EP1161440A1 - PROCEDE DE REPLIAGE DE MOLECULES DE POLYPEPTIDES CONTENANT DES DOMAINES Ig - Google Patents

PROCEDE DE REPLIAGE DE MOLECULES DE POLYPEPTIDES CONTENANT DES DOMAINES Ig

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Publication number
EP1161440A1
EP1161440A1 EP00911054A EP00911054A EP1161440A1 EP 1161440 A1 EP1161440 A1 EP 1161440A1 EP 00911054 A EP00911054 A EP 00911054A EP 00911054 A EP00911054 A EP 00911054A EP 1161440 A1 EP1161440 A1 EP 1161440A1
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EP
European Patent Office
Prior art keywords
polypeptide
domain
polypeptides
protein
chaperone
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00911054A
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German (de)
English (en)
Inventor
Alan Roy MRC U. for Pro. Function and D. FERSHT
Myriam Marlenne Altamirano
Adrian MRC Lab. of Molecular Biology WOOLFSON
Cesar MRC Lab. of Molecular Biology MILSTEIN
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Medical Research Council
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Medical Research Council
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Publication date
Priority claimed from GBGB9906056.8A external-priority patent/GB9906056D0/en
Priority claimed from GBGB9925985.5A external-priority patent/GB9925985D0/en
Application filed by Medical Research Council filed Critical Medical Research Council
Publication of EP1161440A1 publication Critical patent/EP1161440A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation

Definitions

  • the present inv ention relates to a method lor refolding recombinant polvpeptides comprising Ig domains, such as immunoglobulms
  • the present invention provides a method for promoting the folding of a polypeptide comprising at least one Ig domain, which method comprises contacting the polypeptide with a molecular chaperone and at least one foldase. It is especially preferred that the contacting takes place under reducing conditions.
  • the molecular chaperone and. or foldase are immobilised to a solid phase, more preferably both the chaperone and foldase(s ) are immobilised to a solid phase.
  • the solid phase is a matrix. More preferably the matrix is present in a chromatographv column.
  • the molecular chaperone is an hsp ⁇ O chaperonin or fragment thereof having refolding activity, more preferably a molecular chaperone fragment comprising a region consisting of fragments 191 -376, 191 -345 or 191-335 of the sequence of E. coli Gro ⁇ L or a homologue thereof.
  • the foldase is selected from a thiol/disulphide oxidoreductase and a peptidyi- prolyl isomerase.
  • the thiol/disulphide oxidoreductase is selected from E. coli DsbA and mammalian protein disulphide isomerase and the peptidyl prolyl isomerase is independently selected from cyclophilin, parbulen. SurA and FK506 binding proteins.
  • the method of the invention comprises contacting the polypeptide with a molecular chaperone and both a thiol/disulphide oxidoreductase and a peptidyl-prolyl isomerase.
  • a molecular chaperone e.g., a thiol/disulphide oxidoreductase and a peptidyl-prolyl isomerase.
  • the thiol/disulphide oxidoreductase is DsbA and the peptidyl-prolyl isomerase is cyclophilin A.
  • the present invention also provides the use of a molecular chaperone and one or more foldases for promoting the folding of a polypeptide comprising at least one Ig domain.
  • the invention provides a polypeptide comprising at least one Ig domain which polypeptide is obtainable by the method of the invention.
  • Said polypeptide is typically obtained at higher yields and having a higher specific activity than a polypeptide obtained using normal methods of protein expression in. for example, non-mammalian expression systems such as E. coli.
  • a polypeptide of the invention may be used in therapy. .
  • Chaperones. including chaperonins. are polypeptides which promote protein folding by non-enzymatic means, in that they do not catalyse the chemical modification of any structures in folding polypeptides. by promote the correct folding of polypeptides by facilitating correct structural alignment thereof.
  • Molecular chaperones are well known in the art. several families thereof being characterised. The invention is applicable to any molecular chaperone molecule, which term includes, for example, the molecular chaperones selected from the following non-exhaustive group:
  • HSP family HSP70 family. DNA K, DNAJ. HSP60 family (GroEL), ER-associated chaperones.
  • HSP90. Hsc70. sHsps: SecA; SecB. Trigger factor, zebrafish hsp 47. 70 and 90. HSP 47.
  • GRP 94. Cpn 10.
  • BiP. GRP 78.
  • C lp. FtsH Ig invariant chain, mitochondrial hsp70. EBP, mitochondrial m-AAA. Yeast Ydj l , Hsp 104, ApoE. Syc, Hip, TriC family.
  • CCT. PapD and calmodulin see O99/05163 for references).
  • hsp60 heat shock protein 60
  • hsp70 heat shock protein 70
  • Chaperones of the hsp60 class are structurally distinct from chaperones of the hsp70 class.
