of rCH/Fc, amphotericin B or a combination of the two drugs.
  • the present invention relates to chitinase immunoglobulin fusion products comprising chitinase or fragments or variants thereof, preferably a human chitinase or fragments or variants thereof, fused to at least a portion of an immunoglobulin chain, preferably the constant region of a heavy chain.
  • the invention is based upon the discovery that such fusion products retain chitinase biological activity, both in vitro and in vivo, have unexpectedly improved pharmacokinetics when administered in vivo, and have a decreased tendency to precipitate in buffer at protein concentrations of 1 mg/mL or more.
  • fusion of human chitinase to an immunoglobulin constant region provides at least a 10-fold increase in the circulating serum half-life compared to the unfused human chitinase fragment alone.
  • the fusion product retains not only the in vitro chitinase enzymatic and chitin-binding activities of unfused (or wild type) human chitinase, but also exhibits similar or improved in vitro synergy with other non-enzymatic antifungal agents in comparison to unfused human chitinase.
  • chitinase immunoglobulin fusion product means a polypeptide comprising a chitinase product (i.e., a chitinase or a fragment or variant thereof that retains chitinase activity), fused to at least a portion of an immunoglobulin
  • Ig chain or a variant thereof may be fused to either the N-terminus or C-terminus of the chitinase portion.
  • human chitinase, fragments and variants thereof that retain chitinase and/or chitin-binding activity are most preferred, any chitinase may be used, including bacterial, insect and plant chitinases.
  • the cloning and expression of human chitinase cDNA and the biological activities of recombinant human chitinase are described in detail in co- owned, co-pending U.S. Application Serial No. 08/877,599 filed June 16, 1997 and corresponding International Patent Publication No.
  • pMO-218 includes the entire coding region of the human chitinase cDNA (nucleotides 2 to 1399 of SEQ ID NO: 1), which comprises a twenty-one amino acid putative signal sequence followed by 445 encoded amino acids (residues 1 to 445 of SEQ ID NO: 2).
  • the DNA sequence of the chitinase-coding insert of clone pMO-13B (deposited on June 7, 1996 under the terms of the Budapest Treaty with the ATCC under Accession No. 98078) is set forth in SEQ ID NO: 3 and the encoded amino acid sequence is set forth in SEQ ID NO: 4.
  • This clone contains 25 additional nucleotides at the 5' end compared with MO-218; in addition, MO-13B (SEQ ID NO: 3) contains one different nucleotide at nucleotide position 330 (corresponding to nucleotide 305 of MO-218, SEQ ID NO: 1) which changes the encoded amino acid at position 80 of the mature protein from a glycine (in SEQ ID NO: 2) to a serine (in
  • the chitin-binding domain of human chitinase has been determined to reside in the C-terminal 49 amino acids of the protein, while triacetylchitotriose hydrolyzing activity has been observed to reside in the N-terminal portion (amino acids 1-373). See co-owned, co-pending U.S. Application Serial No. 09/267,574 filed
  • the chitinase portion of the chitinase Ig fusion product may be a natural, recombinantly produced or wholly or partially synthetic chitinase or fragment or variant thereof that retains chitinase enzymatic and/or chitin-binding activity.
  • Nariants may comprise chitinase analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added, without loss of the chitinase enzymatic and/or chitin-binding activity. It is contemplated that any conservative amino acid substitution known in the art may be made without affecting the biological activity of the variant.
  • the human chitinase product component of the fusion protein comprises the mature human chitinase amino acid sequence set forth in SEQ ID NO: 2 or 4.
  • the human chitinase product component comprises a polypeptide encoded by: (a) a polynucleotide encoding the mature chitinase amino acid sequence set forth in SEQ ID NO: 2 or 4; or (b) a polynucleotide that hybridizes to the complement of the polynucleotide of claim (a) under stringent conditions.
  • the human chitinase product may also comprise an amino acid sequence equivalent to (e.g., at least about 99%, at least about 98%, at least about 95%, at least about 90%, at least about 85%, at least about 80% identical to) SEQ ID NO: 2 or 4 and include polypeptides with conservative substitutions to the amino acid sequence of SEQ ID NO: 2 or 4.
  • "Conservative" substitutions of one amino acid for another are substitutions of amino acids having similar structural and/or chemical properties, and are generally based on similarities in polarity, charge, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved.
