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WO1995001994A1 - Polypeptides ctla4 recombinants et procede de fabrication - Google Patents

Polypeptides ctla4 recombinants et procede de fabrication Download PDF

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Publication number
WO1995001994A1
WO1995001994A1 PCT/US1994/007685 US9407685W WO9501994A1 WO 1995001994 A1 WO1995001994 A1 WO 1995001994A1 US 9407685 W US9407685 W US 9407685W WO 9501994 A1 WO9501994 A1 WO 9501994A1
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Prior art keywords
ctla4
polypeptide
active
protein
cells
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PCT/US1994/007685
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English (en)
Inventor
George N. Cox
Dickson G. Pratt
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Synergen, Inc.
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Publication date
Application filed by Synergen, Inc. filed Critical Synergen, Inc.
Priority to EP94923387A priority Critical patent/EP0665852A1/fr
Priority to JP7504186A priority patent/JPH08506247A/ja
Priority to AU73265/94A priority patent/AU7326594A/en
Publication of WO1995001994A1 publication Critical patent/WO1995001994A1/fr

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    • 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
    • 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
    • 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
    • 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
    • C07K14/70521—CD28, CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to soluble proteins useful for controlling T cell activation, and more particularly to soluble CTLA4 proteins produced by recombinant DNA methods.
  • T cell activation has been implicated in a number of deleterious conditions, including autoimmune diseases and transplant rejection.
  • Optimal activation of T cells for clonal expansion is believed to require two signals. One is an antigen-specific signal delivered through T cell receptors (TCR) , while the second is an antigen-non-specific or co-stimulatory signal. Chen et al., Cell 71:1093-1102 (1992); Liu et al., Eur. J. Immunol. 22:2855-2859 (1992).
  • the second non-specific signal is determined by a class of T cell regulatory molecules known as co-stimulators that determine whether T cells are activated to proliferate or enter into a state of unresponsiveness known as clonal anergy.
  • B7 a T cell regulatory molecule, is a co-stimulatory protein expressed on the cell surface of antigen presenting cells such as activated macrophages, activated B lymphocytes and dendritic cells as reported in Razi- olf et al., Proc. Natl Acad. Sci. U.S.A. 89:4210-4214 (1992) and Freeman et al., J. Immunol. 139:326-3267 (1992) .
  • B7 is a natural ligand for T cell surface proteins known as CD28 and CTLA4 (cytolytic T-lymphocyte-associated antigen number 4) .
  • CD28 and CTLA4 share substantial ho ology in their amino acid sequences, particularly in the transmembrane and cytoplasmic domains, and are therefore believed to share similar functions in the T-cell co-stimulation pathway.
  • B7 is known to have a greater affinity for CTLA4 compared with CD28.
  • CD28 is constitutively expressed on most T lymphocytes.
  • CTLA4 was determined to be preferentially expressed by activated versus unactivated cytolytic T cells in DNA hybridization experiments described in Brunet et al. , Nature 328:267-270 (1987). It is now known that CTLA4 is expressed by activated cytotoxic T lymphocytes and activated helper T lymphocytes.
  • B7 The interactions of B7 with CD28 and CTI-A4 play an important role in the full activation of T cells in the co-stimulation pathway during an immune response.
  • Neutralization of B7 or CD28 activity prevents T cell proliferation in response to foreign antigens and polyclonal activators such as lectins.
  • Neutralization of B7 activity also prevents T cells from acting as helper cells for the induction of antibody synthesis by B cells.
  • Cytokines that are known to be regulated by the interaction of B7 with CD28 include interleukin-2, tumor necrosis factors alpha and beta, gamma interferon and granulocyte-macrophage colony stimulating factor (Gimmi et al., Proc. Nat'l Acad. Sci. U.S.A. 88:6575-6579 (1991); Linsley et al., J. EXD. Med. 173:721-730 (1991); and Thompson et al., Proc. Nat'l Acad. Sci. U.S.A. 86:1333-1337 (1989)).
  • cytokines Synthesis of these cytokines is not completely inhibited by commonly used immunosuppressive agents such as cyclosporine, which is a fungal metabolite used to suppress the immune system in patients undergoing organ transplants or suffering from autoimmune diseases. Consequently, it is believed agents that effectively inhibit B7 activity could be used as an alternative to cyclosporine therapy or in combination with cyclosporine to provide an additive or synergistic effect in inhibiting T cell proliferation. Because of the similarity between CD28 and CTLA4, it is believed that the interaction of B7 with CTLA4 should also regulate cytokine synthesis in T lymphocytes.
  • CTLA4- Ig fusion protein A fusion protein containing the extracellular domain of CTLA4 joined to the F c heavy chain region of an immunoglobulin molecule has been developed as a possible agent having B7 inhibitory activity.
