WO2008057634A2 - Polypeptides ayant des propriétés antiinflammatoires accrues et cytotoxiques réduites et procédés associés - Google Patents

Polypeptides ayant des propriétés antiinflammatoires accrues et cytotoxiques réduites et procédés associés Download PDF

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Publication number
WO2008057634A2
WO2008057634A2 PCT/US2007/072771 US2007072771W WO2008057634A2 WO 2008057634 A2 WO2008057634 A2 WO 2008057634A2 US 2007072771 W US2007072771 W US 2007072771W WO 2008057634 A2 WO2008057634 A2 WO 2008057634A2
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region
polypeptide
sialic acid
igg
linkage
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PCT/US2007/072771
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English (en)
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WO2008057634A3 (fr
Inventor
Falk Nimmerjahn
Kaneko Yoshikatus
Jeffrey V. Ravetch
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The Rockefeller University
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Priority claimed from PCT/US2006/041791 external-priority patent/WO2007055916A2/fr
Priority claimed from PCT/US2007/008396 external-priority patent/WO2007117505A2/fr
Priority to CA2666308A priority Critical patent/CA2666308C/fr
Application filed by The Rockefeller University filed Critical The Rockefeller University
Priority to EA200970416A priority patent/EA200970416A1/ru
Priority to AU2007317755A priority patent/AU2007317755A1/en
Priority to EP07812601A priority patent/EP2091969A4/fr
Priority to MX2009004399A priority patent/MX2009004399A/es
Priority to US12/447,204 priority patent/US8470318B2/en
Priority to CNA2007800397349A priority patent/CN101528774A/zh
Priority to JP2009534723A priority patent/JP2010512306A/ja
Priority to US11/957,015 priority patent/US20080206246A1/en
Priority to US12/013,212 priority patent/US20090004179A1/en
Publication of WO2008057634A2 publication Critical patent/WO2008057634A2/fr
Publication of WO2008057634A3 publication Critical patent/WO2008057634A3/fr
Priority to IL197920A priority patent/IL197920A0/en
Priority to US13/336,199 priority patent/US20120134988A1/en
Priority to US13/896,070 priority patent/US20130273040A1/en
Priority to US14/624,483 priority patent/US20160176950A1/en
Priority to US15/288,764 priority patent/US20170088608A1/en
Priority to US15/629,056 priority patent/US20170283492A1/en
Priority to US15/629,119 priority patent/US20170320935A1/en
Priority to US16/145,688 priority patent/US20190031737A1/en
Priority to US16/245,053 priority patent/US20190127446A1/en
Priority to US17/121,622 priority patent/US20210101961A1/en
Priority to US17/535,507 priority patent/US20220162290A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification

Definitions

  • the present invention relates to novel method for designing therapeutic polypeptides for treatment of inflammatory diseases.
  • BACKGROUND Although cellular receptors for immunoglobulins were first identified nearly 40 years ago, their central role in the immune response was only discovered in the last decade. They are key players in both the afferent and efferent phase of an immune response, setting thresholds for B cell activation and antibody production, regulating the maturation of dendritic cells and coupling the extraordinar specificity of the antibody response to effector pathways, such as phagocytosis, antibody dependent cellular cytotoxicity and the recruitment and activation of inflammatory cells. Their central role in linking the humoral immune system to innate effector cells has made them attractive immunotherapeutic targets for either enhancing or restricting the activity of antibodies in vivo.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • phagocytosis inflammatory mediator release, clearance of antigen, and antibody half-life
  • ADCC antibody dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • phagocytosis inflammatory mediator release, clearance of antigen, and antibody half-life
  • Antibody constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions.
  • antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, and IgG4; IgAl and IgA2.
  • the heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, d, e, ?, and ⁇ , respectively.
  • human IgGl and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily.
  • the Fc region is central to the effector functions of antibodies.
  • the crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry, 20, 2361-2370 (1981), which is incorporated herein by reference) .
