HK1208881B

HK1208881B - Method for producing soluble fcr as fc-fusion with inert immunoglobulin fc-region and uses thereof

Info

Publication number: HK1208881B
Application number: HK15109569.5A
Authority: HK
Inventors: Petra Rueger; Tilman Schlothauer; Stefan Seeber
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2012-08-02
Filing date: 2013-07-31
Publication date: 2018-06-15

Description

Method for producing soluble FcR as Fc fusion with inert immunoglobulin Fc region and use thereof

Herein is reported a method for the production of soluble Fc receptors as fusion polypeptides with inert immunoglobulin Fc regions that prevent self-aggregation and uses thereof, such as FcR chromatography columns, for the determination of FcR interaction of low affinity antibodies.

Background

Immunoglobulins generally comprise two light polypeptide chains and two heavy polypeptide chains. Each of the heavy and light polypeptide chains comprises a variable region (typically the amino-terminal portion of the polypeptide chain) that contains a binding domain capable of interacting with an antigen. Each of the heavy and light polypeptide chains also comprises a constant region (typically the carboxy-terminal portion). The constant region of the heavy chain mediates binding of the immunoglobulin to, for example, cells with Fc γ receptors (fcyr), such as phagocytes, or with neonatal Fc receptors (FcRn), also known as Brambell receptors, and also mediates binding to certain factors, including factors of the classical complement system, such as component (C1 q).

Hulett and Hogarth (Hulett, m.d. and Hogarth, p.m., adv.immunol.57(1994)1-127) report that the extracellular receptors for the Fc portion of class G immunoglobulins are a family of transmembrane glycoproteins comprising three different receptor types with different binding specificities: fc γ RI, Fc γ RII, and Fc γ RIII. Type I receptors interact with uncomplexed IgG, whereas type II and type III receptors preferably interact with complexed IgG.

Human Fc γ RIII (CD16) exists in two isoforms and two polymorphic forms. The first isoform, Fc γ RIIIa, is a transmembrane molecule encoded by a gene distinct from the second isoform, Fc γ RIIIb, which is a GPI-anchored membrane protein. Polymorphic form V159 has a valine residue at position 159 of the amino acid sequence and polymorphic form F159 has a phenylalanine residue at position 159.

For immunoglobulins of the IgG class, ADCC and ADCP are governed by the involvement of Fc regions in a family of receptors known as Fc-gamma (Fc γ) receptors (Fc γ R). In humans, this family of proteins comprises Fc γ RI (CD64), Fc γ RII (CD32) including isoforms Fc γ RIIA, Fc γ RIIB, and Fc γ RIIC, and Fc γ RIII (CD16) including isoforms Fc γ RIIIA and Fc γ RIIIB (Raghavan and Bjorkman, Annu. Rev. cell Dev. biol.12(1996) 181-220; Abes, et al, Expert Reviews (2009) 735-747). Fc γ R is expressed on a variety of immune cells, and the formation of the Fc/Fc γ R complex recruits these cells to the site of bound antigen, often resulting in signaling and subsequent immune responses such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. Furthermore, while Fc γ RI, Fc γ RIIA/C, and Fc γ RIIIA are activating receptors characterized by an intracellular immunoreceptor tyrosine-based activation motif (ITAM), Fc γ RIIB possesses an inhibitory motif (ITIM) and is therefore inhibitory. Although Fc γ RI binds monomeric IgG with high affinity, Fc γ RIII and Fc γ RII are low affinity receptors, interacting with complexed or aggregated IgG.

IgG binding to the activating and inhibitory Fc γ receptor or the first component of complement (C1q) depends on residues located in the hinge and CH2 domains. 2 regions of the CH2 domain are critical for Fc γ R and complement C1q binding and have unique sequences. Replacement of residues 233-236 and 327, 330 and 331 of IgG1 and IgG2 and IgG4 greatly reduced ADCC and CDC (Armour, et al, Eur. J. Immunol.29(1999) 2613-2624; Shields, et al, J. biol. chem.276(2001) 6591-6604). Idusogene, et al (J.Immunol 166(2000)2571-2575) directed against the therapeutic antibody Rituxan^(R)Mapping of the C1q binding site and showing Pro329Ala substitution reduced Rituximab (Ritu)ximab) the ability to bind C1q and activate complement. Replacement of Pro329 with Ala has been reported to result in reduced binding to Fc γ RI, Fc γ RII and Fc γ RIIIA receptors (Shields, et al, j.biol.chem.276(2001) 6591-.