  • This fusion protein referred to as "CTLA4- Ig fusion protein," is described in Linsley et al., J. EXP. Med. 174:561-569 (1991) and in PCT Patent Publication No. WO 93/00431. According to these publications, the CTLA4-Ig fusion protein is secreted from mammalian cells as a disulfide-linked dimeric protein that aggregates in solution. However, attempts to express the extracellular domain of CTLA4 as an unfused protein in mammalian cells were unsuccessful.
  • the B7 inhibitory activity of the CTLA4-Ig fusion protein has been tested in both in vitro and in vivo experiments.
  • the CTLA4-Ig fusion protein bound to B7 and neutralized its activity.
  • the CTLA4-Ig fusion protein was a better inhibitor of B7 activity than a comparable CD28-Ig fusion protein.
  • the results of these assays are consistent with the previous experiments showing that B7 binds to CTLA4 with greater affinity than CD28.
  • the CTLA4-Ig fusion protein was found to inhibit T cell proliferation, with a half maximal inhibitory dose of 30 ng/ml, in a mixed lymphocyte reaction as reported in Linsley et al., supra.
  • the fusion protein also inhibited the ability of helper T cells to stimulate antibody production by B lymphocytes in an in vitro study described in Linsley et al., J. EXP. Med. 174:561-569 (1991).
  • CTLA4-Ig fusion protein was determined to be immunosuppressive and capable of prolonging survival of pancreatic and heart allografts in mice and rats (Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Nat'l Acad. Sci. U.S.A. 89:11102-11105 (1992)).
  • the administration of CTLA4-Ig resulted in the long term acceptance of pancreatic allografts.
  • the CTLA4-Ig fusion protein has several disadvantages as a therapeutic agent for human disease. Because the fusion protein is a non-naturally occurring molecule, a patient receiving the protein may develop an immune response to the protein. Antigenicity may be more of a problem when patients are taken off the therapeutic agent so they are no longer im unosuppressed and are capable of mounting an immune response against the fusion protein. Therefore, antigenicity may prevent the CTLA-Ig fusion protein from being administered intermittently to patients suffering from chronic diseases. In addition, the half-life of the CTLA4-Ig fusion protein in mice is about 4 days, with significant levels of the fusion protein still detectable in the animals 5 weeks after the cessation of treatment with CTLA4-Ig.
  • CTLA4-Ig fusion protein is expressed in mammalian cells, which is a costly method of producing recombinant proteins.
  • the present invention relates to recombinantly-produced CTLA4 polypeptides that are not fusion proteins containing human Ig molecules.
  • the soluble, recombinant polypeptides contain, as a basic unit, a monomer consisting essentially of the extracellular domain of the CTLA4 receptor protein.
  • the recombinant polypeptides are the product of joining two or more monomers through intermolecular disulfide bonds or through a cross-linking agent, such as polyethylene glycol (PEG) , to form biologically active dimers and other multimers.
  • PEG polyethylene glycol
  • the polypeptides can also be functional derivatives of the monomers and multimers, such as cysteine muteins in which a cysteine is substituted for one or more amino acids or is added to the wild-type CTLA4 amino acid sequence.
  • the substitution is preferably made at residue numbers 79, 80, 81, 109, 110 or 111 of the extracellular domain of the CTLA4 receptor protein as shown in SEQ ID NO.2, while the addition of an extra cysteine is preferably made after residue number 125 from the N-terminal end of the naturally-occurring CTLA4 receptor protein.
  • Other functional derivatives include, for example, pegylated dumbbell molecules in which two cysteine muteins are attached through activated groups on each end of a PEG molecule.
  • the present invention also provides methods of making the recombinant polypeptides. The methods include the steps of:
  • the polypeptide can assume an active tertiary structure.
  • the polypeptide can also be permitted to assume an active quaternary structure.
  • Vectors and host cells useful for the expression of the recombinant CTLA4 polypeptides are also provided.
  • the invention further provides pharmaceutical compositions containing the CTLA4 polypeptides as the active ingredient.
  • the invention also relates to methods for separating the various forms of the recombinantly-produced polypeptides, particularly the separation of monomers and dimers.
  • the invention also relates to methods for separating various dimer species and purifying active dimers from less active dimers. After obtaining active, recombinant polypeptides according to the above methods, the resulting mixture is passed over an ion exchange column, particularly an anion exchange column, followed by passage over a sizing column.
  • the separation methods produce one pool mixture of at least about 90% dimers and a second pool of at least about 85% monomers. These methods also separate active dimers from less active dimers.
  • a biologically active, recombinant CTLA4 protein involved expressing CTLA4 as a fusion protein. More particularly, the fusion protein is described in PCT Publication No. WO 93/00431 as containing the extracellular domain of CTLA4 fused to the heavy chain region of a human immunoglobulin molecule (referred to as • , CTIJA4-Ig ,, protein) . According to this publication, successful expression of the extracellular domain of the CTLA4 receptor protein requires an expression system that permits the protein to form dimers.
  • the unfused or truncated versions of the CTLA4 protein appear not to be expressed in an active form.