  • the Fc region is generated by papain cleavage N-terminal to Cys, 226.
  • IgG has long been appreciated to mediate both pro- and anti-inflammatory activities through interactions mediated by its Fc fragment.
  • Fc-FcyR interactions are responsible for the pro-inflammatory properties of immune complexes and cytotoxic antibodies
  • IVIG intravenous gamma globulin
  • Fc fragments are anti-inflammatory and are widely used to suppress inflammatory diseases.
  • the precise mechanism of such paradoxical properties is unclear but it has been proposed that glycosylation of IgG is crucial for regulation of cytotoxicity and inflammatory potential of IgG.
  • IgG contains a single, N-linked glycan at Asn in the CH2 domain on each of its two heavy chains.
  • the covalently- linked, complex carbohydrate is composed of a core, biantennary penta-polysaccharide containing N- acetylglucosamine (GIcNAc) and mannose (man) . Further modification of the core carbohydrate structure is observed in serum antibodies with the presence of fucose, branching GIcNAc, galactose (gal) and terminal sialic acid (sa) moieties variably found. Over 40 different glycoforms have thus been detected to be covalently attached to this single glycosylation site. Fujii et al . , J. Biol.
  • the invention fills the foregoing need by providing such methods and molecules.
  • the invention provides an isolated polypeptide containing at least one IgG Fc region, having altered properties compared to an unpurified antibody preparation, wherein sialylation of the isolated polypeptide is higher than the sialylation of the unpurified antibody preparation.
  • the isolated polypeptide containing at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a ⁇ 2,6 linkage, and wherein said polypeptide having a higher anti-inflammatory activity as compared to an unpurified antibody.
  • the isolated polypeptide containing at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a ⁇ 2,6 linkage, and wherein said polypeptide having a reduced binding to an Fc activating receptor as compared to an unpurified antibody preparation.
  • the Fc activating receptor is selected from the group consisting of Fc?RIIA, Fc?RIIC and Fc?RIIIA.
  • the instant invention provides a pharmaceutical formulation comprising a polypeptide containing at least one Fc region having a higher antiinflammatory activity, in combination with a suitable carrier or diluent.
  • a method of modulating properties of a polypeptide comprising an Fc region comprising altering the sialylation of the polysaccharide chain of the Fc region.
  • the method comprises: providing an unpurified source of the polypeptide containing at least one Fc region, said unpurified source of the polypeptide containing at least one Fc region comprising a plurality of the polypeptides containing at least one Fc region having a polysaccharide chain comprising a terminal sialic acid connected to a galactose moiety through a ⁇ 2,6 linkage, and a plurality of the polypeptides containing at least one Fc region lacking a polysaccharide chain comprising a terminal sialic acid connected to a galactose moiety through the ⁇ 2,6 linkage; and increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the ⁇ 2,6 linkage to the plurality of the polypeptide containing at least one Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactos
  • Figure 1 is an illustration of MALDI-Tof analysis of SNA + FC linkages.
  • Figure 2 summarizes experiments demonstrating that enrichment of ⁇ 2,6 linkages between sialic acid and galactose improves anti-inflammatory properties of IVIG Fc fragments .
  • Figure 3 summarizes experiments demonstrating that removal of ⁇ 2,6 linkages between sialic acid and galactose attenuates anti-inflammatory properties of IVIG Fc fragments .
  • the inventors have surprisingly found that the cytotoxic and anti-inflammatory response of the IgG Fc domain results from the differential sialylation of the Fc- linked core polysaccharide .
  • the cytotoxicity of IgG antibodies is reduced upon sialylation; conversely, the anti-inflammatory activity of IVIG is enhanced.
  • IgG sialylation is shown to be regulated upon the induction of an antigen-specific immune response, thus providing a novel means of switching IgG from an innate, anti-inflammatory molecule in the steady-state, to a adaptive, proinflammatory species upon antigenic challenge.