Recombinant FcRn and variants thereof for the purification of Fc-containing fusion proteins are reported in WO 2010/048313. High levels of expression and secretion of Fc-X fusion proteins in mammalian cells were reported by Lo et al (Lo, K-M., et al, prot. Eng.11(1998) 495-500). Dumont, F.A., et al (Biodrugs 20(2006)151-160) reported monomeric Fc fusions. Receptor Fc fusion therapeutics, taps, and mimetibdodtm techniques were reported by Huang, c. (curr. opin. biotechnol.20(2009) 592-599). Immunoglobulin fusion proteins are reported in WO 01/03737. Expression and export of anti-obesity proteins as Fc fusion proteins is reported in WO 00/40615.

Summary of The Invention

It has been found that soluble Fc receptors can be produced by expressing the Fc receptor as a fusion polypeptide having an Fc region that does not substantially bind to the fused Fc receptor. The use of fusion polypeptides to express Fc receptors increases the available production of Fc receptors in the form of fusion polypeptides or as isolated receptors. Furthermore, the fusion polypeptides as reported herein provide increased flexibility for combining more than one copy of Fc receptors in a single molecule, e.g. for increased affinity, or for combining different Fc receptors (of different origin or different types or both) in a single molecule.

One aspect as reported herein is a fusion polypeptide according to formula I:

R1-FC-R2 (formula I)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor, and

FC denotes a heavy chain Fc region polypeptide,

wherein R1 or R2 or both are present,

wherein FC does not substantially bind R1 and/or R2.

In one embodiment the fusion polypeptide has formula II

R1-CS1-L1-CS2-FC-CS3-L2-CS4-R2 (formula II)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor,

FC denotes a heavy chain Fc region polypeptide,

CS1 denotes the first cleavage site,

CS2 denotes a second cleavage site which,

CS3 denotes a third cleavage site,

CS4 denotes a fourth cleavage site,

l1 denotes a first intervening amino acid sequence, and

l2 denotes a second intervening amino acid sequence,

wherein R1 or R2 or both are present,

wherein any of CS1, CS2, CS3, CS4 may be present or absent independently of each other,

wherein L1 and L2 may be present or absent independently of each other,

wherein FC does not substantially bind R1 and/or R2.

In one embodiment R1 and R2 are independently from each other selected from the group of human fey receptors, human neonatal Fc receptors, murine Fc receptors, and rabbit neonatal Fc receptors.

In one embodiment the human Fc γ receptor is selected from human Fc γ RI (CD64), human Fc γ RII (CD32), human Fc γ RIIA, human Fc γ RIIB, human Fc γ RIIC, human Fc γ RIII (CD16), human Fc γ RIIIA, and human Fc γ RIIIB.

In one embodiment the human neonatal Fc receptor is a human FcRn.

In one embodiment the murine Fc receptor is selected from the group consisting of murine Fc γ RI (CD64), murine Fc γ RII (CD32), murine Fc γ RIIB, murine Fc γ RIII (CD16), murine Fc γ RIII-2(CD16-2), and murine Fc γ RIV.

In one embodiment the Fc is a variant of a heavy chain polypeptide selected from the group of human IgG heavy chain polypeptides, murine IgG heavy chain polypeptides, rabbit IgG heavy chain polypeptides.

In one embodiment, the FC is a variant of a heavy chain polypeptide selected from the group of human IgG1 heavy chain polypeptide, human IgG2 heavy chain polypeptide, human IgG3 heavy chain polypeptide, human IgG4 heavy chain polypeptide, murine IgG1 heavy chain polypeptide, murine IgG2 heavy chain polypeptide, murine IgG2a heavy chain polypeptide, murine IgG3 heavy chain polypeptide, rabbit IgG heavy chain polypeptide.

In one embodiment, the heavy chain Fc region polypeptide has an amino acid mutation at one or more of positions 234, 235, 236, 237, 238, 239, 253, 254, 265, 266, 267, 268, 269, 270, 288, 297, 298, 299, 307, 311, 327, 328, 329, 330, 331, 332, 434 and 435.

In one embodiment, one or more of the Fc receptors are fey receptors.

In one embodiment, the human IgG1 heavy chain polypeptide has a mutation at one or more of amino acid positions 233, 234, 235, 236, 265, 297, 329, and 331.

In one embodiment, the human IgG1 heavy chain polypeptide has one or more of the amino acid mutations E233P, L234A, L235A, L235E, Δ G236, D265A, N297A, N297D, P329A, P329G and P331S.

In one embodiment, the human IgG1 heavy chain polypeptide has the amino acid mutations L234A and L235A and one or more of E233P, L235E, Δ G236, D265A, N297A, N297D, P329A, P329G and P331S.

