US20100113355A1 - Novel antibody molecules and nucleic acids binding to fungal stress protein hsp90 - Google Patents

Novel antibody molecules and nucleic acids binding to fungal stress protein hsp90 Download PDF

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US20100113355A1
US20100113355A1 US12/597,229 US59722906A US2010113355A1 US 20100113355 A1 US20100113355 A1 US 20100113355A1 US 59722906 A US59722906 A US 59722906A US 2010113355 A1 US2010113355 A1 US 2010113355A1
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sequence
seq
myc
domain
scfv peptide
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Naresh Chennamsetty
Stefan Ewert
Bernard Helk
Bernhardt Trout
Philipp Wechner
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the present invention relates to novel antibody molecules specifically binding to fungal stress protein hsp90, nucleic acids encoding such peptides and pharmaceutical compositions and uses thereof.
  • the antibody fragment also known as Mycograb® (Efungumab) is a fusion protein comprising the V H and V L domains of immunoglobulin connected by a linker peptide.
  • Such antibody fragments are also known as “single chain variable fragment” (scFv).
  • Mycograb® is produced by fermentation in E. coli in the form of inclusion bodies, which are extracted from the cell mass, refolded and subsequently purified by chromatographic steps under denaturing conditions.
  • Multimers in particular high molecular weight aggregates
  • Such aggregates may not be desirable for therapeutic uses.
  • the present invention now provides improved scFv peptides binding to hsp90 fungal stress protein, which have advantageous properties with respect to e.g. folding properties and/or formation of aggregates.
  • the peptides of the invention are thus particularly useful for therapeutic uses.
  • a scFv peptide comprising a V H domain and a V L domain linked by an amino acid spacer, wherein the V H domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64 and the V L domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66 and wherein the scFv peptide comprises an additional feature selected from the group consisting of
  • the V H domain comprises a sequence with at least 90%, 95%, 99% or 100% identity to the sequence of SEQ. ID NO. 64. It is also preferred that the V L domain comprises a sequence with at least 90%, 95%, 99% or 100% identity of the sequence of SEQ. ID NO. 66. It is to be understood that the additional feature is present irrespective of the level of sequence identity. For example, if the additional feature is a substitution of the amino acid at position I 29 then this substitution is present even in embodiments where the V H domain comprises a sequence with only 80% sequence identity to SEQ. ID NO. 64.
  • the scFv peptide comprises the additional feature selected from (a) to (e) or a combination thereof and further comprises the substitution of the cysteine residue in the V H domain at the position corresponding to position 28 with a tyrosine residue.
  • the substitution of the amino acid in the V H domain is selected from the group consisting of: C 28 S, I 29 S, H 68 R, N 85 S, C 97 Y, C 97 S and combinations thereof.
  • the substitution of the amino acid in the V L domain is selected from the group consisting of: V 2 I, V 3 Q, F 10 S, F 14 S, A 39 K, N 76 S and combinations thereof.
  • the scFv peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 12, 16, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 wherein Xaa denotes an amino acid residue other than cysteine and wherein the N-terminal methionine residue may optionally be cleaved off.
  • Xaa is Tyr (Y).
  • Xaa is Ala (A), Leu (L), Ile (I), Val (V), Pro (P) or Met (M); in yet another embodiment Xaa is Phe (F) or Try (W); in yet another embodiment Xaa is Gly (G); in yet another embodiment X is Ser (S) or Thr (T); in yet another embodiment Xaa is Glu (E) or Asp (D); in yet another embodiment Xaa is Gln (O) or Asn (N); in yet another embodiment Xaa is Arg (R), Lys (K) or His (H).
  • the scFv peptide comprises an amino acid sequence selected from the group consisting of SEQ. ID NO. 8, 10, 14, 18 and 22, wherein Xaa denotes the amino acid residue serine and wherein the N-terminal methionine residue may optionally be cleaved off.
  • the scFv peptide further comprises a purification tag, more preferably a sequence of 6 histidine residues at the C-terminus.
  • a scFv peptide consisting of, or consisting essentially of, an amino acid sequence as set forth SEQ ID NO. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 wherein said peptides may optionally comprise a purification tag such as e.g. a His-Tag (e.g. as set forth in SEQ ID NO. 10, 22 or 34).
  • a purification tag such as e.g. a His-Tag (e.g. as set forth in SEQ ID NO. 10, 22 or 34).
  • the purification Tags typically do not contribute to the therapeutic effect of the molecule and may therefore be removed after purification of the scFv fragments of the present invention.
  • a scFv peptide comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62.
  • a scFv peptide consisting of, or consisting essentially of, an amino acid sequence as set forth SEQ ID NO.
  • peptides may optionally also comprise a purification tag such as e.g. a His-Tag (e.g. as set forth in SEQ ID NO. 2, 4 or 20).
  • a purification tag such as e.g. a His-Tag (e.g. as set forth in SEQ ID NO. 2, 4 or 20).
  • the first Met residue of the peptides of the present invention may be also cleaved off in vivo, e.g. by E. coli MAP (methionine amino peptidase) if expressed in E. coli.
  • E. coli MAP methionine amino peptidase
  • the scFv peptides of the present invention comprise two domains linked by an amino acid spacer (the terms “spacer” and “linker” are used interchangeably), e.g. having the amino acid sequence (GGGGS) wherein n is an integer from 1 to 12, e.g. 1, 2, 3, 4 or 5.
  • One of the domains, designated as V H corresponds to the heavy chain part of the antibody fragment (corresponding e.g. to amino acid residues 2 to 122 in the scFv fragment of amino acid sequence set forth in SEQ ID NO. 2 and SEQ ID NO. 30, or amino acid residues 132 to 152 in SEQ ID NO. 32).
  • the other domain, designated as V L corresponds to the light chain part of the antibody fragment (corresponding e.g.
  • VH or the VL domain may be located at the N-terminus of the scFv peptides of the present invention, i.e. the molecules may be linked as follows: VH-linker-VL or VL-linker-VH.
  • the optional pelB signal sequence results in subcellular localisation of the peptide to the periplasmic membrane, when expressed in E. coli , in order to improve solubility of the peptide.
  • a scFv fragment comprising an amino acid sequence as set forth in SEQ ID NO. 30 or 32.
  • a scFv peptide consisting of, or consisting essentially of, an amino acid sequence as set forth SEQ ID NO. 30 or 32 wherein said peptides may optionally also comprise a purification tag such as e.g. a His-Tag.