  • Hydrophobic, polar neutral, polar basic and polar acidic amino acids may be grouped as described in Lehninger [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77], incorporated herein by reference.
  • Hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (acidic) amino acids include aspartic acid and glutamic acid; basic amino acids include lysine, arginine and histidine.
  • the Ig portion of the chitinase Ig fusion product may be an entire Ig chain, either heavy or light, but preferably is a fragment of the constant region of an Ig heavy chain that retains at least the CH2 and CH3 regions.
  • the Ig chain is preferably from a human Ig and may be from any class of Ig, including IgGl, IgG2, IgG3, IgG4, IgM or IgA. However, an artificial consensus sequence rather than a natural Ig sequence may be used.
  • the chitinase is fused to a portion of human IgG4 that consists of two amino acids from the CHI region and the hinge, CH2 and CH3 regions.
  • deletions or substitutions of amino acids may be made within the Ig chain to produce variants that retain the improved half-life advantage of the Ig fusion product.
  • cysteine residues may be deleted or replaced with other amino acids, e.g. alanine or serine, to prevent disulfide crosslinking between Ig portions.
  • the Fc receptor binding site and/or complement (Clq) binding site of the Ig constant region may also be modified by deletion or substitution of amino acid residues.
  • One aspect of the invention provides polynucleotides encoding such chitinase Ig fusion products (including natural, synthetic or partially synthetic DNA or
  • RNA DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, and which encode polypeptides with chitinase and/or chitin-binding activity are also contemplated by the invention.
  • Exemplary stringent hybridization conditions are as follows: hybridization at 42°C in 50% formamide and washing at
  • the invention includes biological replicas (i.e., copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention.
  • biological replicas i.e., copies of isolated DNA sequences made in vivo or in vitro
  • Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating polynucleotides encoding chitinase Ig fusion products of the invention, including any of the DNAs described above, are provided.
  • Preferred vectors include expression vectors in which the incorporated fusion product-encoding cDNA is operatively linked to an endogenous or heterologous expression control sequence and a transcription terminator.
  • procaryotic or eucaryotic host cells are stably transformed or transfected with polynucleotide sequences of the invention or otherwise genetically engineered (e.g., through homologous recombination) in a manner allowing the desired chitinase Ig fusion product to be expressed therein.
  • host cells are particularly useful in methods for the large scale production of chitinase Ig fusion products wherein the cells are grown in a suitable culture medium permitting expression of the desired polypeptide and the desired polypeptide products are isolated, e.g., by immunoaffinity or protein A purification from the cells or from the medium in which the cells are grown.
  • Chitinase Ig fusion products may be partially or wholly chemically synthesized but are preferably produced by recombinant procedures involving host cells of the invention.
  • the use of mammalian host cells may provide post-translational modifications (e.g., myristolation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.
  • chitinase Ig fusion products and therapeutic agents comprising such products to mammalian subjects, especially humans, for the purpose of ameliorating disease states caused by chitin-containing parasites such as fungi is contemplated by the invention.
  • Fungal infections mycoses
  • candidiasis, aspergillosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, histoplasmosis, cryptococcosis, chromoblastomycosis, sporotrichosis, mucormycosis, and the dermatophytoses can manifest as acute or chronic disease and may be treated according to the invention.
  • Candida species including C. tropicalis, C. parapsilosis, C. stellatoidea, C. krusei, C. parakrusei, C. lusitanae, C. pseudotropicalis, C. guilliermondi and C. glabrata, Aspergillus species, including A. fumigatus, A. flavus,
  • Pathogenic fungi cause serious, often fatal disease in immunocompromised subjects, and products of the invention are expected to be particularly helpful in treating infections in these subjects.
  • Cancer patients undergoing chemotherapy, immunosuppressed individuals, and HIV-infected individuals are susceptible to mycoses caused by Candida, Aspergillus, Pneumocystis carinii, and other fungi.
  • Amphotericin B and fluconazole are useful therapeutics for fungal infections, but toxicity associated with these drugs causes serious adverse side effects that limit their usefulness. The mortality of systemic candidiasis is greater than 50% despite amphotericin B treatment.
  • the chitinase Ig fusion products may be concurrently administered with or even conjugated to other conventional (preferably non-enzymatic) antifungal agents, including amphotericin B and the structurally related compounds nystatin and pimaricin; 5-fluorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, itraconazole and tioconazole; allylamines- thiocarbamates, such as tolnaftate, naftifine and terbinafine; griseofulvin; ciclopirox olamine; haloprogin; undecylenic acid; and benzoic acid.