  • the Fc- sialylated IgGs bind to a unique receptor on macrophages that in turn upregulates an inhibitory Fc ⁇ receptor (Fc ⁇ R) thereby protecting against autoantibody-mediated pathology.
  • Fc ⁇ R inhibitory Fc ⁇ receptor
  • the instant disclosure provides an advantageous strategy of creating and selecting IgGs with desired cytotoxic and anti-inflammatory potential.
  • the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Rabat et al . , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference.
  • the "EU index as in Rabat” refers to the residue numbering of the human IgGl EU antibody.
  • the term "native" or “parent” refers to an unmodified polypeptide comprising an Fc amino acid sequence.
  • the parent polypeptide may comprise a native sequence Fc region or an Fc region with pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions).
  • polypeptide refers to any fragment of a protein containing at least one IgG Fc region, including, without limitation, fully functional proteins, such as, for example, antibodies, e.g., IgG antibodies.
  • Fc region is used to define a C-terminal region of an immunoglobulin heavy chain.
  • the "Fc region” may be a native sequence Fc region or a variant Fc region.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the "CH2 domain" of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, MoI Immunol, 22, 161- 206 (1985), which is incorporated herein by reference).
  • the "CH3 domain” comprises the stretch of residues C- terminal to a CH2 domain in an Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of an IgG) .
  • the term "hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) . Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S—S bonds in the same positions .
  • binding domain refers to the region of a polypeptide that binds to another molecule.
  • the binding domain can comprise a portion of a polypeptide chain thereof (e.g., the a chain thereof) which is responsible for binding an Fc region.
  • One exemplary binding domain is the extracellular domain of an FcR chain.
  • a "functional Fc region” possesses at least a partial "effector function” of a native sequence Fc region.
  • effector functions include CIq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • phagocytosis e.g., B cell receptor; BCR
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as herein disclosed
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • a “variant Fc region” as appreciated by one of ordinary skill in the art comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one "amino acid modification.”
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and more preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith, even more preferably, at least about 99% homology therewith.
  • altered glycosylation refers to a polypeptide, as defined above, be it native or modified, in which the carbohydrate addition to the heavy chain constant region is manipulated to either increase or decrease specific sugar components.
  • polypeptides such as, for example, antibodies, prepared in specific cell lines, such as, for example, Lec2 or Lec3, may be deficient in the attachment of sugar moieties such as fucose and sialic acid.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • FcR is a native sequence human FcR.
  • FcR, including human FcR binds an IgG antibody (a gamma receptor) and includes receptors of the Fc?RI, Fc?RII, and Fc?RIII subclasses, including allelic variants and alternatively spliced forms of these receptors .
  • Fc?RII receptors include Fc?RIIA (an "activating receptor") and Fc?RIIB (an "inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc?RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc?RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daron, Annu Rev Immunol, 15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu Rev Immunol, 9, 457-92 (1991); Capel et al . , Immunomethods, 4, 25-34 (1994); and de Haas et al .
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to an in vitro or in vivo cell-mediated reaction in which cytotoxic cells that express FcRs (e.g., monocytic cells such as natural killer (NK) cells and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs e.g., monocytic cells such as natural killer (NK) cells and macrophages
  • NK natural killer
  • any effector cell with an activating Fc?R can be triggered to mediate ADCC.
  • the NK cell expresses Fc?RIII only, whereas monocytes, depending on their state of activation, localization, or differentiation, can express Fc?RI, Fc?RII, and Fc?RIII.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Bolland, Annu Rev Immunol, (2001), which is incorporated herein by reference.
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions.
  • the cells express at least one type of an activating Fc receptor, such as, for example, Fc?RIII and perform ADCC effector function.
  • Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, and neutrophils, with PBMCs and NK cells being preferred.
  • the effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • sialic acid content refers both to the total number of sialic acid residues on an Fc region of a heavy chain of an antibody and to the ratio of sialylated antibodies to asialylated antibodies in an unpurified antibody preparation, unless the phrase is in a context clearly suggesting that another meaning is intended.