In one embodiment, the human IgG1 heavy chain polypeptide has the amino acid mutations L234A and L235A and P329A or P329G.

In one embodiment, the human IgG2 heavy chain polypeptide has a mutation at one or more of amino acid positions 233, 234, 235, 236, 265, and 329.

In one embodiment, the human IgG4 heavy chain polypeptide has a mutation at one or more of amino acid positions 228, 235, 265, and 329.

In one embodiment, the human IgG4 heavy chain polypeptide has one or more of the mutations S228P, L235E, P329A, and P329G.

In one embodiment, the human IgG4 heavy chain polypeptide has mutations S228P and L235E and P329A or P329G.

In one embodiment, the heavy chain Fc region polypeptide has an amino acid mutation at one or more of positions 248, 250, 251, 252, 253, 254, 255, 256, 257, 272, 285, 288, 290, 291, 308, 309, 310, 311, 314, 385, 386, 387, 428, 433, 434, 435, and 436.

In one embodiment, one or more of the Fc receptors is FcRn.

In one embodiment, the human IgG heavy chain polypeptide has a mutation at one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447.

In one embodiment, a human IgG heavy chain polypeptide having reduced binding to FcRn has one or more amino acid alterations at amino acid positions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439, and/or 447.

In one embodiment, a human IgG heavy chain polypeptide having reduced binding to FcRn has the amino acid mutations I253A, H310A, and H435A.

In one embodiment, the intervening amino acid sequence is selected from a first group comprising (G3S)3, (G3S)4, (G3S)5, (G3S)6, (G4S)3, (G4S)4, (G4S)5, (G5S)2, (G5S)3, and (G5S)4, or from a second group comprising an Arg tag, an Avi tag, a His-Avi tag, a His tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin binding peptide, a cellulose binding domain, a chitin binding domain, a GST tag, or an MBP tag, or from a combination of two elements selected from these groups.

In one embodiment, the cleavage site is selected from the group consisting of an IgA protease cleavage site, a granzyme B protease cleavage site, a Tev protease cleavage site, a Precision protease cleavage site, a thrombin cleavage site, a Faktor10a protease site, an Ides protease cleavage site, an enterokinase cleavage site, or a SUMO protease cleavage site.

In one embodiment, the fusion polypeptide does not comprise an additional protease cleavage site but rather an inherent protease cleavage site, such as a papain cleavage site, a pepsin cleavage site, or an Ides protease cleavage site.

One aspect as reported herein is a dimeric fusion polypeptide comprising two fusion polypeptides as reported herein.

In one embodiment the first FC comprises the mutation T366W and optionally the mutation S354C and the second FC comprises the mutations T366S, L368A and Y407V and optionally the mutation Y349C.

In one embodiment the fusion polypeptide is characterized by

a) R1 and R2 of the first and second polypeptides are identical,

b) r1 and R2 of the first fusion polypeptide are identical, R1 and R2 of the second fusion polypeptide are identical but different from R1 and R2 of the first fusion polypeptide,

c) r1 of the first and second fusion polypeptides are the same and R2 of the first and second polypeptides are the same but different from R1,

d) r1 of the first and second fusion polypeptides are the same and neither R2 is present,

e) r1 of the first and second fusion polypeptides are different and neither R2 is present,

f) r2 of the first and second fusion polypeptides are the same and neither R1 is present,

g) r2 of the first and second fusion polypeptides are different and neither R1 is present,

h) r1 of the first fusion polypeptide and R2 of the second polypeptide are different and R2 of the first fusion polypeptide is absent and R1 of the second polypeptide is absent.

One aspect as reported herein is a method for the production of a soluble Fc receptor, said method comprising the steps of:

a) culturing a cell comprising a nucleic acid encoding a fusion polypeptide as reported herein,

b) recovering the fusion polypeptide from the cell or the culture medium,

c) optionally cleaving the fusion polypeptide with a protease,

thereby producing soluble Fc receptors.

One aspect as reported herein is the use of an immobilized fusion polypeptide as reported herein or an immobilized dimeric fusion polypeptide as reported herein as ligand for affinity chromatography.

One aspect as reported herein is the use of an immobilized fusion polypeptide as reported herein or an immobilized dimer fusion as reported herein for determining Fc receptor binding of an antibody.

In one embodiment, the fusion polypeptide is bound to a solid phase.

One aspect as reported herein is a pharmaceutical composition comprising a fusion polypeptide as reported herein.

One aspect as reported herein is the use of a fusion polypeptide as reported herein for the preparation of a medicament.

In one embodiment the medicament is for the treatment of an inflammatory disease.

In one embodiment the disease is a disease characterized by increased antibody levels.