  • amphotericin B and the structurally related compounds nystatin and pimaricin including amphotericin B and the structurally related compounds nystat
  • the chitinase Ig fusion products are expected to improve the effectiveness of these conventional antifungal agents, perhaps by rendering the fungi more susceptible to their action. A reduction in the amount of conventional antifungal agent needed to exert the desired antifungal effect may allow the drugs to be used at less toxic levels.
  • This effect of the chitinase Ig fusion product on the activity of conventional antifungal agents can manifest as classic checkerboard synergy in in vitro tests as indicated, e.g.
  • fractional inhibitory concentration (FIC) index or the fractional fungicidal concentration (FFC) index may manifest as conversion of a fungistatic agent into a fungicidal agent or as a reduction in the resistance of fungi to conventional antifungal agents.
  • Conjugation to non-selective cytotoxic agents may allow selective targeting of these agents through the chitin-binding function of the chitinase Ig fusion products.
  • chitinase Ig fusion products may be administered alone since the products themselves may have antifungal effects.
  • Multimeric chitinase Ig fusion products may provide enhanced antifungal effects and include multimeric products that have been covalently cross-linked by chemical means; recombinantly produced polypeptides comprising multiple chitinase portions linked in tandem with the Ig portion; and dimeric or multimeric chitinase Ig fusion products linked through disulfide bonding of the Ig portions.
  • the administration of multimeric products may also reduce the amount of concurrently administered conventional antifungal agent necessary to exert a desired antifungal effect.
  • the invention contemplates the use of chitinase Ig fusion products in the preparation of a medicament for the prophylactic or therapeutic treatment of fungal infections.
  • therapeutic/pharmaceutical compositions comprising chitinase Ig fusion products for use in methods for treating a mammal, preferably a human, susceptible to or suffering from fungal infections.
  • Such compositions may include a physiologically acceptable diluent or carrier, and may also include other conventional antifungal agents. Dosage amounts indicated would be sufficient to supplement endogenous chitinase activity.
  • compositions of the invention may be administered by various routes depending on the infection to be treated, including via subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intrathecal, topical, oral, or suppository administration.
  • Constant administration includes administration of the different agents together, or before or after each other.
  • the different agents may be administered by the same or different routes.
  • one agent may be administered intravenously while the second agent is administered intramuscularly, intravenously, subcutaneously, orally, topically or intraperitoneally.
  • the different agents may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow all agents to achieve effective concentrations at the site of infection.
  • Example 1 addresses construction of a vector encoding a human chitinase immunoglobulin (Ig) fusion product.
  • Example 2 addresses expression of this vector in COS cells.
  • Example 3 addresses purification and formulation of a human chitinase Ig fusion product.
  • Example 9 addresses construction of a vector encoding a human chitinase Ig fusion product for expression in CHO cells.
  • Plasmids pMO218 (ATCC Accession No. 98077) and pMO13B (ATCC Accession No. 98078) each contain complete human chitinase cDNA and are described in co-owned, co-pending U.S. Application Serial No. U.S. Application Serial No. 08/877,599 filed June 16, 1997 and corresponding International Patent Publication No. WO 97/47752, both of which are incorporated herein by reference in their entirety.
  • Vectors encoding a human chitinase immunoglobulin fusion product (designated rCH/Fc and consisting of essentially full length human chitinase fused at its C-terminus to the N-terminus of the constant region of human IgG4) were constructed from plasmids pMO218 and pMO13B as follows. The C-terminus of the human chitinase cDNA was modified by PCR to delete the termination codon and replace it with a Xhol restriction site.
  • Primers 218-4 (5' TTCAACAGTGGCTGCAGA 3', SEQ ID NO: 5), which binds upstream of a unique Sphl site at about nucleotide 741 of human chitinase, and 713XhoI (5' TACACTCGAGATTCCAGGTGCATTTGC 3', SEQ ID NO: 6), which deletes the putative termination codon of CH and adds the Xhol restriction site, were used to amplify the C-terminal portion of human chitinase.
  • a 50 ⁇ L PCR reaction was carried out with the following reagents: 5 ⁇ L of 10X PCR salt (0.5 M KCL, 100 mM Tris, pH 8.3, 15 mM MgCl j ); 5 ⁇ L of 5 mM dNTP's; 5 ⁇ L of primer 218-4 at 100 ng/mL; 5 ⁇ L of primer 713XhoI at 100 ng/mL, 1 ⁇ L of DNA template (plasmid CH218, also referred to as MO218) diluted 1:50 in TE, pH 8.0); 0.5 ⁇ L of TAQl polymerase and 30 ⁇ L of H 2 0.