  • hsp ⁇ O chaperones appear to form a stable scaffold of two heptamer rings stacked one atop another which interacts with partially folded elements of secondary structure.
  • hsp70 chaperones are monomers of dimers and appear to interact with short extended regions of a polypeptide. Hsp70 chaperones are well conserved in sequence and function.
  • Analogues of hsp70 include the eukaryotic hsp70 homologue originally identified as the IgG heavy chain binding protein (BiP). BiP is located in all eukaryotic cells within the lumen of the endoplasmic reticulum (ER). The prokaryotic DnaK hsp70 protein chaperone in Escherichia coli shares about 50% sequence homology with an hsp70 KAR2 chaperone in yeast. Moreover, the presence of mouse BiP in yeast can functionally replace a lost yeast KAR2 gene.
  • BiP IgG heavy chain binding protein
  • Hsp60 chaperones are universally conserved and include hsp60 homologues from large number of species, including man. They include, for example, the E. coli GroEL polypeptide; Ehrlichia sennetsu GroEL; Trichomonas vaginalis hsp ⁇ O; rat hsp60; and yeast hsp ⁇ O.
  • the present invention relates to fragments of polypeptides of the hsp ⁇ O family.
  • these proteins being universally conserved, any member of the family may be used; however, in a particularly advantageous embodiment, fragments of GroEL. such as E. coli GroEL. are employed, especially those fragments termed minichaperones which are substantially monomeric in solution (see WO98/ 13496). Particularly preferred fragments of E. coli GroEL described in W098/ 13496 are discussed below.
  • the molecular chaperone may moreover be a circular permutation of a chaperone polyopeptide sequence. Circular permutation is described in Graf and Schachman, PNAS(USA) 1996. 93: 1 1591 ; the strategy set forth may be applied generally to most of the polypeptides with close N and C termini.
  • the circularly permutated sequence chaperones or chaperone fragments maintain their protein folding activity. Essentially. the polypeptide is circularised by fusion of the existing N and C termini, and cleavage of the polypeptide chain elsewhere to create novel N and C termini.
  • Chaperone activity may be determined in practice by an ability to refold cyclophilin A but other suitable proteins such as glucosamine-6-phosphate deaminase or a mutant form of indoleglycerol phosphate synthase (IGPS) (amino acid residues 49-252 ) may be used.
  • IGPS indoleglycerol phosphate synthase
  • a rhodanese retolding assav mav also be used Details oi a suitable retolding assay are given below
  • Preferred chaperone poh peptides ot the present invention have protein refolding activity in the absence of adenosme t ⁇ phosphate ot more than 50%. preferably 60%, even more preferably 75%.
  • said refolding activitv being determined by contacting the chaperone polypeptide with an inactivated protein of known specific activity prior to inactivation, and then determining the specific activitv of the said protein after contact with the polypeptide. the %> refolding acti itv being
  • the chaperone activity is determined by the refolding of cyclophilin A. More preferably. 8 M urea denatured c ⁇ cloph ⁇ hn A ( 100 ⁇ M) is diluted into 100 miVI potassium phosphate buffer pH 7 0, 10 mM DTT to a final concentration of 1 ⁇ M and then contacted with at least 1 ⁇ M of said polypeptide at 25°C for at least 5 mm, the resultant cyclophilin A activity being assayed by the method of Fischer et al (1984)
  • chaperone polypeptides of the present invention are monomeric in solution and incapable of multime ⁇ sation in solution Monomeric GroEL minichaperones are disclosed in W098/13496 Typicallv. multime ⁇ sation is prevented by using chaperone polypeptides that lack the interacting domains found outside the apical domain, although it could be achieved by suitable mutations
  • a foldase is an enz ⁇ me which participates in the promotion of protein folding through its enzymatic activity to catalyse the rearrangement or isomerisation of bonds in the folding poKpeptide
  • They are thus distinct from a molecular chaperone. which bind to po peptides in unstable or non-native structural states and promote correct folding without enzvmatic catalv sis of bond rearrangement
  • Manv classes of foldase are known. and they are common to animals, plants and bacteria. They include peptidyl prolyl isomerases and thiol/disulphide oxidoreductases.
  • the invention comprises the use of all foldases which are capable of promoting protein folding through covalent bond rearrangement.
  • a foldase includes one or more foldases.
  • the use of the singular does not preclude the presence of a plurality of the entities referred to. unless the context specifically requires otherwise.
  • Thiol/disulphide oxidoreductase As the name implies, thiol/disulphide oxidoreductases catalyse the formation of disulphide bonds and can thus dictate the folding rate of disulphide-containing polypeptides.
  • the invention accordingly comprises the use of any polypeptide possessing such an activity. This includes chaperone polypeptides. or fragments thereof, which may possess protein disulphide isomerase activity.