  • Antibody fragments comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains FcR binding capability.
  • antibody fragments include linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the antibody fragments preferably retain at least part of the hinge and optionally the CHl region of an IgG heavy chain. More preferably, the antibody fragments retain the entire constant region of an IgG heavy chain, and include an IgG light chain.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256, 495-497 (1975), which is incorporated herein by reference, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567, which is incorporated herein by reference) .
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al . , Nature, 352, 624-628 (1991) and Marks et al . , J MoI Biol, 222, 581-597 (1991), for example, each of which is incorporated herein by reference.
  • the polypeptide containing at least one IgG Fc region may be fused with other protein fragments, including, without limitation, whole proteins.
  • proteins may be fused with the polypeptide of the present invention, including, without limitation, other immunoglobulins, especially, immunoglobulins lacking their respective Fc regions .
  • other biologically active proteins or fragments thereof may be fused with the polypeptide of the present invention, as described, for example, in the U.S. Patent No. 6,660,843, which is incorporated herein by reference. This embodiment is especially advantageous for delivery of such biologically active proteins or fragments thereof to cells expressing Fc receptors.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; Morrison et al .
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the polypeptides containing at least one IgG Fc region include those in which specific amino acid substitutions, additions or deletions are introduced into a parental sequence through the use of recombinant DNA techniques to modify the genes encoding the heavy chain constant region. The introduction of these modifications follows well- established techniques of molecular biology, as described in manuals such as Molecular Cloning (Sambrook and Russel, (2001)).
  • the polypeptides with at least one Fc region will include those polypeptides which have been selected to contain specific carbohydrate modifications, obtained either by expression in cell lines known for their glycosylation specificity (Stanley P., et al .
  • the polypeptide of the present invention can be expressed in a host expression systems, i.e., host cells, capable of N-linked glycosylation .
  • a host expression systems may comprise bacterial, fungal, plant, vertebrate or invertebrate expression systems.
  • the host cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell line, (e.g. CHO-Kl; ATCC CCL-61), Green Monkey cell line (COS) (e.g. COS 1 (ATCC CRL- 1650), COS 7 (ATCC CRL-1651)); mouse cell (e.g. NS/0), Baby Hamster Kidney (BHK) cell line (e.g.
  • CHO Chinese hamster ovary
  • COS Green Monkey cell line
  • NS/0 Baby Hamster Kidney
  • ATCC CRL-1632 or ATCC CCL-IO ATCC CRL-1632 or ATCC CCL-IO
  • human cell e.g. HEK 293 (ATCC CRL-1573)
  • any other suitable cell line e.g., available from public depositories such as the American Type Culture Collection, Rockville, Md.
  • an insect cell line such as a Lepidoptora cell line, e.g. Sf9, a plant cell line, a fungal cell line, e.g., yeast such as, for example, Saccharomyces cerevisiae, Pichia pastoris, Hansenula spp . , or a bacterial expression system based on Bacillus, such as B. subtilis, or Eschericiae coli can be used.
  • Therapeutic formulations comprising the polypeptides containing at least one IgG Fc region can be prepared for storage by mixing the polypeptides of the present invention having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenyl, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and tricresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in a microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly- (methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules ) or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration are sterile.
  • the formulations of the instant invention can be easily sterilized, for example, by filtration through sterile filtration membranes .
  • Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the modified antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly (vinylalcohol) ) , polylactides (see, e.g., U.S. Pat. No.
  • copolymers of L-glutamic acid and y ethyl-L- glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate)
  • poly-D- (-) -3-hydroxybutyric acid While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods .
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity . Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions . Creation of sialylated polypeptides containing at least one IgG Fc region.
  • the polypeptides of the present invention can be further purified or modified so that they have an increased amount of sialic acid compared to unmodified and/or unpurified antibodies. Multiple methods exist to reach this objective.