  • the publication further indicates that the Ig portion of the CTLA4-Ig fusion protein is believed to facilitate dimer formation and to aid in the purification of the fusion protein by conventional protein A affinity chromatography.
  • the present invention is based on the discovery of methods for producing a biologically active, soluble recombinant CTLA4 polypeptides (SCTLA4) that are not Ig fusion proteins.
  • SCTLA4 soluble recombinant CTLA4 polypeptides
  • biologically active refers to polypeptides that exhibit B7 binding activity.
  • the term "consists essentially of” as used herein is intended to encompass a monomer encoded by an amino acid sequence corresponding to the extracellular domain of the wild-type CTLA4 protein or corresponding to the extracellular domain joined to additional amino acids other than an amino acid sequence encoding for a human Ig molecule.
  • the calculated molecular weight of the CTLA4 monomeric form is about 12.5-13.5 kDa.
  • the recombinantly produced sCTLA4 monomer appears as two major bands in the range of about 14-16 kDa on SDS PAGE under non-reducing conditions.
  • the recombinant CTLA4 polypeptides of the present invention can also be in the form of dimers or other multimers, which contain more than one basic monomeric unit.
  • Such multimers, particularly dimers can be formed by joining two or more monomers through intermolecular disulfide bonds or by cross- linking agents such as, for example, polyethylene glycol (hereinafter referred to as "PEG") , other polyethers, EDTA and other linkers known to those skilled in the art.
  • PEG polyethylene glycol
  • the dimeric form produced by two monomers joined by intermolecular disulfide bonds has a calculated molecular weight of about 25 kDa and appears as at least three major bands in the range of about 24-27 kDa on SDS PAGE under non-reducing conditions.
  • the invention provides methods for separating the various dimer forms and purifying the most active dimer form.
  • the nucleic acid sequences useful in the present methods include SEQ ID NO:l and its functional equivalents.
  • the term "functional equivalent(s)" means modified sequences having one or more additions, deletions, or substitutions to the above sequence that do not substantially affect the ability of the sequence to encode a polypeptide having B7 binding activity.
  • modified sequences can be produced by means known in the art, including, for example, site directed mutagenesis.
  • the sequences can be obtained from natural sources, such as the natural DNA sequence encoding the extracellular domain of a CTLA4 receptor protein. Alternatively, the sequence can be produced synthetically according to methods known in the art. Additionally, such DNA sequences can be derived from a combination of synthetic and natural sources.
  • the natural sequences further include cDNA and genomic DNA segments. Methods of obtaining the synthetic and natural DNA sequences are described in PCT Publication No. WO 93/00431, published on January 7, 1993, which is incorporated herein by reference.
  • the vectors can contain one or more of the following operational elements: (1) a promoter; (2) a Shine- Dalgarno sequence and initiator codon; (3) a terminator codon; (4) an operator; (5) a leader sequence to facilitate transportation out of the host cell; (6) a gene for a regulator protein; and (7) any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vectors.
  • EP Application No. 90 113 673.9 which is incorporated herein by reference, discloses several useful vectors and desirable operational elements.
  • the vectors can be transferred into suitable host cells by various methods known in the art, including transfection and transformation procedures. Various transfer methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference.
  • host cells can be either eucaryotic or procaryotic cells. Examples of such host cells include Chinese hamster ovary (CHO) cells, yeast, E. Coli and baculovirus infected insect cells.
  • the host cells described in EP Application No. 90 113 673.9, which is incorporated herein by reference, are also useful in the present methods.
  • the host cells of the present invention can be cultured under conditions appropriate for the expression of the recombinant CTLA4 polypeptide. These conditions are generally specific for the host cell and are readily determined by one of ordinary skill in the art in light of the published literature regarding the growth conditions for such cells. For example, Bergev / s Manual of Determinative Bacteriology. 8th ed. , Williams & Wilkins Co., Baltimore, Maryland, which is incorporated herein by reference, contains information relating to appropriate conditions for culturing bacteria. Similar information relating to culturing yeast and mammalian cells are described in R. Pollack, Mammalian Cell Culture. Cold Spring Harbor Laboratories (1975) , incorporated herein by reference.
  • CTLA4 polypeptides can be confirmed by using anti-CTLA4 antibodies according to assay procedures known in the art, such as Western blotting or ELISA for example. Once expression of the recombinant polypeptides has been confirmed, the polypeptides can then be harvested according to methods known to those skilled in the art.
  • Suitable denaturing agents are those compounds or chemicals that cause a change in the conformation of a protein by disrupting the intermolecular or intramolecular bonds that results in a loss of biological activity without substantially affecting its primary structure.
  • denaturing agents include guanidine hydrochloride and urea.
  • guanidine hydrochloride is used as the denaturant.
  • concentration of guanidine is in the range of about 0.5M to about 6.0M, preferably at least about 6.0M.