In one embodiment the disease is an autoimmune disease.

In one embodiment the disease is rheumatoid arthritis.

Detailed Description

Brief Description of Drawings

FIG. 1 is a plasmid map of a fusion polypeptide expression plasmid.

FIG. 2. papain cleavage analytical SDS-PAGE gels.

FIG. 3.12% Bis Tris Gel +/-DTT; fc γ RIIIaV158-Avi-Fc LALA P239G was cleaved with PreScission protease (lanes 3,8) and IgA protease (lanes 2,7), respectively: non-specific cleavage with PP can be seen (lanes 3, 8).

FIG. 4 isolation and quantification of different glycosylated forms of anti-Her antibody (wild type, top) and glycoengineered anti-Her antibody.

FIG. 5 comparison of affinity columns using Fc γ RIIIaV158 and Fc labeled Fc γ RIIIaV 158.

Figure 6. BIAcore sensorgrams of the response of Fc γ RIIIaV158-Fc LALA P329G fusion polypeptide compared to 40RU of Fc γ RIIIaV158 showed more than 100 response units; FcgRIIIa V158_008 represents a non-cleaved fusion polypeptide, FcgRIIIa V158_ 007 represents a shortened non-functional variant of FcgRIIIa (═ control), and FcgRIIIa V158_ jf323 represents the complete HisAvi marker comprising a functional variant of FcgRIIIa.

Figure 7 sensorgrams for Fc γ receptor V158-Fc LALA P329G fusion polypeptide (figure 7a), Fc γ receptor V158 (figure 7b), cleaved Fc γ receptor V158-Fc LALA P329G fusion polypeptide (figure 7c), non-functional Fc γ receptor V158-Fc LALA P329G fusion polypeptide (figure 7d) (═ control).

Definition of

The term "binding to Fc receptors" is intended to mean binding to Fc receptors, for example, in BIAcore^(R)The binding of the Fc region to the Fc receptor was determined (Pharmacia Biosensor AB, Uppsala, Sweden).

In BIAcore^(R)In an assay, an Fc receptor is bound to a surface and binding of an analyte (e.g., a fusion polypeptide or antibody containing an Fc region) is measured by Surface Plasmon Resonance (SPR). Binding affinity is defined by the terms ka (association constant: rate constant for association of an Fc region fusion polypeptide or conjugate to form an Fc region/Fc receptor complex), KD (dissociation constant: rate constant for dissociation of an Fc region fusion polypeptide or conjugate from an Fc region/Fc receptor complex), and KD (KD/ka). Alternatively, the bound signal of the SPR sensorgram may be directly compared to the reference response signal with respect to resonance signal height and dissociation behavior.

The term "CH 2 domain" refers to the portion of an antibody heavy chain polypeptide that extends from about EU position 231 to EU position 340 (EU numbering system according to Kabat). In one embodiment, the CH2 domain has the amino acid sequence of SEQ ID NO:01 (APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQESTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK). The CH2 domain is unique in that it does not pair closely with another domain. In contrast, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native Fc region. It has been speculated that sugars may provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, mol.Immunol.22(1985) 161-206.

The term "CH 3 domain" refers to the portion of an antibody heavy chain polypeptide that extends from about EU position 341 to EU position 446. In one embodiment, the CH3 domain has the amino acid sequence of SEQ ID NO 02 (GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG).

The term "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, several of which may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.

The term "Fc region" refers to the C-terminal region of an immunoglobulin. The Fc region is a dimeric molecule comprising two disulfide-linked antibody heavy chain Fc region polypeptides (Fc region polypeptide chains). The Fc region may be produced by papain digestion or IdeS digestion or trypsin digestion of the intact (full-length) antibody or may be produced recombinantly.

The Fc region obtainable from a full length antibody or immunoglobulin comprises residue 226(Cys) of the C-terminus of a full length heavy chain and thus comprises a partial hinge region and two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain. It is known from US 5,648,260 and US 5,624,821 that modification of defined amino acid residues in the Fc region results in phenotypic effects.

The formation of a dimeric Fc region comprising two identical or different antibody heavy chain fragments is mediated by non-covalent dimerization of the included CH3 domains (amino acid residues referred to e.g.Dall' Acqua, biochem.37(1998) 9266-9273). The Fc region is covalently stabilized by the formation of disulfide bonds in the hinge region (see, e.g., Huber et al, Nature 264(1976) 415-420; Thies et al, J.mol.biol.293(1999) 67-79). Since the positions of the residues involved in dimerization of the CH3-CH3 domains are located at the inner interface of the CH3 domain, while the residues involved in Fc region-FcRn interactions are located at the outer side of the CH2-CH3 domain, the amino acid residue changes introduced within the CH3 domain to disrupt dimerization of the CH3-CH3 domain interactions have no adverse effect on FcRn binding.