  • 10X PCR salt 0.5 M KCL, 100 mM Tris, pH 8.3, 15 mM MgCl j
  • 5 ⁇ L of 5 mM dNTP's 5 ⁇ L of primer 218-4 at 100 ng/mL
  • 5 ⁇ L of primer 713XhoI at 100 ng/mL
  • the reagents were mixed together, overlaid with lOO ⁇ L of mineral oil and amplified at 94 °C for 4 minutes, followed by one minute at 94 °C, 1 minute at 60 °C, and 1 minute at 72 °C for 31 reaction cycles on a Perkin Elmer Cetus DNA Thermal Cycle machine.
  • the resulting PCR product was purified over a CL6B resin
  • a 607bp Sphl/Xhol fragment of this PCR product encoding the modified C-terminal portion of human chitinase was then joined in a three-way hgation, described below, with an 872bp Hindfll/Sphl DNA fragment encoding the N-terminal portion of human chitinase (including the leader sequence) and the HindlH/XhoI- digested vector pDEF2S/Ig4 which encodes the immunoglobulin constant region of human IgG4.
  • the 872bp HindlH/Sphl DNA fragment was obtained as follows. Approximately 2 ⁇ g of each of pMO218 and pMO13B was digested with Hindlll and Sphl in a 20 ⁇ L reaction that contained 2 ⁇ L of 10X high salt buffer (Boehringer Mannheim (BMB)), 1 ⁇ L each of restriction endonucleases Hindlll (BMB lOU/ ⁇ l) and Sphl (BMB 10 U/ ⁇ L) and 13 ⁇ L of H 2 O. After incubating 1 hour at 37 °C, an 872-bp fragment encoding the N-terminal portion of human chitinase was isolated for each plasmid.
  • BMB 10X high salt buffer
  • pDEF2S/Ig4 was produced as follows.
  • the plasmid pDEF2S was first constructed from pDEF2 (described in US Pat. No. 5,888,809) by digesting pDEF2 with Sail, followed by blunting with Klenow polymerase and reclosure of the plasmid with T4 DNA ligase, thereby destroying the Sail site.
  • the plasmid pDEF2S/Ig4 was constructed from pDEF2S by cloning into the Hindlll-Xbal site a 1217 bp Hindlll - Xbal fragment with the sequence in SEQ ID NO: 7.
  • This fragment contains: a Xhol restriction site, immediately followed by codons corresponding to the final two amino acids of the human immunoglobulin g4 heavy chain CHI domain, immediately followed by cDNA sequence encoding the hinge, CH2, and CH3 regions of the human g4 protein, immediately followed by 330 bp of sequence that is naturally present in the 3' flanking region of the human g4 gene and which contains a polyadenylation signal [Ellison et al., DNA, 1: 11-18 (1981)], followed, 25 bp downstream, by a Xbal restriction site.
  • BMB alkaline phosphatase
  • Fragments were isolated after each of the three digestion reactions by adding 4 ⁇ L of 5X DNA loading dye to each sample, loading 15 ⁇ L onto a 0.8% low melting agarose [LMP agarose, BRL] IX TAE agarose gel, electrophoresing at 90m
  • the three fragments were briefly heated to 65 °C (for 5 minutes) before being ligated at room temperature (-22 °C) for one hour in a reaction mixture containing: 4 ⁇ L of the HindlH/Sphl (N-terminal) fragment from either the MO218 or
  • MO13B plasmid 4 ⁇ L of the PCR amplified Sphl/Xhol (C-ter inal) fragment, 2 ⁇ L of ClP-treated pDEF2S/Ig4 Hindlll/Xhol fragment, 2 ⁇ L of 10X ligase salt (BMB) and 1 ⁇ L of T4 DNA ligase (BMB). After the ligation reaction was complete each reaction was briefly heated to 65 ⁇ C and 15 ⁇ L of the ligation mixture was added to 100 ⁇ L of CaCl 2 competent E. coli cells.