  • any interfering cyanate that may form can be removed by passing the urea solution over an anion exchange column, such as DOWEX 1-X8 (BioRad, Richmond, California) . Cyanate can modify amino groups in the protein and, therefore, should be removed. (Stark, Methods in Enzvmologv 11:125 (1967)) Next, the disulfide bonds are then reduced with a reducing agent.
  • Suitable reducing agents include, for example, beta- mercaptoethanol, dithiothreitol (DTT) and cysteine.
  • DTT is used as the reducing agent.
  • the preferred DTT concentration is 6mM.
  • the free thiols present in the reduced protein are oxidized by the addition of a large excess of an oxidizing agent, preferably a disulfide-containing oxidizing agent such as, for example, oxidized glutathione or cystine.
  • an oxidizing agent preferably a disulfide-containing oxidizing agent such as, for example, oxidized glutathione or cystine.
  • the resulting solution is diluted prior to adding a second reducing agent to catalyze disulfide interchanges.
  • the second reducing agent contains a sulfhydryl (thiol) group such as, for example, DTT, 2-mercaptoethanol, dithioerythritol, cysteine, cystamine.
  • the second reducing agent can also be a disulfide containing compound such as sodium borohydride or any of the Group VIA hydrides having added cystine, oxidized glutathione or any cysteine-containing peptides.
  • the purpose of adding the second reducing agent is to produce an environment in which the CTLA4 recombinant polypeptides assume a variety of 3-dimensional configurations by the formation and breaking of various disulfide or other non-covalent bonds.
  • the denatured and reduced protein is diluted and allowed to refold into monomers and dimers without the addition of additional oxidizing or reducing agents as described in Example 8B.
  • the mixture can be passed over a phenyl sepharose column or, alternatively, over a reverse phase column for further separating the monomers from the dimers.
  • Useful ion exchange columns include, without limitation, Mono Q, Q-Sepharose, Resource Q and Source 15Q columns. Other equivalent separation procedures known to those skilled in the art can also be used to separate the various recombinant CTLA4 forms.
  • the term "functional derivative” can mean an active fragment, an analog or a derivative of a recombinant CTLA4 polypeptide described above that substantially retains the biological activity of the unmodified recombinant CTLA4 polypeptide.
  • such modified polypeptides preferable have an amino acid ho ology of greater than about 40% compared to SEQ. ID. NO. 2, more preferably in excess of 50%, and most preferably in excess of 90%.
  • An amino acid homology of about 99% is particularly useful.
  • one modification can be the substitution or addition of a cysteine to provide a "free cysteine" within the amino acid sequence to produce a "cysteine mutein.”
  • cyste mutein or "CTLA4 mutein,” as used herein, refers to muteins having at least one cysteine that is not involved in an intramolecular or intermolecular disulfide bond.
  • the free cysteine can appear at any amino acid residue that does not substantially interfere with its ability to bind B7.
  • a cysteine is substituted for at least one amino acid appearing at residue number 79, 80, 81, 109, 110, 111, or added after residue number 125 of SEQ.ID.NO.
  • muteins and other derivatives can be prepared by methods well known to those skilled in the art. Such methods include, for example, mutagenic techniques in which nucleotides are substituted or added that encode for a cysteine. A general method is described, for example, in U.S. Patent No. 4,518,584, incorporated herein by reference. Alternatively, the muteins can be synthesized by methods also known to those skilled in the art.
  • the cysteine mutein can be attached to polyethylene glycol (PEG) at a free cysteine to increase its molecular weight and improve its pharmacokinetic properties such as an increased serum half-life.
  • PEG polyethylene glycol
  • Long chain polymer units of PEG can be bonded to the mutein via covalent attachment to the sulfhydryl group of a free cysteine residue on the mutein.
  • PEG polymers with different molecular weights can be used, for example, 5.0 kDa (PEG 5000 ) , 8.5 kDa (PEG 850O ) , 10 kDa (PEG 10>000 ) , and 20 kDa (PEG 20000 ) .
  • the functional or reactive group attached to the long chain PEG polymer is the activating group to which the mutein attaches at the free cysteine site.
  • Suitable activating groups include, for example, maleimide, sulfhydryl, thiol, triflate, tresylate, aziridine, exirane or 5-pyridyl.
  • PEG molecules can also be attached to CTLA4 at free amines using NHS (N-hydroxysuccinimide)-derivatized PEG molecules.
  • CTLA4 conjugates are also contemplated, for example, (1) by attaching a single PEG molecule to a CTLA4 monomer (mono- pegylated) or dimer, for example, at free amines as described in the examples below; (2) by attaching two PEG molecules to a CTLA4 dimer, or (3) by attaching two or more CTLA4 monomers or dimers through a cross-linking moiety, such as PEG, to produce a compound that can be depicted schematically as a "dumbbell.”
  • two or more CTLA4 dimers can be attached through a cross-linking moiety such as PEG to produce a "dimer dumbbell.”