The effector function associated with the Fc region is initiated by the interaction of the Fc region with a cell surface receptor specific for the effector function. In most cases, receptor activation can be achieved with antibodies of the IgG1 isotype, while antibodies of the IgG2 and IgG4 isotypes have no or limited effector function.

Receptors that elicit effector function are Fc receptor types (and subtypes) Fc γ RI, Fc γ RII, and Fc γ RIII. Effector functions associated with the IgG1 isotype can be reduced by introducing specific amino acid changes in the lower hinge region (e.g., L234A and/or L235A that are involved in Fc γ R and C1q binding). In particular, certain amino acid residues located in the CH2 and/or CH3 domains are also associated with circulating half-life of the antibody molecule or Fc region fusion polypeptide in the bloodstream. Circulating half-life is determined by binding of the Fc region to the neonatal Fc receptor (FcRn).

Numbering of amino acid residues in the constant region of the antibody is performed according to the EU index of Kabat (Kabat et al 1991, Sequences of Proteins of immunological interest, U.S. department of Public Health, Bethesda, Md.).

The term "Fc region of human origin" refers to the C-terminal region of an immunoglobulin heavy chain of human origin, which comprises at least part of the hinge region, the CH2 domain and the CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system described in Kabat, E.A. et al, Sequences of Proteins of immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, also known as the EU index.

The term "FcRn-binding portion of an Fc-region" refers to a portion of an antibody heavy chain polypeptide that extends from about EU position 243 to EU position 261, and from about EU position 275 to EU position 293, and from EU position 302 to EU position 319, and from EU position 336 to EU position 348, and from EU position 367 to EU 393 and EU position 408, and from EU position 424 to EU position 440. In one embodiment, one or more of the following amino acid residues are changed by EU numbering according to Kabat: f243, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, a287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, a339, K340, G341, Q342, P343, R344, E427, P346, Q347, V385, C367, V373, F372, Y375, Y391, S375, S382, N338, a339, K340, G341, Q342, P343, R344, P345, P378, Q385, V373, V376, C367, V376, F372, Y375, S384, S9, N187, N425, S26, N380, N425, S26, N380, N425, N380, H425, H436, N380, H425, H393, H380, H425, H420, H380, H382, N380, H.

The polypeptide chain of the wild-type human Fc region of the IgG1 isotype has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：03).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations L234A, L235A has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：04).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations T366S, L368A and Y407V has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：05).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with the mutation T366W has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：06).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations L234A, L235A and T366S, L368A and Y407V has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：07).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations L234A, L235A, and T366W has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：08).

the polypeptide chain of a variant human Fc region of IgG1 isotype with mutation P329G has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：09).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations L234A, L235A and P329G has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：10).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations P239G and T366S, L368A and Y407V has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：11).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations P329G and T366W has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：12).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations L234A, L235A, P329G and T366S, L368A and Y407V has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPKEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：13).

the polypeptide chain of a variant human Fc region of the IgG1 isotype with mutations L234A, L235A, P329G and T366W has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO：14).

the polypeptide chain of the wild-type human Fc region of the IgG4 isotype has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：15).

the polypeptide chain of a variant human Fc region of the IgG4 isotype with mutations S228P and L235E has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：16).

the polypeptide chain of a variant human Fc region of the IgG4 isotype with mutations S228P, L235E and P329G has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：17).

the term "Fc receptor", abbreviated "FcR" refers to a receptor that binds to the Fc region. In one embodiment, the FcR is a native sequence human FcR. Furthermore, in one embodiment, the FcR is one that binds an IgG antibody (fcgamma receptor) and includes receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic and alternatively spliced variants thereof. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcR is reviewed in ravatch and Kinet, Annu.Rev.Immunol 9(1991) 457-492; capel et al, immunolmethods 4(1994) 25-34; de Haas et al, J.Lab.Clin.Med.126(1995) 330-. The term "FcR" herein encompasses other fcrs. The term also includes the neonatal receptor FcRn responsible for transfer of maternal IgG to the fetus (see, e.g., Guyer et al, J.Immuno1.117(1976) 587; Kim et al, J.Immuno1.24(1994) 249).