  • the present invention provides scFv fragments comprising a VH and VL domain and a linker according to the present invention (e.g. as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 30, 32 or 34) having at least one amino acid substituted at one or more of the following positions: C29S, 130X, H69X, N86X, C98X, V139X, V140X, F147X, F151X, A176X, N213X, wherein X denotes an amino acid other than as set forth in SEQ ID NO. 2 (the numbering is as set forth in SEQ ID NO. 2 and corresponding amino acid positions in other mutants can be easily determined).
  • a linker e.g. as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 30, 32 or 34
  • the present invention provides scFv fragments having at least one of the following amino acid substitution: C29S, 130S, H69R, N86S, C98Y or C98S, V1391, V140Q, F147S, F151S, A176K, N213S. It is to be appreciated that numbering of amino acids in relation to this aspect includes the N-terminal methionine residue.
  • a scFv fragment comprising an amino acid sequence as set forth in SEQ ID NO. 24, 26 or 28.
  • a scFv peptide consisting of, or consisting essentially of, an amino acid sequence as set forth SEQ ID NO. 24, 26 or 28 wherein said peptides may optionally also comprise a purification tag such as e.g. a His-Tag.
  • a pharmaceutical composition comprising a scFv peptide according to the present invention, e.g. comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 wherein Xaa is defined as above, in combination with a pharmaceutically acceptable excipient, diluent or carrier. Details of suitable excipients are provided in Remington's Pharmaceutical Sciences and US Pharmacopoeia, 1984, Mack Publishing Company, Easton, Pa., USA.
  • Exemplary excipients include pharmaceutical grade (Ph Eur) Urea and L-Arginine (Ph Eur).
  • a typical formulation of an scFv peptide of the invention is 10 mg of pure scFv peptide, 150 mg of pharmaceutical grade (Ph Eur) Urea and 174 mg L-Arginine (Ph Eur) reconstituted in 5 ml water.
  • An scFv peptide or a pharmaceutical composition of the invention may be administered in a dosage in the range of 0.1 to 10 mg/kg body weight of the patient.
  • a dosage in the range 0.5 to 5 mg/kg body weight is preferred; with a dosage of around 1 mg/kg being particularly preferred.
  • the pharmaceutical composition may be administered orally.
  • the peptides of the present invention are useful in the treatment of fungal infections e.g. as disclosed in WO01/76627 or WO05/102386 each of which is hereby incorporated by reference.
  • the peptides of the present invention are useful in the treatment of systemic fungal infections such as invasive candidiasis or invasive aspergillosis or invasive meningitis e.g. virulent Candida species C. albicans, C. tropicalis and C. krusei and the less virulent species C. parapsilosis and Torulopsis glabrata .
  • the peptides of the present invention are also useful in the treatment of infections by Candida, Cryptococcus, Histoplasma, Aspergillus, Torulopsis, Mucormycosis, Blastomycosis, Coccidioidomycosis, Paracoccidioidomycosis organism or malaria. Accordingly, the present invention provides a method of treating a patient with a fungal infection comprising administering to the patient an effective amount of a scFv peptide of the present invention, e.g. comprising an amino acid sequence as set forth in SEQ ID NO.
  • N-terminal Met may optionally be cleaved off.
  • the present invention provides a composition or a combined preparation comprising a scFv peptide of the present invention, e.g. comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 (the N-terminal Met may optionally be cleaved off), wherein Xaa is defined as above, and a antifungal agent such as e.g. a polyene antifungal or a echinocandin antifungal or an azole antifungal.
  • a antifungal agent such as e.g. a polyene antifungal or a echinocandin antifungal or an azole antifungal.
  • antifungals useful as combination partners of scFv peptides of the present invention include e.g. amphotericin B, derivatives of amphotericin B such as AmBisome, amphotericin-B lipid complex (Abelcet), amphotericin-B colloidal dispersion (Amphocil) and amphotericin-B intralipid emulsion; nystatin; 5-fluorocytosine; caspofungin, anidulafungin, micafungin, LY303366; azoles such as isavuconazole, voriconazole, itraconazole, fluconazole, miconazole, ketoconazole, posaconazole, anidulafungin, micafungin, griseofulvin, terbinafine.
  • amphotericin B derivatives of amphotericin B such as AmBisome, amphotericin-B lipid complex (Abelcet), amphotericin-B colloidal disper
  • the scFv peptide and its combination partner are not packaged as fixed dose combinations.
  • the combined preparations of the present invention may be for simultaneous, separate or sequential use in the treatment of fungal infections.
  • the peptides of the present invention may also be used in combination with more than one antifungal agent, e.g. with amphotericin B and 5-fluorocytosine, a fungin and Amphotericin B or an echinocandin plus azole.
  • the present invention provides a method of treating a patient with a fungal infection comprising administering to the patient an effective amount of a scFv peptide of the present invention, e.g. comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 (the N-terminal Met may optionally be cleaved off), wherein Xaa is defined as above, and at least one of the antifungal agents described above.
  • Preferred combination partners are amphotericin B or derivatives of amphotericin B, caspofungin, anidulafungin, micafungin, voriconazole, itraconazole. The combination partners may be administered simultaneously, separately or sequentially.
  • the fungus causing the infection is resistant or partially resistant against an antifungal combination partner of the peptides of the invention.
  • the peptides of the present invention are also useful in the treatment of cancer, or a condition involving raised levels of TNF ⁇ and/or IL-6 such as autoimmune diseases or sepsis e.g. as disclosed in WO06/003384 or WO07/077,454 (PCT/GB2007/00.0029) each of which is hereby incorporated by reference.
  • the peptides of the present invention are useful in the treatment of leukemia such as e.g.
  • the present invention provides a method of treating a patient with a cancer disease or a condition involving raised levels of TNF ⁇ and/or IL-6 (e.g.
  • a scFv peptide of the present invention comprising administering to the patient an effective amount of a scFv peptide of the present invention, e.g. comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 (the N-terminal Met may optionally be cleaved off), wherein Xaa is defined as above.
  • the autoimmune disease is Crohn's disease, rheumatoid arthritis, ulcerative colitis or systemic lupus erythermatosus.
  • the peptides of the present invention are useful for combination therapies with anticancer agents.
  • suitable anticancer agents include doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel, cisplatin, imatinib (Gleevec®), paclitaxel, cytarabine or hydroxyurea.
  • the present invention provides a composition or a combined preparation comprising a scFv peptide of the present invention, e.g. comprising an amino acid sequence as set forth in SEQ ID NO.