  • thiol/disulphide oxidoreductases are generally referred to as protein disulphide isomerases (PDIs). PDI interacts directly with newly synthesised secretory proteins and is required for the folding of nascent polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells.
  • PDIs protein disulphide isomerases
  • Enzymes found in the ER with PDI activity include mammalian PDI. yeast PDI, mammalian ERp59. mammalian prolyl-4-hydroxylase. yeast GSBP and mammalian T3BP. A. niger PdiA and yeast EUGI (see WO99/05163 for references).
  • equivalent proteins exist, such as the DsbA protein of E. coli.
  • Other peptides with similar activity include, for example, p52 from T. crazi.
  • These polypeptides, and other functionally equivalent polypeptides. are included with the scope of the present invention. as are derivatives of the polypeptides which share the relevant activity (see below).
  • the thiol/disulphide oxidoreductase according to the invention is selected from mammalian PDI or E. coli DsbA.
  • PPIs Peptidyl-prolyl isomerases
  • PPIs Peptidyl-prolyl isomerases
  • Known examples include cyclophilin. parbulen. SurA and FK506 binding proteins FKBP51 and FKBP52 (see WO99/05163 for references).
  • PPI is responsible for the cis-trans isomerisation of peptidyl-prolyl bonds in polypeptides. thus promoting correct folding.
  • the invention includes any polypeptide having PPI activity. This includes chaperone polypeptides. or fragments thereof, which may possess PPI activity.
  • the derivatives which may be used according to the present invention include splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants, glycosylation variants and other covalent derivatives of molecular chaperones or foldases which retain the functional properties of molecular chaperones.
  • peptidyl-prolyl isomerases and or thiol/disulphide oxidoreductases are examples of oxidoreductases.
  • Exemplary derivatives include molecules which are covalently modified by substitution. chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Such a moiety may be a detectable moiety such as an enzyme or a radioisotope.
  • Fu ⁇ her included are naturally occurring variants of molecular chaperones or foldases found within a particular species, whether mammalian, other vertebrate, yeast. prokaryotic or otherwise. Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of a molecular chaperone or foldase.
  • Derivatives also include variants of naturally occurring chaperones/foldases. which may, for example, have been engineered or selected from libraries of mutagenised libraries derived from naturally occurring forms.
  • the components of the combination according to the invention may comprise derivatives of molecular chaperones or foldases. including variants of such polypeptides which retain common structural features thereof.
  • Variants which retain common structural features can be fragments of molecular chaperones or foldases.
  • Fragments of molecular chaperones or foldases comprise smaller polypeptides derived from therefrom.
  • smaller polypeptides derived from the molecular chaperones or foldases according to the invention define a single feature which is characteristic of the molecular chaperones or foldases. Fragments may in theory be almost any size, as long as they retain the activity of the molecular chaperones or foldases described herein.
  • a fragment When applied to chaperone molecules, a fragment is anything other that the entire native molecular chaperone molecule which nevertheless retains chaperonin activity.
  • a fragment of a chaperonin molecule remains monomeric in solution. Preferred fragments are described below.
  • chaperone fragments are between 50 and 200 amino acids in length, preferably between 100 and 200 amino acids in length and most preferably about 150 amino acids in length.
  • fragments With respect to molecular chaperones of the GroEL/hsp-60 family, a preferred set of fragments have been identified which possess the desired activity. These fragments are set forth in our copending international patent application W098/13496 and in essence comprise any fragment comprising at least amino acid residues 230-271 of intact GroEL. or their equivalent in another hsp ⁇ O chaperone. Preferably, the fragments should not extend beyond residues 150-455 or 151 -456 of GroEL or their equivalent in another hsp ⁇ O chaperones.
  • the fragments comprise the apical domain of GroEL. or its equivalent in other molecular chaperones. or a region homologous thereto as defined herein.
  • the apical domain spans amino acids 191 -376 of intact GroEL. This domain is found to be homologous amongst a wide number of species and chaperone types.
  • the fragments are selected from fragments consisting essentially of residues 191-376, 191 -345. 191 -335 or 193-335 of the sequence of intact GroEL.
  • Derivatives of the molecular chaperones or foldases also comprise mutants thereof. including mutants of fragments and other derivatives, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain the activity of the molecular chaperones ⁇ r foldases described herein.
  • conserv ative amino acid substitutions may be made substantially without altering the nature of the molecular chaperones or foldases. as may truncations from the 5' or 3' ends.
  • Deletions and substitutions may moreover be made to the fragments of the molecular chaperones or foldases comprised by the invention.
  • Mutants may be produced from a DNA encoding a molecular chaperone or foldase which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids.
  • substitutional. deletional or insertional variants of molecular chaperones or foldases can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of the relevant molecular chaperone or foldase.