The term "Fc γ receptor", abbreviated "Fc γ R" or "fcgamma" refers to any member of the family of proteins that bind the Fc region of IgG antibodies and are encoded by the Fc γ R gene. In humans, this family includes, but is not limited to: fc γ RI (CD64), including isoforms Fc γ RIA, Fc γ RIB and Fc γ RIC; fc γ RII (CD32), including isoforms Fc γ RIIA (including allotype H131 and R131), Fc γ RIIB (including Fc γ RIIB-1 and Fc γ RIIB-2), and Fc γ RIIc; and Fc γ RIII (CD16), including isoforms Fc γ RIIIA (including allotypes V158 and F158; Swiss-Prot inlet P08637; N-terminus-MRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQ-C-terminus; SEQ ID NO:18) and Fc γ RIIIb (including allotype Fc γ RIIB-NA1 and Fc γ RIIB-NA2) (see, e.g., Jefferis et al, Immunol. Lett.82(2002)57-65, incorporated by reference in their entirety); and Fc γ R isoforms or allotypes. The Fc γ R may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse Fc γ rs include, but are not limited to, Fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16), and Fc γ RIII-2(CD16-2), and either Fc γ R isoforms or allotypes. The amino acid residues involved in the Fc region-Fc γ R interaction were 234-239 (lower hinge region), 265-269(B/C loop), 297-299(D/E loop) and 327-332(F/G loop) (Sondermann et al, Nature 406(2000) 267-273). Amino acid mutations that result in reduced binding/affinity to Fc γ RI, Fc γ RIIA, Fc γ RIIB and/or Fc γ RIIIA include N297A (with reduced immunogenicity and extended half-life binding/affinity) (routlegge et al, Transplantation 60(1995) 847; Friend et al, Transplantation 68(1999) 1632; Shield et al, J.biol.chem.276(1995)6591), residue 233-. Some exemplary amino acid substitutions are described in US 7,355,008 and US 7,381,408.

The term "neonatal Fc receptor," abbreviated "FcRn," refers to a protein that binds the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may be from any organism including, but not limited to, human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as the heavy and light chains. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and β -2-microglobulin. The interacting amino acid residues of the Fc region with FcRn are near the junction of the CH2 and CH3 domains. The Fc region-FcRn contact residues are all within a single IgG heavy chain. The amino acid residues involved are amino acid residues 248, 250, 257, 272, 285, 288, 290, 291, 308, 311 and 314 (all in the CH2 domain) and amino acid residues 385, 387, 428 and 433, 436 (all in the CH3 domain). Amino acid mutations that result in increased binding/affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al, j.biol.chem.276(2001) 6591).

Amino acid residues of neonatal Fc receptors that are conserved among species are histidine residues at positions 310 and 435 in the Fc region. These residues contribute to the pH dependence of the FcRn interaction in the Fc region (see, e.g., Victor, G. et al, Nature Biotechnol.15(1997) 637-640); dall' Acqua, W.F. et al J.Immunol.169(2002) 5171-5180). Mutations in the Fc region that reduce the interaction with FcRn can reduce antibody half-life.

The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain, e.g., from position about 216 to position about 230 of the EU numbering system according to Kabat. The hinge region is typically a dimeric molecule consisting of two polypeptides having the same amino acid sequence. The hinge region typically comprises about 25 amino acid residues and has flexibility that allows the antigen binding region to move independently. The hinge region can be subdivided into three domains: upper, middle and lower hinge regions (Roux et al, J.Immunol.161(1998) 4083).

The term "lower hinge region" of an Fc region refers to a stretch of amino acid residues immediately C-terminal to the hinge region, i.e., residues 233 to 239 of the Fc region according to EU numbering of Kabat.

The term "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Wild-type human Fc regions include native human IgG1Fc region (non-a and a allotypes), native human IgG2Fc region, native human IgG3Fc region, and native human IgG4Fc region, as well as naturally occurring variants thereof.

The term "pharmaceutical composition" refers to a formulation that is in a form such as to allow the biological activity of the active ingredient contained therein to be effective, and which does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is to be administered.

"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition other than an active ingredient that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The term "polypeptide" refers to a naturally occurring or synthetically produced polymer composed of amino acids joined by peptide bonds. Polypeptides of less than about 20 amino acid residues may be referred to as "peptides", while molecules consisting of two or more polypeptides or comprising one polypeptide of more than 100 amino acid residues may be referred to as "proteins". The polypeptide may also comprise non-amino acid components, such as sugar groups, metal ions or carboxylic acid esters. The non-amino acid component may be added by the cell in which the polypeptide is expressed and may vary with the cell type. A polypeptide is defined herein in terms of its amino acid backbone structure or a nucleic acid encoding its amino acid backbone structure. Additions (e.g., sugar groups) are not generally specified, but may be present.