  • Xaa is defined as above, and a anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel, cisplatin, imatinib, paclitaxel and hydroxyurea.
  • a scFv of the present invention e.g.
  • peptide comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 (the N-terminal Met may optionally be cleaved off), wherein Xaa is defined as above, and at least one of the anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel, cisplatin, imatinib, paclitaxel and hydroxyurea.
  • nucleic acid molecules encoding scFv peptides as described and improved nucleic acid constructs which are particularly useful for expressing such scFv peptides e.g. in E. coli .
  • the nucleic acid constructs of the present invention for instance lead to improved expression of the scFv peptides in E. coli , e.g. with respect to homogeneity and titer of the expressed scFv peptide.
  • the nucleic acid molecule further comprises the sequence (taa) n located at the 3′ end of the sequence encoding the scFv peptide wherein n is 1 or 2.
  • a nucleic acid molecule comprising a sequence encoding a V H domain comprising a sequence having at least 80% sequence identity to the sequence of SEQ ID NO. 64 and a V L domain comprising a sequence having at least 80% sequence identity to the sequence of SEQ ID NO. 66 and further comprising the sequence (taa) n located at the 3′ end of the sequence encoding the V H or V L domains wherein n is 1 or 2.
  • the present invention provides a nucleic acid molecule, e.g. a DNA or RNA molecule, comprising a nucleotide sequence as set forth in SEQ ID NO. 3, 5, 11, 15, 19, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61 wherein nnn denotes a codon coding for an amino acid other than Cys.
  • nnn may code for Tyr such as e.g. TAT.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO.1, 3, 5, 11, 15 or 19.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO. 7, 9, 13, 17 or 21 wherein nnn denotes a codon coding for a serine residue.
  • nucleic acid sequences can be readily modified without altering the encoded amino acid sequence.
  • nucleic acid molecules which have (i) at least 80% identity, preferably at least 90%, 95%, 99% or 100% identity to SEQ. ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61; or (ii) hybridize under high stringency conditions to the nucleic acid molecules having a sequence as set forth in SEQ ID NO.
  • high stringency conditions is readily understood by the skilled person and may refer, e.g., to washing in 6 ⁇ SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos). Suitable ranges of such stringency conditions for nucleic acids of varying compositions are described in Krause and Aaronson (1991) Methods in Enzymology, 200:546-556.
  • the present invention provides a vector molecule comprising a nucleotide sequence of a nucleic acid molecule of the invention, e.g. as set forth in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61.
  • such vector molecule is suitable for expressing the nucleic acid molecules as set forth in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61. in e.g. E. coli .
  • suitable expression vectors are readily known to the skilled person.
  • An example of suitable vector includes for instance pGEX or pET.
  • Another embodiment provides a host cell, e.g. E. coli , comprising such a vector molecule.
  • a method for producing a scFv peptide of the present invention e.g. comprising an amino acid sequence as set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 62 (the N-terminal Met may optionally be cleaved off), wherein Xaa is defined as above which comprises culturing a host cell having incorporated therein an expression vector containing under control of suitable transcriptional control elements a nucleic acid sequence of a nucleic acid molecule of the invention e.g. as described in SEQ ID NO.
  • the percentage “identity” between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schulffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-PLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402) using default parameters.
  • the BLAST algorithm can be accessed on the Internet using the URL http://www.ncbi.nlm.nih.gov/blast/.
  • FIG. 1 is a diagram showing schematically the sequence of the wild type Mycograb scFv peptide and Mycograb mutants. Stop codons of the nucleic acid molecules encoding the respective peptides are also shown at the C-terminal end.
  • FIG. 2 is a diagram showing the principle of the ELISA assay of Example 2.
  • FIG. 3 is a graph in which the black bars show yield after solubilization with NLS of all investigated mutants.
  • the error bars show the Standard deviation for samples analyzed twice.
  • the white bars are a graphic representation of mass balance after NLS refolds of all investigated mutants. Mutants are ranked according to increasing refolding recovery values.
  • FIG. 4 shows graphs indicating the recovery after refolding for 5 selected mutants when urea and DTT was used as solubilizing agent (A) and when GuHCl and DTT were used as solubilizing agent (B).
  • White bars Recovery when mass of protein found in the IB.SOL solution was used for calculation (equ. 1)
  • Black bars Refolding recovery when mass of protein found in the IB.RES solution is used for calculation.
  • FIG. 5 is a graph showing the time required for a visible beginning clarification of a solubilization solution after addition of 4% NLS (white bars) and the time required until no further clarification was observed (black bars) for all tested mutants. Mutants are ranked according to the start time in ascending order.
  • FIG. 6 is a variability chart for the response start of solubilization and indicates number of cysteines, number of linker elements and if the heavy (vh) or light chain (vl) fragment was at the N-terminus.
  • Mutant Myc 106 had the fastest solubilization start but contained 5 cysteines.
  • FIG. 7 shows chromatograms as an overlay of REF end samples of Mutants MYC 135, Myc 130, Myc 133, Myc 119, Myc 123 wild type and Myc 116.
  • FIG. 8 shows chromatograms as an overlay of REF end samples of Mutants MYC 134, Myc 137, Myc 138, Myc 106 Myc 136, Myc 123 and Myc 139.
  • FIG. 9 shows scaled estimates and a prediction profiler of the following parameters: linker length, number of cysteines and Vh/Vl arrangement for the response retention time of mutants.
  • the scaled estimates predict to what extent the retention time would shift when the parameter is increased from centerpoint (the red number in the prediction profiler plot on the x-axis) to a higher level.
  • FIG. 10 is a plot of linker length versus retention time measured in RP-HPLC (RPC2) for tested mutants. The early retention time of MYC 130 compared with the other mutants is highlighted.
  • FIG. 11 shows a normalized overlay of all REF.END samples from FIGS. 7 and 8 for estimation of peak area from peak 2.
  • FIG. 12 shows a RP-HPLC 2 chromatogram overlay of a REF.End sample of MYC 119 solubilized with 8M urea+DTT, 8M urea, 6M GuHCl+ DTT and 6M GuHCl dilution was 1:50 with a buffer containing 20 mM Tris, 2 mM cysteine, 1% NLS, pH 9.0.
  • FIG. 13 is an RPC 2 chromatogram of a REF.End sample of MYC 119 after solubilization with 6M urea and 5 mM DTT and subsequent refolding by a 1:10 dilution.
  • FIG. 14 is an image of a gel following SDS Page analysis of REF.IM and REF.END sample of MYC 1.19 after solubilization with 6M urea and refolding by a 1:10 and 1:50 dilution, respectively.