  • a PEG molecule containing two activating groups can be used such as, for example, PEG bis- maleimide (a PEG molecule containing a maleimide activating group on each end of the molecule) or bis-NHS-PEG (a PEG molecule containing an NHS group at each end of the molecule) .
  • PEG bis- maleimide a PEG molecule containing a maleimide activating group on each end of the molecule
  • bis-NHS-PEG a PEG molecule containing an NHS group at each end of the molecule
  • the carrier can contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the pharmaceutical compositions can be prepared by methods known in the art, including, by way of an example, the simple mixing of reagents. Those skilled in the art will know that the choice of the pharmaceutical carrier and the appropriate preparation of the composition depend on the intended use and mode of administration.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations can be stored either in a ready-to-use form or in a form that requires reconstitution prior to administration.
  • storage of the formulations is at temperatures conventional for such pharmaceuticals, including room temperature or preferably 4°C or lower, such as -70°C.
  • the formulations can be stored and administered between a pH range of about 5 to 8, preferably at about physiological pH.
  • the recombinant CTLA4 polypeptides and their functional derivatives can be used for a variety of purposes.
  • the recombinant polypeptides can be used as immunogens to produce polyclonal or monoclonal antibodies according to methods known in the art such as described, for example, in Harlow & Lane, Antibodies: A Laboratory Manual (1988) , incorporated herein by reference.
  • CTLA4 is immunosuppressive
  • the recombinant CTLA4 polypeptides are first denatured and, if desired, reduced prior to their use as an immunogen to produce anti-CTLA4 antibodies.
  • Such antibodies can, in turn, be used to detect CTLA4 receptor proteins on the cell surface of T cells or for in vivo uses such as imaging or to inhibit the binding of B7 to such receptor proteins according to procedures well known in the art.
  • the recombinant polypeptides of the present invention can also be used as research reagents to detect the presence of B7 or to purify B7 according to procedures known in the art.
  • the recombinant polypeptides can be labelled with a marker prior to being exposed to a sample suspected of containing the ligand to be detected.
  • the polypeptides can also be attached to a solid support for the purification of B7.
  • the recombinant polypeptides and functional derivatives thereof can be used to prevent, suppress or treat disorders associated with inappropriate T cell activation and proliferation. Accordingly, the present invention provides methods for the therapy of disorders associated with such deleterious T cell activation and proliferation.
  • the therapeutic methods of the present invention are accomplished by administering to a patient an effective amount of a recombinant CTLA4 polypeptide of the present invention or a functional derivative thereof to inhibit deleterious T cell activation.
  • the active ingredient is preferably formulated into a pharmaceutical composition as previously described.
  • the term "patient” refers to any animal having T cells that are capable of being co-stimulated by B7, including humans.
  • the recombinant CTLA4 polypeptides and their functional derivative are also referred to as the "active ingredient(s) .”
  • an effective dosage depends on a variety of factors known to those skilled in the art, including the species, age, weight, and medical condition of the patient, as well as the type of disorder to be prevented, suppressed or treated, the severity of the condition, the route of administration and the active ingredient used. A skilled physician or veterinarian can readily determine and prescribe an effective amount of the active ingredient. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the active ingredient. Thereafter, the dosage is increased by small increments until the optimum or desired effect is attained without causing significant harm or deleterious side effects. Preferably, the daily dosage is in the range of about 10-2000 mg per human patient.
  • the compounds and pharmaceutical compositions of the present invention can be administered orally or parenterally by any means known in the art, including, for example, by intravenous, subcutaneous, intraarticular or intramuscular injection or infusion. To achieve and maintain the desired effective dose, repeated administration may be desirable.
  • the frequency of dosing will depend on several factors such as, for example, the formulation used, the type of disorder, the individual characteristics of the patient, and the like. Those skilled in the art can readily determine the appropriate frequency based on such factors.
  • HuT 78 DNA sequences encoding the extracellular domain of the CTLA- 4 protein were cloned from a human T cell leukemia cell line, Hut 78, using the Polymerase Chain Reaction (PCR) technique.
  • the HuT 78 cell line (catalogue # TIB 161) was obtained from the American Type Culture Collection in Rockville, MD.
  • the Hut 78 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 5 x 10 "5 M 2-mercaptoethanol, 100 U/ml penicillin, and 100 ug/ l streptomycin at 4xl0 5 cells/ml.
  • the cells were activated by the addition of 5ng/ml phorbol 12- myristate 13-acetate (catalogue #P-8139, Sigma Chemical Company, St. Louis, MO), 1 ⁇ g/ml PHA-L (catalogue # L-4144, Sigma Chemical Company, St. Louis, MO), 5 ng/ l IL-2 (R&D Systems, Minneapolis, MN) and grown for an additional 49 hours.
  • 9xl0 6 cells were washed in phosphate buffered saline (PBS) , pelleted, frozen immediately in liquid nitrogen and stored at -70°C overnight.