The term "amino acid sequence tag" refers to a sequence of amino acid residues linked to each other by peptide bonds having specific binding properties. In one embodiment, the amino acid sequence tag is an affinity or purification tag. In one embodiment, the amino acid sequence tag is selected from the group consisting of an Arg tag, a His tag, an Avi tag, a His-Avi tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a GST tag, and an MBP tag. In one embodiment, the amino acid sequence tag is selected from the group consisting of SEQ ID NO:19(RRRRR), SEQ ID NO:20(RRRRRR), SEQ ID NO:21 (Avi-tag), SEQ ID NO:22 (His-Avi-tag), SEQ ID NO:23 (hhhhhhhh), SEQ ID NO:24(KDHLIHNVHKEFHAHAHNK), SEQ ID NO:25(DYKDDDDK), SEQ ID NO:26(DYKDHDGDYKDHDIDYKDDDDK), SEQ ID NO:27(AWRHPQFGG), SEQ ID NO:28(WSHPQFEK), SEQ ID NO:29(MDVEAWLGAR), SEQ ID NO:30(MDVEAWLGARVPLVET), SEQ ID NO:31(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), SEQ ID NO:32(EQKLISEEDL), SEQ ID NO:33(KETAAAKFERQHMDS), SEQ ID NO:34(KRRWKKNFIAVSAANRFKKISSSGAL), SEQ ID NO:35 (cellulose binding domain), SEQ ID NO:36 (cellulose binding domain), SEQ ID NO:37(TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ), SEQ ID NO:38(GST tag) and SEQ ID NO:39(MBP marker).

The term "enzymatic cleavage site" refers to a sequence of amino acid residues linked to each other by peptide bonds that can be specifically cleaved by a protease. In one embodiment, the protease is IgA protease, granzyme B, Tev protease, PreScission protease, thrombin, factor 10a, Ides protease, or enterokinase.

The term "IgA protease" refers to a protease derived from Neisseria gonorrhoeae (Neisseria gonorrhoeae) having a recognition site comprising one of the following sequences, wherein "↓" denotes the position of the cleaved bond:

Pro-Ala-Pro↓Ser-Pro(SEQ ID NO:40),

Pro-Pro↓Ser-Pro(SEQ ID NO:41),

Pro-Pro↓Ala-Pro(SEQ ID NO:42),

Pro-Pro↓Thr-Pro(SEQ ID NO:43),

Pro-Pro↓Gly-Pro(SEQ ID NO:44),

Pro-Arg-Pro-Pro↓Thr-Pro(SEQ ID NO:45),

Val-Val-Ala-Pro-Pro↓Ala-Pro(SEQ ID NO:46),

Val-Val-Ala-Pro-Pro↓Ser-Pro(SEQ ID NO:47),

Val-Val-Ala-Pro-Pro↓Thr-Pro(SEQ ID NO:48),

Val-Val-Ala-Pro-Pro↓Gly-Pro(SEQ ID NO:49),

Pro-Arg-Pro-Pro↓Thr-Pro(SEQ ID NO:50),

Ala-Pro-Pro-Ala↓Ala-Pro(SEQ ID NO:51),

Pro-Arg-Pro-Pro↓Ala-Pro(SEQ ID NO:52),

Pro-Arg-Pro-Pro↓Ser-Pro(SEQ ID NO:53),

Pro-Arg-Pro-Pro↓Gly-Pro(SEQ ID NO:54).

the term "linker" or "peptide linker" as used in this application refers to a peptide linker of natural and/or synthetic origin. They constitute a linear amino acid chain, of which the 20 naturally occurring amino acids are monomeric building blocks. The chain has a length of between 1 and 50 amino acids, preferably between 1 and 28 amino acids, particularly preferably between 3 and 25 amino acids. The linker may comprise a repeating amino acid sequence or a sequence of naturally occurring polypeptides, such as polypeptides having hinge function. The linker has the function of ensuring that the peptide conjugated to the anti-CD 4 antibody can exert its biological activity by allowing the peptide to fold and present correctly. Preferably, the linker is a "synthetic peptide linker" designed to be rich in glycine, glutamine and/or serine residues. These residues are, for example, arranged in small repeating units of up to five amino acids, such as GGGGS, QQQQG or SSSSG. This small repeat unit can be repeated two to five times to form a multimeric unit. At the amino-terminus and/or the carboxy-terminus of the multimeric unit, up to six additional arbitrary, naturally occurring amino acids may be added. Other synthetic peptide linkers consist of a single amino acid that is repeated between 10 and 20 times, and may contain up to six additional arbitrary, naturally occurring amino acids at the amino terminus and/or the carboxy terminus. All peptide linkers can be encoded by nucleic acid molecules and thus can be expressed recombinantly.