  • Lanes 1-8 non reducing SDS Page
  • lanes 9-14 reducing SDS Page.
  • R reducing
  • n-r non reducing
  • FIG. 15 is an RP-HPLC chromatogram overlay (RPC 2) of an inclusion body sample from mutant MYC 119 after solubilization with 6 M urea (black) and 4% NLS (blue).
  • RPC 2 RP-HPLC chromatogram overlay
  • FIG. 16 shows images of: left gel: Reducing (r) SDS-Page for Mutants MYC 118, 119, 130, 133, 134, 135 and 137; and right gel: Non-reducing (n-r) SDS Page of the same samples
  • FIG. 17 shows images of: left gel: Reducing SDS-Page for Mutants MYC 106, 123 wt, 136, 138, 139 and 140; and right gel: Non-reducing SDS Page of the same samples
  • FIG. 18 is an overlay of SEC HPLC 0.5% NLS chromatograms of REF.End samples for the mutants Myc 118, Myc 119, Myc 130, Myc 133 and Myc 135. IBs from these mutants were isolated at bench scale.
  • FIG. 19 is an overlay of SEC HPLC 0.5% NLS chromatograms of REF.End samples for the mutants. Myc 134, Myc 136, Myc 137, Myc 138, Myc 139, Myc 140, Myc 106 and Myc 123 wild type. IBs from these mutants were isolated in the pilot plant.
  • FIG. 20 shows a scatter plot and linear regression (continuous line) of measured MW versus theoretical MW of REF.End samples for all mutants. The 95% confidence interval for the fit is also shown (dashed line). The dot at top left shows MYC 130. The dots within the dashed lines are within the 95% CI and therefore not significantly different from the wildtype. The dots below both dashed lines represent mutants with lower average MW than predicted and the dots above both dashed lines represent mutants where a higher average MW was measured than predicted.
  • FIG. 21 shows SEC-HPLC (formulation) chromatograms for REF.END samples of Myc 119, Myc 106-origami, Myc 123 wt and Myc 137 after UFDF against 50 mM Tris, pH 9.0 buffer. Samples were taken after each volume reconstitution. Sample prior to UFDF treatment (5), after 1 st ‘buffer exchange step (2), 2 nd buffer exchange step (3), 3 rd (4) and last (5) step.
  • FIG. 22 shows RP-HPLC 2 chromatograms of REF.IM, REF.3T and REF.END samples for all tested mutants:
  • SEQ ID NO. 9 is MycoC29X-6H-TAA, e.g.: Myc113, MycoC29S-6H-TAA
  • E. coli host cells are transformed with the expression vector and cultivated in submers culture.
  • expression of scFv is induced by derepression or activation of the inducible promoter (i.e. tac, trc or T7-lac promoter).
  • the inducible promoter i.e. tac, trc or T7-lac promoter.
  • This induction leads to accumulation of scFv in the host cell, resulting in production of insoluble inclusion bodies mainly made of aggregated scFv.
  • cells are harvested by centrifugation and disrupted.
  • the insoluble inclusion bodies are subsequently isolated by gravimetric means.
  • the scFv peptides are accumulated in the form of inclusion bodies within the E. coli cells.
  • inclusion bodies are isolated and the product is extracted by solubilization and refolding. Purification to over 95% purity is achieved by ion exchange chromatography and immobilized metal affinity chromatography (IMAC).
  • MYC123 wild type Mycograb
  • mutant Mycograb peptides were detected in an ELISA using the peptide epitope of hsp 90 as antigen.
  • Mycograb or mutant Mycograb became bound to biotinylated peptide, which in turn was bound to streptavidin-coated microtitre plates.
  • a scrambled peptide was used as a control sequence.
  • Detection was accomplished using a peroxidase conjugated anti-His antibody, which binds to the His region of the MYC123 protein.
  • the peroxidase reacted with the ABTS substrate to produce a green substance, the absorption of which was measured at 405 nm.
  • the absorption at 405 nm is proportional to the activity of MYC 123 in the solution.
  • the activity was determined from the 6-point calibration curve for a reference standard and was indicated as % activity compared with the reference.
  • FIG. 2 The principle of the ELISA is depicted in FIG. 2 in which Streptavidin 1 is coated on a plate and is bound to biotin 2.
  • the biotin 2 is, in turn, bound to the Hsp 90 peptide 3 which is located in the Hsp90 binding site 4 of the MYC 123 scFv peptide 5.
  • the scFv peptide 5 has a His tag 6 to which becomes bound the Anti-His-Peroxidase detection antibody 7.
  • the ELISA utilised was a direct detection assay where Mycograb or mutant Mycograb was captured using a streptavidin surface microplate coated with a biotinylated antigenic peptide (Biotin-NKILKVIRKNIVKK—epitope sequence from candidal Hsp90). The presence of Mycograb or mutant Mycograb was then detected using an anti-His tag antibody conjugated to horse radish peroxidase. ABTS, a substrate for the horse radish peroxidise, was then added to the wells, and the concentration of Mycograb present was proportional to the absorbance measured at 405 nm. The activity of samples of Mycograb or mutant Mycograb was determined directly from a standard curve generated using the pre-existing Mycograb drug product reference material.
  • Streptawell High Bind microtitre plates were supplied by Roche (Cat No.11989685001). Assays were performed using a Bio-Rad Model 680 microplate reader. Hardware control was performed using the Microplate Manager Software version 5.2.1 (Bio-Rad, USA). Data analysis was performed using Microsoft Excel.
  • Blocking buffer Stock 1 (5% w/v BSA in Milli-Q water)
  • Blocking buffer Stock 2 (Wash Buffer+5% w/v BSA)
  • Blocking buffer Stock 2 1 mL was added to 49 mL of Wash buffer. Made fresh for each experiment.
  • a 2 mg/ml solution of the custom synthesised antigenic peptide solution was made up by weighing out 10 mg of peptide and dissolving it in 5 ml of Milli-Q water. 50-100 ⁇ l aliquots were dispensed into 1.5 ml Eppendorf tubes and stored frozen at ⁇ 80° C. for up to one year.
  • Antigenic peptide Working Solution (4 ⁇ g/mL peptide in Peptide Diluting Buffer) 25 ⁇ L of Antigenic peptide solution (2 mg/mL) was added to 12.475 mL of Peptide Diluting Buffer to give a 4 ⁇ g/mL solution. Made fresh for each experiment.