  • the fragments, mutants and other derivative of the molecular chaperones or foldases preferably retain substantial homology with the native molecular chaperones or foldases.
  • "homology” means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function.
  • homology is used to refer to sequence identity.
  • the derivatives of molecular chaperones or foldases preferably retain substantial sequence identity with native forms of the relevant molecular chaperone or foldase.
  • Substantial homology where homology indicates sequence identity, means more than 40% sequence identity, preferably more than 45% sequence identity and most preferably a sequence identity of at least 50%.. 60% or more, as judged by direct sequence alignment and comparison.
  • Homology comparisons can be conducted by eye. or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the skilled person can identify suitable homologues by, for example, carrying out a search of online databases using all or part of a molecular chaperone/foldase sequence as a query sequence.
  • a search of the S issprot database using the BlastP program Ver 2.0.8 (default settings) (Jinghui Zhang et al.. 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucleic Acids Res. 25:3389-3402) and amino acids 191 to 376 of E. coli GroEL as the query sequence identified well over a hundred homologous sequences, many of which gave homology scores of at least 50% identity.
  • Homologues identified include members of the hsp ⁇ O chaperonin family which includes the eubacterial GroEL, mitochondrial hsp60 and chloroplast cpn60. Other specific homologues together with their database accession numbers are detailed in W098/13496.
  • sequence similarity may be defined according to the ability to hybridise to a complementar ⁇ - strand of a nucleotide sequence encoding any of the chaperone or foldases mentioned above, such as E. coli GroEL. E. coli DsbA or mammalian cyclophilin A.
  • sequences are able to hybridise with high stringency.
  • Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinal" ⁇ ' skill in the field.
  • the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5"C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature.
  • the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
  • high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na ' at 65-68°C.
  • High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC. 5x Denhardt's. 1 % SDS (sodium dodecyl sulphate). 0.1 Na ⁇ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - O. lx SSC. 0.1 % SDS.
  • Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridisation temperature in lx SSC. 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC. 0.1 % SDS.
  • the contact between the Ig domain-containing polypeptides and the molecular chaperone and foldase occurs with the molecular chaperone and/or foldase immobilised on a solid support.
  • solid supports include beads, "chips " , resins, matrices, gels, and the material forming the walls of a vessel. Matrices, and in particular gels, such as agarose gels, may conveniently be packed into columns.
  • a particular advantage of solid phase immobilisation is that the reagents may be removed from contact with the polypeptide(s) with facility.
  • Solid phase materials for use in batch or to be packed into columns are widely available - see for example Sigma ' s 1999 reagent catalogue entitled "Biochemicals. organic compoounds and diagnostic reagents " which includes a range of activated matrices suitable for coupling polypeptides such as cyanogen bromide activated matrices based on sepharose/ agarose.
  • Molecular chaperones/foldases may be immobilised to a solid phase support such as by covalent means or otherwise.
  • a variety of methods for coupling polypeptides to solid phase supports are known in the art.
  • molecular chaperones and/or preferably foldase polypeptides may be attached to a solid phase support using a method which comprises a reversible thiol blocking step. This is important where the peptide contains a disulphide. An example of such a method is described below.
  • the disulphides are reduced using a reducing agent such as DTT (dithiothreitol). under for example an inert gas, such as argon, to prevent reoxidation.
  • a reducing agent such as DTT (dithiothreitol).
  • an inert gas such as argon
  • the polypeptide is cyanylated. for example using NCTB (2-nitro. 5-thiocyanobenzoic acid) preferably in stoichiometric amounts, and subjected to controlled hydrolysis at high (non-acidic) pH. for example using NaHC0 3 .
  • the pH of the hydrolysis reaction is preferably between 6.5 and 10.5 (the pK of DsbA is 4.0). more preferably between 7.5 and 9.5. and most preferably around about 8.5.
  • the thiols are thus reversibly protected.
  • the polypeptide is then brought into contact with the solid phase component, for example at between 2.0 and 20.0 mg polypeptide/ml of solid component, preferably between 5.0 and 10.0 and most preferably around about 6.5 mg.
  • the coupling is again carried out at a high (non-acidic) pH. for example using an NaHCO 3 coupling buffer.
  • the pH of the coupling reaction is preferably between 6.5 and 10.5. more preferably between 7.5 and 9.5. and most preferably around about 8.5.
  • the remaining active groups may be blocked, such as with ethanolamine. and the uncoupled polypeptide removed by washing.
  • Thiol groups may finally be regenerated on the coupled polypeptide by removal of the cyano groups, for example by treatment with DTE or DTT.
  • the present invention provides a method for promoting the correct folding/refolding of a polypeptide comprising at least one Ig domain which method involves the use of a combination of a molecular chaperone and a foldase.