  • the source of unpurified polypeptides such as, for example, IVIG
  • lectin which is known to bind sialic acid.
  • lectins display different affinities for ⁇ 2,6 versus oc2,3 linkages between galactose and sialic acid.
  • selecting a specific lectin will allow enrichment of antibodies with the desired type of linkage between the sialic acid and the galactose.
  • the lectin is isolated from Sambuccus nigra.
  • SNA Sambuccus nigra agglutinin
  • MAA Maakia amurensis
  • a fraction of the polypeptides containing at least one IgG Fc region having a desired linkage between the galactose and the sialic acid will be retained in the column while a fraction lacking such linkage will pass through.
  • the sialylated fraction of the polypeptides containing at least one IgG Fc region can be eluted by another wash with a different stringency conditions.
  • one may employ an enzymatic reaction with a sialyltransferase and a donor of sialic acid as described, for example, in the U.S. Pat. No. 20060030521.
  • sialyltransferase enzymes useful in the claimed methods are ST3Gal III, which is also referred to as (X- (2, 3 ) sialyltransferase (EC 2.4.99.6), and OC- (2, 6) sialyltransferase (EC 2.4.99.1).
  • Alpha- (2, 3) sialyltransferase catalyzes the transfer of sialic acid to the Gal of a Gal- ⁇ -1, 3GIcNAc or Gal- ⁇ - 1,4GIcNAc glycoside (see, e.g., Wen et al . , J. Biol. Chem. 267: 21011 (1992); Van den Eijnden et al . , J. Biol. Chem. 256: 3159 (1991)) and is responsible for sialylation of asparagine-linked oligosaccharides in glycopeptides .
  • the sialic acid is linked to a Gal with the formation of an OC- linkage between the two saccharides.
  • Bonding (linkage) between the saccharides is between the 2-position of NeuAc and the 3-position of Gal.
  • This particular enzyme can be isolated from rat liver (Weinstein et al . , J. Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al . (1993) J. Biol. Chem. 268: 22782-22787 ; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401) and genomic (Kitagawa et al . (1996) J. Biol. Chem. 271: 931-938) DNA sequences are known, facilitating production of this enzyme by recombinant expression .
  • ⁇ - (2, 6 ) sialyltransferase Activity of ⁇ - (2, 6 ) sialyltransferase results in 6- sialylated oligosaccharides, including 6-sialylated galactose.
  • the name " ⁇ - (2, 6 ) sialyltransferase” refers to the family of sialyltransferases attaching sialic acid to the sixth atom of the acceptor polysaccharide.
  • Different forms of a- ( 2, 6 ) sialyltransferase can be isolated from different tissues. For example, one specific form of this enzyme, ST ⁇ Gal II, can be isolated from brain and fetal tissues. Krzewinski-Recchi et al . , Eur. J. Biochem. 270, 950 (2003).
  • cell culture conditions can be manipulated to change the sialylation rate. For example, to increase the sialic acid content, production rate is decreased and osmolality is generally maintained within a lower margin suitable for the particular host cell being cultured. Osmolality in the range from about 250 mOsm to about 450 mOsm is appropriate for increased sialic acid content.
  • This and other suitable cell culture conditions are described in, e.g., U.S. Patent No. 6,656,466. Patel et al .
  • host cells such as, for example, immortalized human embryonic retina cells
  • a nucleic acid encoding a sialyltransferase such as, for example, an ⁇ -2, 3- sialyltransferase or an ⁇ -2, 6-sialyltransferase, operably linked to a promoter, such as, for example, a CMV promoter.
  • the ⁇ -2, 3-sialyltransferase may be the human ⁇ -2, 3- sialyltransferase, known as SIAT4C or STZ (GenBank accession number L23767), and described, for example, in the U.S. Pat. No. 20050181359.