  • Control Article material was diluted in Sample Diluting buffer to give 5 mL of a 5 ⁇ g/mL top concentration sample. This solution was then used to generate two-fold serially diluted samples each in a final volume of 2 mL over a concentration range of 5-0.156 ⁇ g/mL.
  • the plate wells were then washed 3 ⁇ 30 sec with 200 ⁇ l of PBS 0.1% (v/v) Tween 20 buffer on a Thermo WellWash AC.
  • Mycograb and mutant Mycograb samples were prepared prior to loading by diluting down to 5 ⁇ g/ml in 20 mM Tris buffer pH 7.8, 0.1% (w/v) BSA from the resuspended Mycograb vial stock solution.
  • Mycograb® samples were then prepared from this initial 5 ⁇ g/ml solution by serial dilution ⁇ 2 down to 0.15625 ⁇ g/ml with 20 mM Tris buffer pH 7.8, 0.1% (w/v) BSA. All the individual dilutions were performed in either 1.5 ml Eppendorf tubes (VWR Cat No 211-2139) or 7 ml Bijoux containers (VWR Cat. No. 215-0328), depending on the amount required for the experiment. 100 ⁇ l of each dilution sample was then loaded onto the plate in triplicate. A control set of blank wells containing 100 ⁇ l 20 mM Tris pH 7.8, 0.1% (w/v) BSA was also included in Row H.
  • mouse monoclonal Anti-His HRP conjugate (Sigma A7058) was loaded into each well at a concentration of 1:2000 in 0.1% (w/v) BSA PBS-0.1% (v/v) Tween 20 and left for 1 hour at room temperature.
  • Samples 1, 3 and 4 are process intermediates (not final drug substance) obtained after prepurification of inclusion bodies, refolding and removal of detergent NLS by Dowex chromatography or diafiltration).
  • Sample 2 is an original wild type drug product produced by Biomeva/Thymoorgan and used in Phase III trials. The specification for the original drug product was 75-125% of the reference standard. All samples were judged as active (binding).
  • the antimycotic activity of MYC123 using Cryptococcus neoformans as model organism, was determined. This assay measures antifungal activity and may mimic the action of MYC 123 in the clinical setting.
  • MYC123 The MIC of MYC123 was determined by broth micro dilution according to the National Committee for Clinical Laboratory Standards document M27-A2 (2002). Briefly: RPMI medium was inoculated with 10 3 CFU/ml of C. neoformans . MYC 123 was added in decreasing concentrations to the medium (1024 ⁇ g/ml, 512 ⁇ g/ml, 256 ⁇ g/ml . . . ). The MIC plates were incubated at 37° C. for 72 h. The endpoints were determined as the concentration to produce optically clear wells (MIC-0) and the concentration resulting in a prominent decrease in turbidity 50% growth inhibition, MIC-2) compared with the growth control.
  • Safety cabinet A SAB plate was inoculated with C. neoformans and incubated for 48-72 hr at 35° C. The plate was sealed with parafilm.
  • RPMI RPMI was prepared. Antifungal agents were prepared according to NCCLS methodology (M27-A2)—total of 11 concentrations in RPMI growth medium. Concentrations were at 2 ⁇ the final concentration required for single MIC
  • Inoculum Preparation Direct colony suspension.
  • a direct colony suspension of Cryptococcus neoformans was made from a 48-72 hr old plate into RMPI medium. This was adjusted to 0.5 MacFarlands standard (approx 1 ⁇ 10 6 -5 ⁇ 10 6 cfu/ml). A 1:50 dilution was made. A further 1:20 dilution (approx 1 ⁇ 10 3 -5 ⁇ 10 3 cfu/ml, 2 ⁇ inoculum required) was made
  • the MIC plates were incubated at 37° C. for 72 hrs.
  • the inoculums suspension were serially diluted and 10 ⁇ l of the dilutions were plated out onto a SAB plate and incubated at 37° C. for 72 hrs.
  • Samples 1 and 3-6 were process intermediates (not final drug substances) obtained after prepurification of inclusion bodies, refolding and removal of detergent NLS (by Dowex chromatography or diafiltration).
  • Sample 2 was on original wild type drug product produced by Biomeva/Thymoorgan and used in Phase III trials.
  • the aim of the Mycograb mutants was to obtain a mutant scFv peptide with improved structural properties compared with the wild type Mycograb. It was believed that through point mutations, especially the replacement of free cysteine by tyrosine, aggregation and formation of incorrect disulfide bonds during down stream processing should be reduced. It was also believed that exchanging the orientation of the heavy chain fragment with the light chain fragment and removing the HIS-Tag is be beneficial for formation of a native 3D structure of the Mycograb molecule.
  • constructs were sequenced prior to fermentation.
  • the fermentation was scaled up to deliver enough material for inclusion body (IB) isolation and for further downstream processing.
  • the expression constructs of the mutants were purified according to the adapted Biomeva process until the refold end step.
  • IB Inclusion body isolation 4 L of fermentation broth obtained in LVA (Labor Mits GmbH) from shake flask culture of Mutants Myc 118, 119, 130, 133 and Myc 135 were disintegrated with a high pressure homogenizer (LAB 40-15 RBFI) in RPP4 at 700 Bar for 2 cycles of 15 min each.
  • the IBs were separated from the cell debris at lab scale with a bottle centrifuge at 10 000 rpms for 20 min at 4° C. IBs were washed twice with water for laboratory use (WFL) and afterwards, a 20% (w/v) suspension in WFL was prepared. The suspension was stored in aliquots at ⁇ 20° C.
  • IBs from mutants Myc 134, 137, 138, 106, 136, 139, 140 and Myc 123 (wt) were isolated at pilot scale in RPP4 because fermentation of these mutants was done at 30 L scale in bioreactors. IBs were separated from cell debris with a disc stack centrifuge. A 20% suspension (w/v) in WFI was prepared. This suspension was stored in aliquots at ⁇ 20° C.
  • Solubilization with NLS (according to adapted Biomeva process). Solubilization of the 20% IB suspension was done by dilution with WFL to a protein concentration of 8 mg/ml followed by a 1:2 dilution with 100 mM Tris/Base, 4% NLS, pH 9.0 buffer. The solution was stirred at room temperature in a beaker until clarification but at least for 30 min. The time until start and end of clarification was recorded.
  • Refolding with NLS was done by 1:4 dilution of the solubilization solution with a 50 mM Tris/Base buffer. The final concentration of NLS was 0.5%. Refolding was initiated by addition of 50 ⁇ M CuCl 2 . Samples were taken and immediately submitted for RPC2 analysis prior and after CuCl 2 addition, then approximately 24, 48, 72 and 96 hours after CuCl 2 addition.