  • the combination of a molecular chaperone and a foldase provides a synergistic effect on protein folding which results in a greater quantity of active, correctly folded protein being produced than would be expected from a merely additive relationship.
  • one or more of the components used to promote protein folding in accordance with the present invention is immobilised on a solid support.
  • molecular chaperones and foldases may be used in solution. They may be used in free solution, but also in suspension, for example bound to a matrix such as beads, for example sepharose beads, or bound to solid surfaces which are in contact with solutions, such as the inside surfaces of bottles containing solutions, test tubes and the like.
  • the method of the present invention is used to assist in refolding recombinantly produced Ig domain-containing polypeptides. which are obtained in an unfolded or misfolded form.
  • recombinantly produced polypeptides ma ⁇ ' be contacted with a molecular chaperone and a foldase to unfold, refold and or reactivate recombinant polypeptides which are inactive due to misfolding and/or are unfolded as a result of there extraction from the host cells in which the ⁇ were expressed (such as from bacterial inclusion bodies ).
  • Such a process may also be termed "reconditioning " .
  • the method of the invention may be employed to maintain the folded conformation of Ig domain-containing polypeptides. for example during storage, in order to increase shelf life. Under storage conditions, many proteins lose their activity, as a result of disruption of correct folding. The presence of molecular chaperones, in combination with foldases. reduces or reverses the tendency of polypeptides to become unfolded and thus greatly increases the shelf life thereof.
  • the method of the invention may be used to promote the correct folding of Ig domain- containing polypeptides which, through storage, exposure to denaturing conditions or otherwise, have become misfolded.
  • the invention may be used to recondition Ig domain-containing polypeptides.
  • immunoglobulins in need of reconditioning may be passed down a column to which is immobilised a combination of a molecular chaperone and a foldase in accordance with the invention.
  • beads having immobilised thereon such a combination may be suspended in a solution comprising the immunoglobulins in need of reconditioning.
  • the components of the combination according to the invention may be added in solution to the immunoglobulins in need of reconditioning.
  • the present invention also provides a method for altering the structure of an Ig domain- containing polypeptide.
  • Structural alterations include folding, unfolding and refolding.
  • the effect of the alterations is preferably to improve the yield, specific activity and/or quality of the molecule. This may typically be achieved by resolubilising. reconditioning and/or reactivating incorrectly folded molecules post-synthesis.
  • reconditioning and “reactivating” thus encompass in vitro procedures.
  • Particular examples of in viiro procedures may include processing polypeptides that have been solubilised from cell extracts (such as inclusion bodies) using strong denaturants such as urea or guanidium chloride.
  • refold reactivate
  • recondition reactivating an inactiv e protein, perhaps denatured using urea, may have an unfolded structure. This inactive protein may then be refolded with a polypeptide of the invention thereby reactivating it. In some circumstances there may be an increase in the specific activity of the refolded/reactivated protein compared to the protein prior to inactivation' denaturation: this is termed "reconditioning".
  • the molecule is typically an unfolded or misfolded polypeptide which is in need of folding. Alternatively, however, it may be a folded polypeptide which is to be maintained in a folded state.
  • the polypeptide contains at least one disulphide linkage (or two cysteine residues capable of forming such as linkage under suitable conditions).
  • the invention envisages at least two situations.
  • a first situation is one in which the polypeptide to be folded is in an unfolded or misfolded state, or both. In this case, its correct folding is promoted by the method of the invention.
  • a second situation is one in which the polypeptide is substantially already in its correctly folded state, that is all or most of it is folded correctly or nearly correctly.
  • the method of the invention serves to maintain the folded state of the pohpeptide by affecting the folded/unfolded equilibrium so as to favour the folded state. This prevents loss of activity of an already substantially correctly folded polypeptide.
  • a polypeptide may be unfolded when at least part of it has not yet acquired is correct or desired secondary or tertiary structure.
  • a polypeptide is misfolded when it has acquired an at least partially incorrect or undesired secondary or tertiary structure.
  • Techniques are known in the art for assessing poh'peptide structure - such as circular dichroism.
  • Contacting of the Ig domain-containing polypeptides w ith the chaperone/foldase combination preferably occurs under reducing conditions, such as in the presence of a combination of oxidised glutathione ( GSSG) and glutathione ( GSH) w hich act as a redox buffer system and prevent formation of disulphide bonds present in the oxidised state.
  • GSSG oxidised glutathione
  • GSH glutathione
  • a particularly convenient method for contacting the molecular chaperone/foldase combination with the Ig domain-containing polypeptides involves incubating the Ig domain-containing polypeptides with the molecular chaperone/foldase combination, whereby the chaperone and foldase are immobilised to sepharose/agarose beads, in a tube. such as an eppendorf tube, in a procedure known as a batch incubation.
  • the tube contents are gently mixed for typically at least 5 minutes, preferably at least 1 to 3 hrs. before allowing the beads to settle by. for example, gravity or low speed centrifugation.