  • PBS phosphate buffered saline
  • RNA/cDNA hybrids After denaturing the RNA/cDNA hybrids at 95°C for 1 minute the temperature was lowered to 60°C and 0.5 ⁇ l (2.5 units) of "AmpliTaq DNA Polymerase” (Perkin-Elmer Corporation, Norwalk, CT) was added and the temperature raised to 72°C for 1 minute.
  • the PCR was performed in an Ericomp "Twinblock” thermal cycler (San Diego, CA) with 29 additional cycles comprising 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C. The PCR amplification was completed with a 10 minute incubation at 72°C.
  • the reaction mixture was extracted with phenol once, followed by precipitation with ethanol and subsequently digested with Ndel and Hindlll restriction endonucleases.
  • the digested DNA was put through a spin column to remove small DNA fragments and a small portion (equivalent to about one twentieth of the original PCR reaction) was ligated to Ndel-HindiII cut pT88IQ, a Tac promoter expression plasmid, and inserted into E. coli host strain DH5-alpha (available from GIBCO BRL, Gaithersberg, MD) .
  • the lad region in the replacement also carries the laclq mutation — a single base substitution which results in an increase in lac repressor production (Muller-Hill et al., Proc. Nat'l Acad. Sci. U.S.A.) 59:1259-1264 (1968)).
  • laclq mutation a single base substitution which results in an increase in lac repressor production
  • This sequence contains an Ndel site (underlined) at the start codon for expression and a polylinker containing recognition sites for BamHl, Xmal, Kpnl, Sail, Sad, BstBI, Spel and SacII.
  • the pT5T::sCTLA4 construct was inserted into E. coli host strain HMS174/DE3 (obtained from Dr. F. William Studier, Brookhaven National Laboratory, Upton, NY) , and plasmid DNA prepared from 3 colonies. The plasmid DNAs were sequenced to verify that the SCTLA4 DNA sequences were correct and had been inserted correctly.
  • CTLA4 cDNAs obtained in the present study contained a threonine residue, and not an alanine residue, at amino acid position 111 of the protein sequence (SEQ ID NO:2) . This result confirms the corrected nucleotide sequence of human CTLA4 reported by Linsley et al., J. Ex . Med. 174:561-569 (1991) .
  • Preliminary expression of the SCTLA4 protein was performed by growing pT5T: :sCTLA-4 in HMS174/DE3 in Luria Broth containing 12ug/ml tetracycline to an OD ⁇ of 1.0 and inducing expression of sCTLA-4 by adding isopropyl beta-D thiogalactopyranoside (IPTG, catalogue #1-5502, Sigma Chemical Company, St. Louis, MO) to a concentration of ImM. Cells were harvested at two hours post- induction. Small aliquots of whole cells were boiled in SDS sample buffer containing 5% 2-mercaptoethanol for 2 minutes and run on a 14% polyacrylamide SDS gel.
  • IPTG isopropyl beta-D thiogalactopyranoside
  • the most prominent band visible upon staining the gel with coomassie blue was approximately the 14 kDa sCTLA-4. This band was absent in a control culture lysate prepared from HMS174/DE3 that contained pT5T with a different gene (interleukin-6) in it.
  • E. coli strain HMS174/DE3 containing plasmid pT5T::sCTLA4 was grown at 37°C in 10 L of complex medium ( 40 g/L NZ-amine HD, 4 g/L KH 2 P0 4 , 1 g/L MgS0 4 -7H 2 0, 1 g/L Na 2 S0 4 , 0.3 g/L Na 3 citrate-2H 2 0 , 50 g/L glycerol, 10 mg/L thiamine HC1, 2 ml/L trace minerals, 0.05 ml/L Mazu DF-204 and 15 mg/L tetracycline) until the optical density at A660 was 10.
  • complex medium 40 g/L NZ-amine HD, 4 g/L KH 2 P0 4 , 1 g/L MgS0 4 -7H 2 0, 1 g/L Na 2 S0 4 , 0.3 g/L Na 3 citrate-2H 2 0 , 50 g/L glyce
  • the cells were induced by adding 0.24 grams of IPTG ( a final concentration of 0.1 mM) to the culture.
  • the cells were grown for an additional 6.5 hours and then harvested by centrifugation.
  • the cell pellet was stored frozen at -20°C until use.
  • the loose pellets were resuspended in breaking buffer and spun again for 60 minutes at 10,000 rpm and the supernatant decanted. A two-phase pellet was observed: the lower pellet was white and the loose upper pellet was beige. The pellets were frozen at -20°C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of samples from the two pellets showed that the majority of the sCTLA-4 was present in the lower white pellet and that the upper beige pellet was composed primarily of E. coli membrane proteins.
  • SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Example 2 To remove the E. coli membrane proteins, the frozen pellets of Example 2 were thawed and resuspended in 1.5 liters of breaking buffer by homogenization with a Polytron PT 3000 mixer
  • the preferred concentration of guanidine-HCl in the refold mixture was determined to be between 0.6M and 4M.