  • Refolding after solubilization with Urea, GuHCl Refolding of a Mycograb solution after solubilization with 8M urea or 6M GuHCl+/ ⁇ DTT was performed by 1:50 dilution with a buffer containing 20 mM Tris/Base, 1% NLS and 2 mM Cystin at pH 9.0.
  • dilution of a Mycograb solution after solubilization with urea by 1:10 with a buffer containing 20 mM Tris/Base, 0.5M L-arginine and 2 mM Cystin at pH 9.0 was also performed.
  • the refold solution was stirred for 96 hrs at 4° C. or 24 hrs at RT, respectively.
  • a refold kinetic was recorded for mutant Myc 119 in order to determine the required refolding time.
  • a sample of a 20% IB suspension of Myc119 was solubilized as described above. Refolding was performed as described above, but samples were taken at respective time intervals and analyzed by RPC 2.
  • NLS removal by UF/DF from refolding solution Mutants Myc 119, Myc 137, Myc 106 and Myc 123 (wt) were solubilized and refolded as described above. After refolding, a buffer exchange of REF.END solution was performed with an Amicon stir cell with 10 kDa molecular weight cut-off. NLS concentration after each turn over volume was determined with RP-HPLC. 50 ml of REF.END solution were concentrated to 25 ml and then filled up again to 50 ml with diafiltration buffer. This procedure was carried out 4 times. Aggregation tendency after NLS removal was measured with SEC-HPLC running with formulation buffer.
  • SDS Page was performed using NuPAGE 4-12 BisTris gels and MOPS as running buffer. The run time was 65 minutes at 200 volt. A mass of 0.2-0.4 ⁇ g Mycograb was applied on each lane. After electrophoresis, the gels were stained with silver. For reducing SDS Page, 100 mM DTT was added to the sample.
  • Table 5 gives a description of the analytical methods listed in Table 4 that were used for evaluation of the mutants. A comment is included describing the specificity of the particular assay.
  • Protein concentration prior and after solubilization and refolding of IB's was measured with the titer assay. As this assay measures all soluble protein present in the sample, mass balance should yield 100%. Mass balance for solubilization was calculated using equ. 1.
  • mg RPC1 IB.SOL is the mass of protein in the solubilization solution' of the IB's calculated from concentration measurement by RPC 1 method and volume of IB SOL solution.
  • mg RPC1 IB.RES is the mass in the 20% IB suspension calculated from concentration measurement by RPC 1 method and volume of the solution after resuspension of the isolated IB's in DI.
  • Mass balance for refolding was calculated using equ.2 and is expressed as % recovery. IB's solubilized with either 4% NLS, 8 M urea+/ ⁇ 5 mM DTT or 6M GuHCl+/ ⁇ 5 mM DTT were diluted and refolded as described in 3.3.1 and 3.3.2, respectively.
  • mg RPC1 Ref.END is the mass of protein according to concentration measurement with RP-HPLC found in the refolding solution times volume of refolding solution.
  • mg RPC 1 IB.SOL is the mass of protein found in the IB solubilisate, % REF is the recovery after refolding.
  • Table 6 shows the recovery after solubilization with NLS of the IB_RES suspension and recovery after refolding calculated from analytical method RPC I for all tested mutants. Also shown is the protein concentration in the IB_RES solution and protein concentration in the refolding solution determined by analytical method RPC I.
  • Table 7 shows protein concentration determined by RPC I of IB_RES, IB_SOL and REF.END samples after solubilization with urea (SOL:urea) and solubilization with GuHCl (SOL:GuHCL). Refolding time was 96 hrs at 4° C. The dilution factor was 500 and 50 for IB_RES and IB_SOL, respectively to yield the REF.END solution.
  • Mass balance of solubilization was exceeding 100% for 10 of the 12 investigated mutants. This could be due to the fact that the IB suspension was a crude sample type and eventually, the IB's were not completely dissolved when the sample was taken, leading to an inhomogeneous solution and thus underestimating total protein concentration. Data variability is high, with a relative standard deviation for 6 mutants analyzed twice ranging from 2.6% (Myc 138) to 42.9% (Myc 133).
  • % Recovery after refolding was between 72% (Myc 138) and 99% (Myc 134). The expected recovery is 100% (similar to recovery after solubilization). Recoveries after refolding were all lower than 100% and scatter not as much as for recovery after solubilization. This indicates that estimation of protein concentration in IB.SOL and REF.end samples is more accurate than in IB.RES samples. However, calculation of recovery primarily serves as a control for solubilization and refolding experiments.
  • the recovery varies from 44% to 230% reflecting the problems with measurement of protein concentration especially in IB.SOL and IB.RES samples, possibly due to insufficient homogenization prior to sampling.
  • Solubilization time with NLS was studied for all mutants. The time until the solution started to become clear and the time until no further clarification could be observed was recorded and is illustrated in FIG. 5 .
  • FIG. 6 shows a variability chart where START [min] of clarification is plotted versus the 3 categories: alignment of heavy or light chain fragment at the N-terminus, number of linker elements and cysteine residues.
  • Myc 134 indicated with 1 in FIG. 6 , data points in category with 4 Cysteines scatter around earlier solubilization start times than compared with data points in the category with 5 cysteines.
  • FIG. 7 An overlay of REF.END samples from all investigated mutants including the wild type (MYC 123) is shown in FIG. 7 and FIG. 8 .
  • Mutant Myc 116 was screened earlier in lab DSP-DEV 1 and was included in the overlay for comparison reasons.
  • FIG. 7 chromatograms of REF.END samples generated from IB's isolated at bench scale and in FIG. 8 , chromatograms of REF.END samples generated from IB's isolated at larger scale in the pilot plant are shown.
  • Retention time varies greatly with molecule construct. There is no trend of retention time (reflecting hydrophobicity) increasing with linker length, as would be expected.
  • One linker element consists of four Glycines and one Serine residue. Glycine is hydrophobic in contrast to Serine, which is hydrophilic. However, the 4 ⁇ higher Glycine content in the linker seems not to significantly increase the hydrophobicity as measured by retention time in RPC 2.
  • Retention time of Myc 130 is shorter than for the rest of the mutants. 10 amino acids were replaced by amino acids of more hydrophilic (5 serines) nature, thus decreasing the hydrophobicity of the molecule and consequently resulting in earlier retention time. Excluding this data point from statistical analysis results in a model showing significant difference in retention time when the orientation of the VL is N-terminal compared to an orientation when its C-terminal. Retention time increases by 0.41 min when the VL element is located N-terminal compared with when its located C terminal.