  • the Ig domain-containing polypeptides in aqueous solution are then simply decanted off.
  • Another convenient method involves placing a solid phase matrix such as sepharose beads, to which the chaperone and foldase are immobilised, in a chromatographv column, applying a sample comprising the polypeptide to be refolded to the top of the column and eluting the polypeptide through the column using a suitable buffer at a suitable rate.
  • a solid phase matrix such as sepharose beads, to which the chaperone and foldase are immobilised
  • Polypeptides comprising at least one Ig domain
  • Ig domain is well known in the art and also includes Ig-like domains.
  • An Ig domain comprises a motif termed an immunoglobulin fold which typically contains two broad sheets of antiparallel ⁇ strands, generally bridged by a conserved disulphide bond (see Biochemistry. 1995. L. Stryer. Freeman. p370).
  • Polypeptides comprising at least one Ig domain or Ig-like domain are generally members of the so-called immunoglobulin superfamily (for a review see Hunkapiller and Hood, 1989). Many members of the immunoglobulin superfamily are present as cell surface molecules where they may be monomeric. dimers or higher oligomers.
  • Variants and engineered pohpeptides include alternative- ⁇ spliced iso forms and ditterentiallv post-translationally modified forms (e g hav ing different ghcosv lation patterns)
  • pohpeptides may be part of multi-protein complexes (such as hetero- or homodimers or other multime ⁇ c forms), the precise stoichiometrv of which ma ⁇ be varied
  • Variants and engineered polypeptides may comprise mutations such as insertions and/or deletions They may also comprise chemically modifications
  • Polypeptides suitable tor retolding using the method ot the invention may also include polypeptides comprising core structural motifs similar to those seen in Ig folds such that they fold using a similar pathway to an Ig domain
  • a suggested pathway for Ig domain folding is described in Clarke et al 1999
  • polypeptides comprising at least one Ig domain include immunoglobulms such as antibodies, or fragments thereof.
  • MHC polypeptides such as MHC class I and class II polypeptides.
  • T cell receptors including CD3 ⁇ , ⁇ . ⁇ ), CD4.
  • N-CAM and receptors tor growth factors such as the NGF receptor, PDGF receptor, FGF receptor and VEGF receptor
  • pohpeptides comprising one or more Ig-hke domains are listed in Halabv and Mornon. 1998 (see also reterences therein) These include antibiotic proteins such as actinoxantin. bacterial proteins including chaperones and enzymes, carcinoembry omc antigen and related antigens c ⁇ tok ⁇ ne receptors, mammalian enzymes such as phosphatases and tyrosine kinases. proto-oncogenes. extracellular matrix proteins such as collagen, superoxide dismutase. immunoglobulms. immunoglobulin receptors. cell surface antigens ot the immune system such as B7, CD2. CD3., CD7, CD22, ICAM-1. ICAM-2.
  • Polypeptides comprising at least one Ig domain have been identified for a variety of species of organisms and thus polypeptides comprising at least one Ig domain may originate from vertebrates such as mammals, invertebrates, such as insects, nematodes and marine sponges, plants, prokaryotes such as eubacteria. and viruses.
  • the polypeptide comprising at least one Ig domain is an antigen presenting molecule, more preferably an antigen presenting molecule involved in T cell recognition of microbial antigens such as lipid and glycolipid antigens.
  • the polypeptide is a member of the CD1 family, non-polymorphic, MHC class 1 -like molecules that associate non-covalently with ⁇ 2-microglobulin.
  • the Ig domain-containing polypeptides to be processed by the method of the invention is typically obtained from cell extracts of host cells expressing recombinant Ig domain- containing polypeptides or their precursors.
  • recombinant proteins may be obtained from any source such as in vitro translation systems.
  • Host cells include prokaryotes such as E. coli. yeast and insect cells (the baculovirus system is capable of very high level protein expression). Expression of the polypeptide comprising at least one Ig domain in the host cell is preferably at high levels to maximise yield.
  • Solubilised cell extracts may optionally be partially purified by, for example, a variety of affinity chromatographv techniques prior to contacting with the chaperone/foldase combination according to the method of the invention.
  • the starting material for the refolding/reconditioning method of the invention is typically denatured polypeptides in solutions of agents such as urea' guanidium chloride.
  • soluble polypeptide samples may be specifically denatured by the addition of appropriate denaturing agents prior to refolding.
  • the untreated polypeptides samples may be dialysed against a suitable refolding buffer prior to contact with the chaperone/foldase combination if required.
  • the refolded polypeptides are typically desalted by dialysis against a suitable storage buffer and/or the use of a desalting column into a suitable storage buffer.
  • Suitable buffers include 25 mM sodium phosphate, 150 mM NaCl and 0.1% PEG 6000 (pH 7.4).