  • the bottle containing the SCTLA4 refold mixture was left at 4°C for 2 days to allow the sCTLA4 to refold into its proper conformation. At this time, 50 ml of the refold mixture was removed and tested for biological activity. Before testing, the refold mixture was centrifuged for 15 minutes in a JA-20 rotor at 8,000 rpm in a J2-21 centrifuge (Beckman Instruments, Palo Alto, CA) to remove any precipitate that had formed during the refolding procedure. Twenty-five ml of the supernatant was then dialyzed against 4 liters of 50 mM NaCl, 20 mM Tris-HCl, pH 8.
  • HSA human serum albumin
  • a broad protein peak eluting at about 450-600mM NaCl also contained sCTLA4.
  • This peak was composed primarily of sCTLA4 monomers and . a few higher molecular weight forms of sCTLA4 which probably are disulfide crosslinked. This material may represent SCTLA4 forms that are aggregated.
  • the monomeric and dimeric forms of SCTLA4 were confirmed by electrophoresing samples of the column fractions on 14% polyacrylamide SDS gels under non-reducing conditions.
  • the dimeric form of SCTLA4 migrated as 2-3 bands in the 24-27 kDa molecular weight range.
  • the monomeric form of SCTLA4 migrated as 2-3 bands in the 14-16 kDa molecular weight range.
  • pool B was more potent than pools A and C in inhibiting lymphocyte proliferation.
  • the maximal inhibition seen with pool B in this experiment was 73%.
  • the dose of pool B that inhibited lymphocyte proliferation by 50% was about 1 ug/ml.
  • the doses of pools A and C that inhibited lymphocyte proliferation by 50% were about 10 ug/ml. No significant inhibition of lymphocyte proliferation was seen with HSA.
  • SCTLA4 proteins in pools A, B and C were inactivated during mixing of the samples .
  • co- stimulatory molecules other than CTLA4 / s ligand, B7 were expressed on the surfaces of one subject's antigen-presenting cells.
  • Other co-stimulatory molecules that are distinct from B7 are known to exist (Razi-Wolf et al., Proc. Nat'l Acad. Sci. (U.S.A.) 89:4210-4214 (1992); Liu et al., Eur. J. Immunol..
  • the B7 gene was cloned from the human Raji B cell line (ATCC No. CCL 86) .
  • mRNA was isolated from 3 x 10 6 Raji cells using a Micro-FastTrack mRNA Isolation Kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions.
  • cDNA copies of one tenth of the mRNA were made using a cDNA Cycle Kit (Invitrogen, San Diego) .
  • PCR conditions were 30 cycles of ( 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C) , followed by a 10 minute incubation at 72°C.
  • the supernatants were aspirated and the cells resuspended in 500 microliters of ice cold FACS media. Labeled cells were analyzed for positive fluorescence using a flow cytometer. Cell lines F9, C12 and H9 were positive for B7 expression using this assay.
  • the parent, non-transfected CHO cells induced 20 pg/ml IL-2.
  • Jurkat cells alone produced 20 pg/ml IL-2.
  • the C12 cell line was chosen for further assay development. Control experiments showed that PHA was required to induce IL-2 production above background levels. Titration experiments showed that IL-2 production increased in a dose-dependent way with increasing numbers of C12 cells per well in the bioassay.
  • CTLA4 was refolded and dimers purified from the Q-Sepharose column essentially as outlined in refold procedure 1. At least three dimer species could be discerned by non-reducing SDS-PAGE. The correct or most active dimer species constituted a minor amount of the total dimer species in this experiment. Fractions containing predominantly dimer species (Fractions 89-109) were pooled, concentrated to 222 ml, and applied to an S-100 sizing column as described in refold procedure 1. CTLA4 dimers eluted as a major protein peak, comprising fractions 33-41, followed by a smaller shoulder peak, comprising fractions 42-54.
  • Con-A-induced liver damage is detectible within 8 hours and results from polyclonal activation of T cells by macrophages in the presence of Con A. Liver damage is measured by release of specific liver enzymes, including serum glutamate pyruvic transaminase (SGPT) , into the blood stream.
  • SGPT serum glutamate pyruvic transaminase
  • each CTLA4 monomer contains a single PEGylated lysine residue. Small amounts of both a PEGylated product migrating at 46000 Da and unPEGylated monomer were also detected after reduction of the 65,000 Da minor PEGylated product. It is likely that these are the products from small amounts of high MW (> 67000 Da) PEGylated species contaminating the 65,000 Da product.