  • FIG. 9 shows the scaled estimates and a prediction profiler of the model with number of cysteines, linker length and chain fragment orientation as factors and retention time as response.
  • the area ratio of monomer/dimer peak to aggregate peak was determined by normalizing peak 1 to the same peak maximum.
  • the peak area of peak 2 after normalization was ranked according to increasing size using visual area estimation.
  • the normalized overview is shown in FIG. 11 .
  • Mutants Myc 118, 119, 130 and Myc 133 were dissolved with 7.6M Urea+/ ⁇ DTT and 5.6M GuHCl+/ ⁇ DTT and refolding was initiated by dilution in refolding buffer.
  • the monomer was expected to elute at approximately 10.5 min. Peaks eluting earlier are not identified and were not observed in refolds with 0.5% NLS. The huge peak 2 indicates strong aggregation. Similar elution profiles were obtained for all REF.End samples after urea/GuHCl solubilization.
  • FIG. 15 shows an overlay of HPLC chromatograms.
  • Reducing and non-reducing SDS-Page was performed to determine impurities and aggregates content in REF.End samples. With reducing SDS-Page Mycograb species appeared as monomeric and dimeric band and host cell impurity content in the sample could be distinguished from aggregated species when compared to a non-reducing SDS-Page gel. Non-reducing SDS Page showed Mycograb monomers, dimers and aggregates. Comparing a non-reduced SDS-Page silver stain gel with a reducing SDS-Page analysis, the amount of aggregated species could be evaluated semi-quantitatively. Reducing and non-reducing SDS-Page gels of REF.END samples of all tested mutants are shown in FIG. 16 and FIG. 17 .
  • the gel in FIG. 16 on the left side shows a reducing SDS-Page of REF.End samples from mutants MYC 118, 119, 130, 133, 134, 135, 137.
  • the band at 30 kDa is monomeric Mycograb and it is predominant in all samples. According to the migration of the monomeric band, Mycograb expressed in mutant MYC 134 seems to have higher molecular weight than the other mutants analyzed on the gel. The same but to a lesser extend was detected for MYC 135. According to Table 9, MYC 134 has the highest theoretical molecular weight among the mutants shown in FIG. 16 , followed by MYC 135.
  • FIG. 17 shows a reducing and non reducing SDS Page of REF.End samples from mutants MYC 106, 136, 138, 139, 140 and the wild type, MYC 123. Differences in MW for the different constructs could be determined according to different migration of the monomeric band. The bands in lanes 5 and 7 appeared at a slightly lower MW than the bands in lanes 2, 3, 6 and 4 which was in agreement with the theoretical MW listed in Table 9.
  • the average molecular weight ranged from 48.6 kDa to 65.8 kDa.
  • the broadness of the peaks reflects the heterogeneity of species in the sample. Though approximately 80% of the product was monomeric in REF.End samples, dimers and higher MW species were present as well as non-product related impurities, resulting in a broad elution peak.
  • the elution profile of Myc 130 sticks out because of increased fronting compared with the other investigated samples. This might have been due to increased heterogeneity in the sample because of the construct's nature (more hydrophilic) or due to an accidentally different sample treatment.
  • Samples shown in FIG. 18 were prepared simultaneously and stored at 4° C. for 5 days prior to analysis.
  • Samples shown in FIG. 19 were prepared simultaneously and stored at 4° C. over night days prior to analysis. Samples seemed to be stable at 4° C. as MW of the constructs were in a similar range.
  • Fronting of Myc 130 may have been due to higher amount of aggregated species compared with the other investigated samples.
  • SDS-page of Myc 130 did not indicate a higher impurity content and increased heterogeneity of the sample.
  • Other factors leading to fronting in SEC such as column overloading and increased temperature during analysis can be excluded as all samples were analyzed on the same day.
  • Theoretical MW was plotted versus MW determined by SEC and a linear relationship with a correlation coefficient of 0.77 was found when the data point for Myc 130 was excluded.
  • assay variability has to be taken into account. Additionally, from FIG. 19 it can be seen that the mass of injected protein was not always the same as peak area for some of the samples. Formation of covalent aggregates should be decreased for mutants with 4 cysteines as compared with Mycograb with 5 cysteines because no free cysteine in mutants with 4 cysteines is available after formation of intermolecular SS bridges. Intermolecular covalent aggregates are formed during refolding even with a mutant with only 4 cysteines but it would be likely that the amount is lower than for a mutant with 5 cysteines.
  • the NLS concentration was lowered by an average factor of 5 from the REF.End solution with ultra/diafiltration using a stir cell.
  • the total buffer volume used during diafiltration divided by the retentate volume is the diafactor and was 2.5.
  • the elution buffer contained 0.5M urea buffer but no NLS to suppress aggregation.
  • Increase in molecular weight was used as a measure of tendency to aggregate and allows the mutants to be compared.
  • Mutants Myc 137, Myc 106, Myc 119 and the wild type Myc 123 were selected for a first set of experiments.
  • Table 10 shows the molecular weight (MW) in kDa determined by SEC-HPLC running in formulation buffer and concentration of NLS (%) determined by RP-HPLC for REF.End samples from mutants MYC 119, 137, 106 and MYC 123 before and after UFDF. The NLS reduction factor was calculated from NLS concentration in the sample prior to UFDF (#2) divided by the concentration of NLS after UFDF (#3). The % increase MW based on SEC HPLC 0.5% NLS was calculated from the MW of the REF.END sample (#1) and the MW after UFDF (#3) for the respective Mutant.
  • the sample prior to UFDF still contained 0.5% NLS. As the sample migrated through the column, NLS was more strongly retarded than the protein and consequently aggregation occurred. Therefore this analytical method was not suited to determine molecular weight of samples containing NLS.
  • MYC 123 has a lower aggregation tendency than MYC 119 based on these data.
  • the analytical SEC HPLC may have an influence on the protein structure and on the formation of aggregates.
  • increase of MW is calculated from data obtained from two different analytical methods and it cannot be assessed if the MW of a sample is similar when it is determined with the two different methods.
  • the mutants are ranked according to increase in MW after NLS removal together with the mutations. It has to be noted that the two mutants with a 3 ⁇ linker element have significantly lower % of MW increase compared with mutants with a 4 ⁇ linker element.