  • the activity of the refolded/reconditioned polypeptide comprising at least one Ig domain preferably has at least 30% activity relative to wild type polypeptide (for example an equivalent antibody secreted by a hybridoma). which has been treated in the same way, more preferably at least 40. 45 or 50%> activity. Activity may conveniently be assessed using, for example, an ELISA.
  • Polypeptides comprising at least one Ig domain which polypeptides are produced by the method of the present invention may be used in a variety of applications both therapeutic and non-therapeutic.
  • the ⁇ ' may be used in scientific research, such as assay systems including antibody-antigen assays and proteomic chip systems. They may also be used in therapeutic applications requiring the use of Ig-domain containing polypeptides. include the treatment or amelioration of infectious, neoplastic, immunological, autoimmune, degenerative, vascular, inflammatory, endocrine, drug or toxin related or other disease or acute, subacute or chronic conditions. As a specific example, they may be used in immunoregulation.
  • soluble Ig-domain containing polypeptides via the administration of soluble Ig-domain containing polypeptides to individuals which interact with specific ligands or their soluble or cell membrane bound receptors, or have an indirect effect on cellular function, for example in immunosuppression.
  • cell adhesion such as in the prevention of tumour cell metastasis). or on isolated body organs (such as in organ transplantation or artificial tissue such as skin, renal or hepatic substitutes).
  • Another specific use is in cancer therapy using therapeutic compounds linked to antibodies or fragments thereof (such as ADEPT).
  • Further therapeutic uses include prophylaxis, either directly or indirectly, for example in the prevention of infectious, neoplastic.
  • immunological immunological (immunomodulation such as the manipulation of B and T cells and their subsets), autoimmune, degenerative, vascular, inflammatory, endocrine, drug or toxin related or other disease or acute, subacute or chronic conditions.
  • Polypeptides obtainable by the method of the invention may also be used in immunotherapy and/or to treat a disease associated with a defect in an Ig domain containing polypeptide.
  • Other applications include medical diagnosis, for example in protein array "chip" devices which assay levels of soluble Ig-domain containing and other types of soluble molecules in liquid biological samples such as serum, urine, saliva, cerebrospinal fluid and ascitic fluid.
  • Polypeptides produced by the method of the invention may also be used in self-assembling molecular system devices such as when they are linked by covalent or other means to other molecules such as DNA tags to pattern the Ig-domain containing polypeptides onto surfaces at the molecular scale.
  • compositions of the invention may preferably be combined with various components to produce compositions of the invention.
  • the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use).
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition of the invention may be administered by direct injection.
  • the composition may be formulated for parenteral. intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the CD1 antigens are non-polymorphic. MHC class 1 -like molecules that associate non- covalently with ⁇ 2-microglobulin.
  • the CD 1 proteins are a family of antigen-presenting molecules that allow T cells to recognise a range of antigens including lipids, glycolipids, peptides, and other types of antigens derived from microbial and viral pathogens. These include mycobacterium tuberculosis, a pathogen that is of global significance and which is responsible for considerable morbidity and mortality.
  • the mini-chaperone (191-345 peptide fragment from E. coli GroEL). is cloned and expressed in E. coli as a fusion protein containing a 17-residue N-terminal histidine tail (Zahn et al.. 1996).
  • the mini-chaperone is immobilised on agarose gel beads as previously reported (Altamirano et al.. 1997) except that NHS-activated Sepharose-4 Fast Mow (Pharmacia Biotech. Sweden) is used This activ ated gel.
  • Human PPI (peptidyl-proh l en -tra -isomerase) is expressed and purified as described (Jasanoff et al . 1994) with some minor modifications B ⁇ efl ⁇ a plasmid carrying the gene of fusion protein GST-PPI is used to transform the E coli C41 D3 strain (Miroux and Walker.
  • the cell pellet is resuspended in buffer (50 mM sodium phosphate, pH 7 5. 100 mM NaCl.
  • the E coli dsbA gene is amplified by PCR using dsbA-Fo and dsbA-Ba primers, based on its known sequence
  • the amplified whole expressed gene, including its signal peptide is digested with Xcol and BamHI and cloned into the high expression plasmid pC ⁇ 820 (Lewis et al . 1993)
  • the pMA14 (pCE820--- ⁇ - b-4) is purified and the sequence is confirmed by standard sequencing techniques
  • the dsbA gene product is overproduced in the E coli C41 D3 strain (Miroux and Walker.
  • DsbA is reduced with 5 mM DTT. in 25 mM MES-K+ buffer pH 6.0 for 1 h; DTT is then removed by dialysis under Ar to avoid reoxidation. DsbA is then cyanylated under Ar with NTCB (2-nitro-5-thiocyanate benzoate) (Altamirano. et al., 1989; Altamirano et al.. 1992) at a final concentration of 5 mM. The reaction is practically instantaneous and it is apparent from the appearance of a yellow colour from the departing group, the anion 2- nitro-5-thiobenzoate.