  • the nucleic acid encoding the sialyltransferase may be introduced into the host cell by any method known to a person of ordinary skill in the art. Suitable methods of introducing exogenous nucleic acid sequences are also described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual (3 rd Edition), Cold Spring Harbor Press, NY, 2000. These methods include, without limitation, physical transfer techniques, such as, for example, microinjection or electroporation; transfections, such as, for example, calcium phosphate transfections ; membrane fusion transfer, using, for example, liposomes; and viral transfer, such as, for example, the transfer using DNA or retroviral vectors .
  • the polypeptide containing at least one IgG Fc region may be recovered from the culture supernatant and can be subjected to one or more purification steps, such as, for example, ion-exchange or affinity chromatography, if desired. Suitable methods of purification will be apparent to a person of ordinary skill in the art.
  • sialylation methods can lead to production of the polypeptides containing at least one IgG Fc region with an extremely high level of sialylation.
  • an enzymatic reaction followed by affinity chromatography may be used for IVIG source of the polypeptides containing at least one IgG Fc region.
  • polypeptides containing at least one IgG Fc region can be purified and analyzed in SDS-PAGE under reducing conditions.
  • the glycosylzation can be determined by reacting the isolated polypeptides with specific lectins, or, alternatively as would be appreciated by one of ordinary skill in the art, one can use HPLC followed by mass spectrometry to identify the glycoforms . (Wormald, MR et al., Biochem 36:1370 (1997). To describe the instant invention in more details, several non-limiting illustrative examples are given below.
  • the anti-platelet antibodies derived from the 6A6 hybridoma, expressed as either an IgGl, 2a or 2b switch variant in 293 cells as previously described (6), were analyzed by mass spectroscopy to determine their specific carbohydrate composition and structure. These antibodies contain minimal sialic acid residues. Enrichment of the sialic acid containing species by Sambucus nigra lectin affinity chromatography yielded antibodies enriched 60-80 fold in sialic acid content.
  • sialylation of IgG2b displaying an in vivo activity comparable to that of asialylated IgGl.
  • sialylation of IgGl reduces its already low binding affinity for its activation receptor FcyRlll by a factor of 7 thereby generating a physiologically inactive antibody.
  • sialylation of the Asn 297 linked glycan structure of IgG resulted in reduced binding affinities to the subclass-restricted activation FcyRs and thus reduced their in vivo cytotoxicity.
  • C57BL/6 and NOD mice were purchased from the Jackson Laboratory (Bar Harbor, ME) .
  • FcyRIIB ⁇ / ⁇ mice were generated in the inventors' laboratory and backcrossed for 12 generations to the C57BL/6 background.
  • KRN TCR transgenic mice on a C57BU6 background (K/B) were gifts from D. Mathis and C. Benoist (Harvard Medical School, Boston, MA) and were bred to NOD mice to generate K/BxN mice.
  • Female mice at 8-10 weeks of age were used for all experiments and maintained at the Rockefeller University animal facility. All experiments were done in compliance with federal laws and institutional guidelines and have been approved by the Rockefeller University (New York, NY) .
  • 6A6 antibody switch variants were produced by transient transfection of 293T cells followed by purification via protein G as described. Nimmerjahn and Ravetch, Science 310, 1510 (2005) . Sialic acid rich antibody variants were isolated from these antibody preparations by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) agarose (Vector Laboratories, Burlingame, CA). Enrichment for sialic acid content was verified by lectin blotting (see below). Human intravenous immune globulin (IVIG, 5% in 10% maltose, chromatography purified) was purchased from Octapharma (Hemdon, VA) . Digestion of human IVIG was performed as described. Kaneko Y.
  • SNA Sambucus nigra agglutinin
  • IVIG was digested by 0.5 mg/ml papain for 1 hr at 37 0 C, and stopped by the addition of 2.5 mg/ml iodados .
  • Fab and Fc resulting fragments were separated from non-digested IVIG on a HiPrep 26/60 S-200HR column (GE Healthcare, Piscataway, NJ) , followed by purification of Fc and Fab fragments with a Protein G column (GE Healthcare) and a Protein L column (Pierce, Rockford, IL) . Fragment purity was checked by immunoblotting using anti-human IgG Fab or Fc-specific antibodies.