  • plasmid DNA Five ⁇ L of plasmid DNA was digested in a 20 ⁇ L reaction containing 2 ⁇ L of 10X salt, 12 ⁇ L H 2 O and l ⁇ L each of restriction endonucleases Hindlll and Xhol, incubated at 37° C for about one hour, then loaded on a 0.8% agarose IX TAE gel. Digestion reactions that yield a band of the proper size, approximately 2.5 kb, were assumed to contain the proper insert.
  • Miniprep DNA preparations designated nos. pDEF2S/CH13B/Ig4-l through -10 (or 13B-1 through
  • 13B-10) represented 13B/Ig4 isolates and miniprep DNA preparations designated nos.
  • pDEF2S/CH218/Ig4-l 1 through -20 represented 218Ag4 isolates.
  • Plasmids 13B-1, 13B-10, 218-13 and 218-17 were sequenced to confirm that the plasmids were free of PCR errors that would change the amino acid sequence of the protein.
  • Plasmids pDEF2S/CH218/Ig4-13 (or pCH218/lg4) and pDEF2S/CH13B/Ig4-l (or pCH13B/Ig4) were transiently transfected into COS cells as follows using DEA dextran chloroquine method, and supernatant containing the recombinant protein was collected and assayed for chitinase enzymatic activity and for protein concentration as determined by ELISA.
  • COS cells were split 1:5 prior to transfection such that they would be between 40-50% confluent on the day of transfection.
  • each plate was washed with 10 mLs of PBS.
  • tranfection media was aspirated and replaced by 6 mLs of 10% DMSO in PBS for about one minute.
  • the cells were then washed in 10 mLs of phosphate buffered saline (PBS) and were incubated overnight in DMEM plus 10% FBS. The following day the cells were washed twice with 10 mLs PBS, and 10 mLs of serum free media was added to the plate. Cells were further incubated for three days at 37°C.
  • PBS phosphate buffered saline
  • rCH/Fc chitinase enzymatic activity, chitin-binding activity, pharmacokinetics and in vitro and in vivo activity of the purified product, called rCH/Fc, was determined as described in Example 4 to 8 below.
  • EXAMPLE 3 Purification and formulation of human chitinase Ig fusion product
  • the COS cell supernatant containing the recombinant fusion product was purified as follows over a protein A column in a single step purification procedure that yielded > 90% pure protein. Briefly, the supernatant (about 9 mLs) was concentrated over a 3 OK centrifugal filter column (UFN2BTK40 Millipore Corp., Bedford, MA) to one mL. The concentrated media was then loaded at 0.5-1.0 cm/min onto a Protein A Sepharose FF column (Pharmacia) that had been equilibrated with IX
  • CMF-PBS (2.7 mM KC1, 1.5 mM KH 2 PO 4 , 137 mM ⁇ aCl, 8.1 mM ⁇ a ⁇ HPOJ.
  • the column was washed with equilibration buffer, CMF-PBS, until the A 2g0 profile returned to baseline. Then the column was washed with 20 mM phosphate, pH 7.2. This low ionic strength wash was necessary prior to elution at a low pH (3.5) because concentrated human chitinase precipitates at low pH ( ⁇ 4.5) in the presence of salt.
  • the human chitinase Ig fusion product was eluted with 40 mM citrate, pH 3.5, into fraction tubes containing 1/10* volume of 1 M ⁇ a phosphate, pH 8.0, as a neutralizing buffer, and the peak eluate was pooled and dialysed in CMF-PBS. The column may then be stripped with 100 mM citrate, pH 3.0, to remove any remaining protein (which is not used). The resulting purified chitinase immunoglobulin fusion product was designated rCH Fc.
  • the concentration of fusion product present in the rCH/Fc samples can be determined by quantifying the human IgG portion of the fusion using an ELISA. Microtiter plates (Immulon 4, Dynatech) were coated overnight at 2-8"C with 50 ⁇ L/well of 1 : 1000 goat anti-human IgG (Jackson ImmunoResearch Cat. No. 109-005-
  • Samples were tested at four dilutions in media: an initial dilution of 1 :20 or 1 :40, with three subsequent serial 5-fold dilutions.
  • Standards and samples 50 ⁇ L/well were incubated in anti-human IgG-coated wells for 90 minutes at room temperature or for 30 minutes at 37 ° C. After aspirating samples, plates were washed three times with wash buffer.
  • Goat antihuman Fc fragment specific-HRP Jackson ImmunoResearch Cat. No. 109-035-098
  • 50 ⁇ L of a 1 : 10,000 dilution in wash buffer was added per well and incubated for 60 minutes at room temperature or for 30 minutes at 37°C.