  • Uncoupled DsbA is removed by washing with five cycles of alternately high and low pH buffer solution (Tris- HC1 0.1 M pFI 7.8 containing 0.5 M NaCl followed by acetate buffer, 0.1M. pH 4 plus 0.5 M NaCl).
  • the gel is finally washed with 5-10 gel volumes of refolding buffer (see below) and SH groups regenerated by treatment with DTT.
  • the gel is washed with ten times gel volume of refolding buffer. After this, the immobilised DsbA protein is oxidised as detailed under experimental protocol. The coupling efficiency of this procedure is higher than 95 %.
  • the gel is stable for at least one year when stored at 4°C in 100 mM sodium phosphate pH 7.0. containing 2 mM EDTA.
  • CDlA/ ⁇ 2M 250 mg is dissolved (with the help of a douncer) in a minimum volume of freshly made 6 M guanidinium chloride prepared in 0.1M potassium phosphate buffer (pH 8) and adjusted to pH8 with an appropriate volume of 4N NaOH. It is then, reduced with 0.1 M DTT and left for 2 h in light-free conditions at 25°C to ensure the completeness of the reaction.
  • the fluorescence and CD spectrum of reduced and denatured CDlA/ ⁇ 2M are the typical ones for a denatured protein.
  • the ternary refolding matrix is obtained by mixing equal concentrations of mini- chaperone. DsbA protein and PPI.
  • the matrix buffer is equilibrated with a freshly made pH 8 buffer prepared with 50 mM Tris-HG (pH 8). 0.3 VI L-arginine hydrochloride. 8 mM oxidised glutathione. 1 mM sodium EDTA and 0.2 M PMSF (refolding buffer).
  • the denatured and reduced human CD IA heavy chain and ⁇ 2M light chain are then mixed together in a molar ratio of 1 :4 (CDlA: ⁇ 2M). The mixture is then added very slowly (dropwise).
  • Each of the three refolding proteins (mini-chaperone or DsbA or PPI) used is also individually tested and a control experiment is also made using refolding buffer alone.
  • the refolding buffer b ⁇ itself produced less than 5% of soluble protein, the majority of which was aggregated.
  • PPI-agarose gave a yield of about 10% of soluble protein, but it was mainly aggregated.
  • DsbA-agarose gave a 10-15% yield of soluble protein, only 1-5% of which was monodisperse (Table I).
  • the binary refolding matrix of miniGroEL and DsbA gave a high yield of protein, of which 74% was monodisperse.
  • the ternary matrix (miniGroEL/DsbA/PPI-agarose) solubilises essentially all the sample (98%). of which 87% appears as a single symmetrical chromatographic peak at the retention time corresponding to its expected molecular mass (Table I).
  • Fluorescence and CD spectrum analysis revealed profiles that were typical for that of a refolded protein.
  • Oxidative refolding chromatography should also enable the production in vitro of other immunoglobulin supergene tamih complexes, both natural and artificial. Empty and hgand-full soluble soluble CD1. MHC and other Ig superfamih molecules, their engineered and naturalh occurring Var e forms and their complexes, are themselves likely to be of potential therapeutic, proph ⁇ lactic, general industrial, diagnostic, scientific and biotechnological importance.

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Abstract

L'invention concerne un procédé pour favoriser le pliage d'un polypeptide comprenant au moins un domaine Ig, le procédé consistant à mettre le polypeptide en contact avec un chaperon moléculaire et une foldase.
EP00911054A 1999-03-17 2000-03-16 PROCEDE DE REPLIAGE DE MOLECULES DE POLYPEPTIDES CONTENANT DES DOMAINES Ig Withdrawn EP1161440A1 (fr)

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GBGB9906056.8A GB9906056D0 (en) 1999-03-17 1999-03-17 Method
GBGB9925985.5A GB9925985D0 (en) 1999-11-02 1999-11-02 Refolding method
GB9925985 1999-11-02
PCT/GB2000/000987 WO2000055183A1 (fr) 1999-03-17 2000-03-16 PROCEDE DE REPLIAGE DE MOLECULES DE POLYPEPTIDES CONTENANT DES DOMAINES Ig

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US8790667B2 (en) 2010-03-05 2014-07-29 Becton Dickinson France Surface immobilized chaperones
WO2016073791A1 (fr) 2014-11-05 2016-05-12 Genentech, Inc. Procédés de production de protéines à deux chaînes dans des bactéries
JP6770966B2 (ja) 2014-11-05 2020-10-21 ジェネンテック， インコーポレイテッド 細菌における２鎖タンパク質の生成方法
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