  • the F4/80 antibody was from Serotec (Oxford, UK).
  • the Ly 17.2 antibody was from Caltag (Burlingame, CA) .
  • Sheep anti- glomerular basement membrane (GBM) antiserum was a gift from M. P. Madaio (University of Pennsylvania, Philadelphia, PA) .
  • Soluble Fc receptors containing a C-terminal hexa-hisitidine tag were generated by transient transfection of 293T cells and purified from cell culture supernatants with Ni-NTA agarose as suggested by the manufacturer (Qiagen) .
  • IVIG was treated with neuraminidase and the composition and structure of the resulting preparation was analyzed by mass spectroscopy. No detectable sialic acid containing glycans remained after neuraminidase treatment.
  • IgG preparations were then tested for their ability to protect mice from joint inflammation induced by passive transfer of KxN serum, an IgG 1 immune complex-mediated inflammatory disease model. De-sialylation with neuraminidase abrogated the protective effect of the IVIG preparation in the KXN serum induced arthritis model. This loss of activity was not the result of reduced serum half-life of the asialylated IgG preparations or the result of changes to the monomeric composition or structural integrity of the IgG. Removal of all glycans with PNGase had a similar effect and abrogated the protective effect of IVIG in vivo.
  • IVIG Preparation of IVIG with an increased content of sialic acid Since sialic acid appeared to be required for the antiinflammatory activity of IVIG, the basis for the high dose requirement (1 g/kg) for this anti-inflammatory activity could be the limiting concentration of sialylated IgG in the total IVIG preparation.
  • the IVIG was fractionated on an SNA- lectin affinity column to obtain IgG molecules enriched for sialic acid modified glycan structures. These sialic acid enriched fractions were tested for protective effects in the KxN serum transfer arthritis model as compared to unfractionated IVIG.
  • the polyclonal IgG in IVIG may also contain O and N linked glycans on the light chains or heavy chain variable domains that can be sialylated
  • Fc fragments were generated from unfractionated and SNA fractionated IVIG and tested for their in vivo activity. As observed for intact IgG, SNA-purified Fc fragments were enhanced for their protective effect in vivo when compared to Fc fragments generated from unfractionated IVIG. In contrast, Fab fragments displayed no anti-inflammatory activity in this in vivo assay.
  • the high dose requirement for the antiinflammatory activity of IVIG can be attributed to the minor contributions of sialylated IgG present in the total preparation. Enrichment of these fractions by sialic acid binding lectin chromatography consequently increased the anti-inflammatory activity.
  • mice are first sensitized with sheep IgG together with adjuvant and four days later injected with a sheep anti-mouse glomerular basement membrane preparation (nephrotoxic serum, NTS). Briefly, mice were pre-immunized intraperitoneally with 200 ⁇ g of sheep IgG (Serotec) in CFA, followed by intravenous injection of 2.5 ⁇ l of NTS serum per gram of body weight four days later.
  • a sheep anti-mouse glomerular basement membrane preparation nephrotoxic serum, NTS.
  • Blood was collected from non-treated control mice four days after the anti-GBM anti-serum injection, and serum IgG was purified by Protein G (GE Healthcare, Princeton, NJ) and sepharose-bound sheep IgG column, generated by covalently coupling sheep IgG on NHS-activated sepharose column (GE Healthcare, Princeton, NJ), affinity chromatography.
  • Protein G GE Healthcare, Princeton, NJ
  • sepharose-bound sheep IgG column generated by covalently coupling sheep IgG on NHS-activated sepharose column (GE Healthcare, Princeton, NJ), affinity chromatography.
  • mice IgG2b anti-sheep IgG antibodies (NTN immunized). Kaneko Y. et al . , Exp. Med., 203:789 (2006).