  • Example 11B Fractions 9 and 10 (doubly PEGylated dimers) , fractions 11 and 12 (mono-pegylated dimers) and fractions 14 and 15 (unreacted dimers) fro Example 11B were pooled separately, concentrated and assayed for activity in the IL-2 production bioassay described in Example 7. Results, shown in Table 9, indicate that mono- pegylated CTLA4 had an IC 50 approximately 3-4X greater than that of unpegylated CTLA4 (400 ng/ml versus 100 ng/ml) . The IC 50 for di-pegylated CTLA4 was about 6X greater than that of unpegylated CTLA4 .

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Abstract

La présente invention porte sur des polypeptides CTLA4 produits par recombinaison et conservant une activité de liaison B7, sans pour autant résulter de la fusion entre le CTLA4 et les gènes Ig humains. Les polypeptides CTLA4 de la présente invention comportent généralement une séquence d'acide aminé correspondant au domaine extra-cellulaire de la protéine réceptrice du CTLA4. En outre, l'invention concerne des dérivés fonctionnels des polypeptides CTLA4, y compris des mutéines et des conjugués contenant du polyéthylène glycol. L'invention porte également sur la préparation des polypeptides CTLA4 recombinants et sur les procédés de séparation des formes monomères et dimères des polypeptides recombinants ainsi que les formes des dimères actives et moins actives.
PCT/US1994/007685 1993-07-09 1994-07-08 Polypeptides ctla4 recombinants et procede de fabrication WO1995001994A1 (fr)

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US6051227A (en) * 1995-07-25 2000-04-18 The Regents Of The University Of California, Office Of Technology Transfer Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
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US6750334B1 (en) * 1996-02-02 2004-06-15 Repligen Corporation CTLA4-immunoglobulin fusion proteins having modified effector functions and uses therefor
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US7094874B2 (en) 2000-05-26 2006-08-22 Bristol-Myers Squibb Co. Soluble CTLA4 mutant molecules
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
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US7452535B2 (en) 2002-04-12 2008-11-18 Medarex, Inc. Methods of treatment using CTLA-4 antibodies
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US5855887A (en) * 1995-07-25 1999-01-05 The Regents Of The University Of California Blockade of lymphocyte down-regulation associated with CTLA-4 signaling
US7229628B1 (en) 1995-07-25 2007-06-12 The Regents Of The University Of California, Office Of Technology Transfer Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US6051227A (en) * 1995-07-25 2000-04-18 The Regents Of The University Of California, Office Of Technology Transfer Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US5811097A (en) * 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US6750334B1 (en) * 1996-02-02 2004-06-15 Repligen Corporation CTLA4-immunoglobulin fusion proteins having modified effector functions and uses therefor
US7405288B2 (en) 1998-03-06 2008-07-29 Diatech Pty. Ltd. V-like domain binding molecules and polynucleotides encoding therefor
US7166697B1 (en) 1998-03-06 2007-01-23 Diatech Pty. Ltd. V-like domain binding molecules
EP0947582A1 (fr) * 1998-03-31 1999-10-06 Innogenetics N.V. Une structure polypeptidique utilisable comme un squelette
US8883984B2 (en) 1998-12-23 2014-11-11 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
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US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
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US8491895B2 (en) 1998-12-23 2013-07-23 Amgen Fremont Inc. Methods of treating cancer with human monoclonal antibodies to CTLA-4
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US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
US7700556B2 (en) 2000-05-26 2010-04-20 Bristol-Myers Squibb Company Methods of treatment using CTLA4 mutant molecules
US8785398B2 (en) 2000-05-26 2014-07-22 Bristol-Myers Squibb Company Methods of treatment using CTLA4 mutant molecules
US7094874B2 (en) 2000-05-26 2006-08-22 Bristol-Myers Squibb Co. Soluble CTLA4 mutant molecules
US9758565B2 (en) 2000-05-26 2017-09-12 Bristol-Myers Squibb Company Methods of treatment using CTLA4 mutant molecules
US7439230B2 (en) 2000-05-26 2008-10-21 Bristol-Myers Squibb Company Methods of treatment using CTLA4 mutant molecules
US10370428B2 (en) 2000-05-26 2019-08-06 Bristol-Myers Squibb Company Methods of treatment using CTLA4 mutant molecules
US7452535B2 (en) 2002-04-12 2008-11-18 Medarex, Inc. Methods of treatment using CTLA-4 antibodies
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US9062111B2 (en) 2005-12-07 2015-06-23 Medarex, L.L.C. CTLA-4 antibody dosage escalation regimens
US9573999B2 (en) 2005-12-07 2017-02-21 E. R. Squibb & Sons, L.L.C. CTLA-4 antibody dosage escalation regimens
US8216996B2 (en) 2006-03-03 2012-07-10 Ono Pharmaceutical Co., Ltd. Multimer of extracellular domain of cell surface functional molecule
US8574872B2 (en) 2006-03-03 2013-11-05 Ono Pharmaceutical Co., Ltd. Multimer of extracellular domain of cell surface functional molecule
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EP0665852A1 (fr) 1995-08-09
AU7326594A (en) 1995-02-06
CA2144319A1 (fr) 1995-01-19

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