  • TMB tetramethylbenzidine
  • buffered substrate 0.1 M sodium acetate, 0.015% H 2 O 2 pH 5.5
  • the reaction was stopped by the addition of 50 ⁇ L of 15% H 2 SO 4 and the absorbance at 450 run (A ⁇ ,,) was determined.
  • the data for standards were fitted to a polynomial equation.
  • concentration of IgG in the test samples was determined using the curve-fit equation, the A ⁇ , and the sample dilution.
  • the rCH/Fc product could be readily formulated in CMF-PBS (2.7 mM KC1, 1.5 mM KH 2 PO 4 , 137 mM NaCl, 8.1 mM Na ⁇ HPO ⁇ pH 7.4) at concentrations as high as 10 mg/mL.
  • CMF-PBS 2.7 mM KC1, 1.5 mM KH 2 PO 4 , 137 mM NaCl, 8.1 mM Na ⁇ HPO ⁇ pH 7.4
  • unfused (wild type) human chitinase which was formulated in 20 mM Tris, pH 7.5, 150 mM NaCl, 5% glycerol, was difficult to formulate because even 1 mg/mL concentrations precipitated out of solution.
  • EXAMPLE 4 Chitinase enzymatic activity assay
  • Serum samples to be tested were initially diluted 1 : 1000 in dilution buffer (0.2 M sodium acetate, 0.1% BSA, 0.05%
  • Assay standards comprised 70 mL aliquots of the fluorescent reaction product 4- methylumbelliferone (Signma M 1381), over the concentration range of 0.20-50 nM in 4-fold increments. Standards were tested in duplicate. A standard curve was constructed by fitting the fluorescence units versus standard concentration data to a straight line. The chitinase activity in the sample was determined in the linear response region for each sample and expressed as nmol/min/mL.
  • the rCH/Fc product obtained as described in Example 3 above had a specific activity of approximately 25-32 ⁇ g/min/mg.
  • the chitin binding activity of rCH/Fc was evaluated as follows. 200 ⁇ g of rCH13B/Fc or rCH218/Fc were diluted to 500 mL with equilibration buffer (20 mM Tris pH 8.0, 500 mM NaCl). A chitin column was prepared with 500 mL of chitin beads (New England Biolabs) in a minicolumn. After the beads settled, the column was washed with 5 mL of 1% SDS followed by 5 mL of equilibration buffer. The test sample was added to the column and the flow through fraction was collected.
  • equilibration buffer 20 mM Tris pH 8.0, 500 mM NaCl
  • the column was then washed with 5 mL of equilibration buffer and two fractions (the first 1 mL and the remaining 4 mL) were collected.
  • the chitin-bound molecules were eluted from the column with 2 mL of 1% SDS in two fractions (the first 1 mL and the remaining 1 mL). All of the fractions were run on a 12% polyacrylamide gel, and results showed that a band of the molecular weight expected for rCH/Fc was observed only in the eluted fractions and not in the wash fractions, indicating that the rCH/Fc bound to the chitin beads in the column.
  • mice received a single intravenous injection into the tail vein of 5.0 mg/kg of this fusion product. At 0, 0.5, 1, 2, 4, 6, 8, 16 and 24 hours after injection, whole blood was collected by cardiac puncture and placed in Microtainer® (Beckton-Dickinson) red cap tubes for serum collection by centrifugation at 6000 rpm for 2 or 3 minutes. Urine was also collected at the same time points. Samples were placed in Eppendorf ® tubes and refrigerated at 4-6 °C until all time points were collected. Enzymatic activity was determined for each sample as described above in Example 4.
  • Protein concentration was also determined for each sample as derived from the A 280 values and extinction coefficient.
  • the serum half-life of the CH/Ig4 fusion was determined to be about 29.9 hours using the Winonlin model #1.
  • the experiment was repeated with male 6-8 week old CD-I mice (5 per time point) administered a dose of 77 mg/kg rCH Fc (using a solution of 11.24 mg/mL rCH/Fc) by either an intravenous or an intraperitoneal route. Chitinase activity and protein concentration were assayed in serum samples collected via cardiac puncture at 0, 1, 2, 4, 6, 8, 16, 24, 28, 32 and 48 hours after injection. The resulting pharmacokinetic data are set forth below in Table 1.