  • % molecular weight Linker VH-VL MUTANT increase HIS tag length alignment cysteins MYC 123_Wt 180 YES 3X VH N-terminal 5 MYC 106 199 YES 3X VH N-terminal 4 origami MYC 137 312 NO 4X VH N-terimnal 4 MYC 119 397 NO 4X VL N-terimnal 5
  • FIG. 21 shows an overlay of the SEC HPLC chromatograms obtained from the sample prior to UFDF and after each volume reconstitution.
  • the shape of the elution peaks did not significantly change with reduction of NLS concentration. Consequently, the MW of the sample also remained constant with reduction of NLS. This might be an indication that there is a limit in the concentration of surfactant below which aggregation is initiated but does not proceed further.
  • the impact of the analytical method on the MW of the sample is not known and may be the reason why all samples have similar MW.
  • c retentat is the concentration of NLS in the retentate
  • c feed is the concentration of NLS in the feed solution.
  • R is the retention, the fraction of solute that is retained by the membrane.
  • VCF is the volume concentration factor and N is the diavolume which is the total buffer volume introduced to the operation during diafiltration divided by the retentate volume.
  • the discrepancy between the theoretical and the measured concentration of NLS in the retentate is an indication for retention of NLS by the membrane greater than 0. This might be due to interaction between NLS and the protein, membrane and/or other components. The ability to deform can also cause unwanted retention.
  • a correctly folded construct with 5 cysteines forms an S—S bond between Cys 23 and Cys 97 which corresponds to T3 and T9 respectively.
  • the bond T3 T9 is located in the light chain.
  • the other disulfide bond is on the heavy chain between Cys 159 and Cys 224, corresponding to T12 and T17.
  • the 5 th cysteine is located at Cys28 and corresponds to the T4 peptide.
  • a construct with 4 cysteines always lacks the Cys 28 residue, the correct S—S bridges are similar as for a construct with 5 cysteines.
  • Mutants 118, 119, 130, 135, 133, 134, 137 and C28Y+HIS (106) as well as C28Y-HIS (108) were analyzed in lab AL1.
  • Mutants 106 origami, 136, 138, 139, 140 and the wild type 123 were analyzed in analytical lab AL2 with a different device.
  • the sensitivity of the mass spectrometer in AL1 is higher than that in AL2 and therefore, a semi-quantitative analysis of mutants analyzed in AL2 could not be obtained. However, it was possible to determine if correctly formed SS bridges are present.
  • a ‘correctly’ folded Mycograb with 5 cysteines should give a significant signal for free SH at the T4 peptide and no signal corresponding to other free SH groups. Additionally, a strong signal for the correct disulfides T3-T9 and T12-T17 is expected and incorrect SS bonds should not be present. Lastly, no intermolecular SS bonds should be present.
  • a ‘correctly’ folded Mycograb with 4 cysteines should not have any free SH groups. Only the correct S—S bonds T3-T9 and T12-T17 should be detected. Additionally, no intermolecular SS bonds should be present.
  • Table 12 shows that neither the wild type nor any of the mutants gave strong signals for the correct S—S bonds only. It has to be considered that the REF.End sample consists of a population of differently folded and covalently aggregated species, so that a mixture of all possible combinations of disulfide bonds and free SH groups is present. However, a promising mutant should at least show significant signals for both of the correct S—S bridges which is the case for MYC 137.
  • Mutants Myc 118, 119, 130, 133, and 137 gave stronger signals for correct S—S bonds than the wild type. However, it has to be considered that Myc 123 was analyzed with a different mass spectrometer of lower sensitivity and hence signal intensities cannot be compared. Signals from peptides of Myc 123 can be compared with signals from mutants 106 origami, 136, 138, 139 and 140. For none of these constructs, correct disulfide bridges were obtained. Only signals for incorrect disulfides were found.
  • Mutant Myc 130 showed strong signals for the T12-T17 SS bond, but the second correct disulfide was not found.
  • MW determined with SEC HPLC is very dependent on the buffer matrix in the sample.
  • a couple of SEC HPLC methods had to be established with a running buffer similar to the buffer of the sample.
  • the matrix dependency of MW determination makes comparison of MW across process steps difficult (Table 10).
  • the most beneficial effect of the mutations can be attributed to the removal of the 5 th cysteine.
  • the number of correct disulfide bonds was increased compared with constructs with 5 cysteines. Additionally, solubility of the IBs was enhanced compared with constructs with 5 cysteines.
  • the tendency of the peptides to aggregate after NLS removal may be increased with the number of linker elements.

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CN105652011B (zh) * 2014-11-20 2017-09-22 深圳市安群生物工程有限公司 检测人HSP90α‑2蛋白的荧光免疫层析试纸及其制备方法
CN113845591B (zh) * 2018-10-30 2023-10-31 迈威(上海)生物科技股份有限公司 Hsp90抗体及其在抗真菌感染中的应用
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Publication number Priority date Publication date Assignee Title
US10487151B2 (en) * 2014-07-23 2019-11-26 Ohio State Innovation Foundation Methods and compositions related to antibody fragments that bind to tumor-associated glycoprotein 72 (TAG-72)
US11787872B2 (en) * 2014-07-23 2023-10-17 Ohio State Innovation Foundation Methods and compositions related to antibody fragments that bind to tumor-associated glycoprotein 72 (TAG-72)

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CR11059A (es) 2009-11-20
US20100166758A1 (en) 2010-07-01
CA2685136A1 (en) 2008-11-06
EP2152746A1 (en) 2010-02-17
WO2008132134A1 (en) 2008-11-06
BRPI0810827A2 (pt) 2014-10-29
CN101679517A (zh) 2010-03-24
EA200901441A1 (ru) 2010-04-30
AR066311A1 (es) 2009-08-12
AU2008244308A1 (en) 2008-11-06
IL201445A0 (en) 2010-06-16
US20090136484A1 (en) 2009-05-28
WO2008132152A1 (en) 2008-11-06
EP2144932A1 (en) 2010-01-20
ECSP099765A (es) 2009-12-28
US7722869B2 (en) 2010-05-25
KR20100017387A (ko) 2010-02-16
CL2008001202A1 (es) 2008-12-19
PE20090728A1 (es) 2009-07-02
WO2008132134A9 (en) 2009-07-09
MA31384B1 (fr) 2010-05-03
TN2009000442A1 (en) 2011-03-31
GT200900276A (es) 2010-05-21
JP2010524485A (ja) 2010-07-22
MX2009011595A (es) 2009-12-08
TW200902714A (en) 2009-01-16

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