  • Mouse IgG2b antibodies are deposited in the glomerulus together with the NTS antibodies and result in an acute and fulminant inflammatory response by the IgG2b mediated activation of FcyRIV on infiltrating macrophages. In the absence of pre-sensitization inflammation is not observed, indicating that the mouse IgG2b anti-sheep IgG antibodies are the mediators of the inflammatory response.
  • the analysis of the pre and post immunization IgGs confirmed that the changes in the N-glycan structure were specific to the terminal sialic acids moieties.
  • the mouse IgG2b anti-sheep antibodies that were deposited in the glomeruli, previously shown to be responsible for engagement of the FcyRIV bearing, infiltrating macrophages displayed reduced sialic acid content as compared to the pre-immunized controls .
  • the footprint histogram of the enriched galactose-sialic acid structures with in vivo anti-inflammatory activity were compared to histograms from sialic acid linkage standards, a2-3 sialyllactose (Figure IB) and a2-6 sialyllactose (Figure 1C) .
  • the signature peaks of the standards are identified by arrows, shown by arrows for a2-3 ( Figure IB) or a2-6 ( Figures IA and 1C), respectively, and compared to the peaks obtained from the sample.
  • EXAMPLE 7 ENRICHMENT OF IVIG FC FRAGMENTS IN CC2,6 LINKAGES BY IN VITRO GLYCOSYLATION IMPROVES ANTI-INFLAMMATORY PROPERTIES OF IVIG.
  • mice received either 0.66 mg of a 2-6 sialylated Fes (black triangles) or 0.66 mg a 2-3 sialylated Fes (red triangles) .
  • IVIG was treated with linkage specific sialidases (SAs), and the digestion verified by lectin blotting ( Figure 3A) .
  • the top panel shows positive Sambucus nigra lectin (SNA) staining for a 2-6 linkages in IVIG (left lane), and a 2-3 SA tx IVIG (center lane), but not in a 2-3,6 SA tx IVIG (right lane) .
  • the middle panel is a dot blot for a2-3 sialic acid linkages (MAL I), displaying positive staining for the fetuin positive control only; 100 ⁇ g protein are loaded per dot.
  • the bottom panel shows coomassie loading control. 10 ⁇ g/lane are shown in the blot and gel.
  • mice were given lg/kg of IVIG preparations prior to 200 ⁇ l of K/BxN sera.
  • footpad swelling was observed in mice administered K/BxN sera (white circles) over the course of a week, as measured by clinical scoring.
  • IVIG treated mice showed minimal swelling (black triangles), as did mice treated with a2-3 SA tx IVIG (white triangles), while mice receiving a2-3,6 SA tx IVIG (red squares) were not protected from footpad swelling.

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Abstract

L'invention concerne un polypeptide contenant au moins une région IgG Fc, ladite région IgG Fc étant glycosylée avec au moins une fonction galactose reliée à une fonction acide sialique terminale respective par une liaison α 2, 6, et ledit polypeptide ayant une activité antiinflammatoire accrue en comparaison d'un anticorps non purifié.
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US12/447,204 US8470318B2 (en) 2005-11-07 2007-07-03 Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods
CNA2007800397349A CN101528774A (zh) 2006-10-26 2007-07-03 具有增强的抗炎性和降低的细胞毒性特性的多肽以及相关方法
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US11/957,015 US20080206246A1 (en) 2006-04-05 2007-12-14 Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods
US12/013,212 US20090004179A1 (en) 2005-11-07 2008-01-11 Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods
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US13/336,199 US20120134988A1 (en) 2005-11-07 2011-12-23 Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods
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US15/288,764 US20170088608A1 (en) 2005-11-07 2016-10-07 Polypeptides With Enhanced Anti-Inflammatory And Decreased Cytotoxic Properties And Relating Methods
US15/629,119 US20170320935A1 (en) 2005-11-07 2017-06-21 Polypeptides With Enhanced Anti-Inflammatory And Decreased Cytotoxic Properties And Relating Methods
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