  • AUC area under serum-concentration-time curve extrapolated to infinity
  • cL total body clearance
  • MRT mean residence time half-life: serum half-life for terminal elimination phase
  • mice Female Balb/c mice, 6-8 weeks old, were injected intravenously in the tail vein with 0.5 mg/kg, 5.0 mg/kg and 50 mg/kg recombinant human chitinase. For each dose group, mice were terminally bled at 0.01, 0.25, 1, 8 and 24 hours after injection (2 animals were used per time point per dosage) and serum samples were assayed for chitinase activity and concentration.
  • AUC area under curve to time infinity
  • Vss steady state volume of distribution
  • cL clearance
  • Minimum inhibitory concentrations i.e., the lowest concentration of test compound which produces no visible growth (a clear tube)
  • MFCs minimum fungicidal concentrations
  • FIC or FFC index of less than 1 is considered to show synergy, while an FIC or FFC index of 0.5 or less is considered to show potent synergy.
  • “Indifference” means that the first test compound does not affect the activity of the second test compound at all (i.e., the full MIC or MFC is required).
  • rCH Fc When tested in combination with amphotericin B, rCH Fc showed quite potent synergy with amphotericin B on all of the C. neoformans strains, for both growth inhibition and for killing.
  • the 6 week old male CD-I mice (average weight 29.6 g) were infected by intravenous inoculation with 9 x 10 4 CFU of a clinical isolate of Candida albicans. Treatment began four days after infection.
  • mice received either phosphate buffered saline (diluent controls) administered intraperitoneally; rCH/Fc (in PBS in a volume of 0.53 ml) administered intraperitoneally at a dose of 200, 80 or 20 mg/kg once daily for 12 days; amphotericin B (AmB, PharmaTek, in sterile 5% dextrose in a 0.2 mL volume) administered intraperitoneally at a dose of 3 mg/kg three times weekly for 2 weeks at days 4, 6, 8, 11 13 and 15 post infection (6 doses total); or both AmB and rCH Fc.
  • Another control group of 10 mice received no fungal challenge and the 200 mg/kg rCH Fc . treatment regimen.
  • the rCH/Fc-treated groups showed no significant difference in mortahty or residual spleen or kidney fungal load compared to untreated controls.
  • the AmB and rCH/Fc combination-treated groups showed no significant difference in mortahty but showed a dramatic reduction in residual spleen fungal infection. All survivors of the group given AmB and 200 mg/kg rCH/Fc were free of residual infection in the spleen, and survivors of the group given AmB and either 80 or 20 mg/kg rCH/Fc were combinations were 90% free of residual spleen infection. In comparison, all survivors of the groups treated with AmB alone showed some residual spleen infection.
  • Plasmids for expression of human chitinase Ig fusion product in CHO cells, pDEF24/CH 13B/Ig4 or pDEF24/CH218/Ig4, were constructed as follows.
  • Plasmids pDEF2S/CH13B/Ig4-l and pDEF2S/CH218/Ig4-13 were digested in a 20 ⁇ L reaction containing approximately 2 ⁇ g DNA, 2 ⁇ L of 10X salt, 1 ⁇ L each of the restriction endonucleases Notl and Xbal and 12 ⁇ L of H 2 O. Each reaction digest was incubated at 37° C for one hour and DNA fragments were isolated on a 0.8% LMA IX TAE gel. The NotllXbal fragment containing the chitinase gene was ligated with a
  • Notl I Xbal fragment from a pDEF24-derived vector (pDEF30) treated with alkaline phosphatase (CIP) as described above in Example 1, and the ligation products were used to transform CaCl 2 competent cells as described above in Example 1.
  • the pDEF30 vector is the same as pDEF24 except that it includes a 321bp fragment from pDCl (described in U.S. Patent No. 5,888,809, incorporated herein by reference) containing a BGH poly A region.
  • pDEF24 was derived from pDEF14 (also described in U.S. Patent No.

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Abstract

Cette invention a trait à des produits de fusion chitinase-immunoglobuline ayant une demi-vie sérique améliorée inattendue ainsi que des propriétés améliorées en matière de formulation.
PCT/US2000/026960 1999-09-30 2000-09-28 Produits de fusion chitinase-immunoglobuline WO2001023430A2 (fr)

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US20050176108A1 (en) * 2003-03-13 2005-08-11 Young-Min Kim Physiologically active polypeptide conjugate having prolonged in vivo half-life
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