Fusion polypeptides as reported herein

It has been found that soluble Fc receptors can be produced by expressing the Fc receptor as a fusion polypeptide having an Fc region that does not substantially bind to the fused Fc receptor.

The term "does not substantially bind to an Fc receptor" means that the Fc region to which the Fc receptor is fused does not bind to the Fc receptor to such an extent that aggregates are formed.

One aspect as reported herein is a fusion polypeptide according to formula I

R1-FC-R2 (formula I)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor, and

FC denotes a heavy chain Fc region polypeptide,

wherein R1 or R2 or both are present,

wherein FC does not substantially bind R1 and/or R2.

One aspect as reported herein is a fusion polypeptide according to formula II

R1-CS1-L1-CS2-FC-CS3-L2-CS4-R2 (formula II)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor,

FC denotes a heavy chain Fc region polypeptide,

CS1 denotes the first cleavage site,

CS2 denotes a second cleavage site which,

CS3 denotes a third cleavage site,

CS4 denotes a fourth cleavage site,

l1 denotes a first intervening amino acid sequence, and

l2 denotes a second intervening amino acid sequence,

wherein R1 or R2 or both are present,

wherein L1 and L2 may be present or absent independently of each other,

wherein FC does not substantially bind R1 and/or R2.

The Fc receptor contained in the fusion polypeptides reported herein may be any Fc receptor from any species, including but not limited to human, mouse, rat, rabbit, and monkey.

In one embodiment the Fc receptor is selected from the group comprising Fc γ -receptors and neonatal Fc receptors. In one embodiment the Fc receptor is a human Fc γ -receptor, a human neonatal Fc receptor, a murine Fc γ -receptor, and a rabbit neonatal Fc receptor.

In one embodiment the human Fc γ -receptor is selected from the group consisting of human Fc γ RI (CD64), human Fc γ RII (CD32), human Fc γ RIIA, human Fc γ RIIB, human Fc γ RIIC, human Fc γ RIII (CD16), human Fc γ RIIIA, and human Fc γ RIIIB.

In one embodiment the human neonatal Fc receptor is a human FcRn.

The Fc region comprised in the fusion polypeptides reported herein should not substantially bind to any Fc receptor to which it is fused.

In one embodiment, the fusion polypeptide has substantially no effector function, which makes it a desirable candidate for applications in which some effector function is unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/elimination of effector function. For example, Fc receptor (FcR) binding assays may be performed to ensure that the fusion polypeptide lacks fcyr binding (and thus may lack ADCC activity). The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc RII and Fc RIII. FcR expression on hematopoietic cells is summarized in table 3 on page 464 of ravatch, j.v. and Kinet, j.p., annu.rev.immunol.9(1991) 457-492. Non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I.et al, Proc. nat' l Acad. Sci. USA 83(1986) 7059-; U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al, J.Exp.Med.166(1987) 1351-1361). Alternatively, non-radioactive assay methods may be utilized (see, e.g., ACTI for flow cytometry)^TMNonradioactive cytotoxicity assay (Celltechnology, Inc. mountain View, CA) and CytoToxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells that can be used in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or in addition, mesh may be assessed in vivo, for example in animal models such as those disclosed in Clynes, R. et al, Proc. Natl. Acad. Sci. USA 95(1998)652-ADCC activity of the molecule (1). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, int. immunol.18(2006: 1759-.

The affinity and binding properties of an Fc region to its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc region/FcR interactions (i.e., specific binding of an Fc region to an Fc γ R), including, but not limited to, equilibrium methods (e.g., enzyme linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA)) or kinetics (e.g., enzyme-linked immunosorbent assay (ELISA)) orAnalysis) and other methods, such as indirect binding assays, competitive inhibition assays, Fluorescence Resonance Energy Transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels on one or more components being examined and/or utilize a variety of detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, W.E. editor, Fundamental Immunology, 4 th edition, Lippincott-Raven, Philadelphia (1999).

In one embodiment, the FC is an FC region of the subclass IgG4 of human origin or of the subclass IgG1, IgG2 or IgG3 of human origin, modified in such a way that no FC γ receptor (e.g., FC γ RIIIa) binds. In one embodiment, the FC is of human origin, in particular from the FC region of the subclass human IgG4, or from a mutated FC region of the subclass human IgG 1. In one embodiment, FC belongs to the subclass human IgG1 with mutations L234A and L235A. In one embodiment, the FC belongs to the subclass human IgG4 with the mutation S228P. While IgG4 showed reduced Fc γ receptor (Fc γ RIIIa) binding, antibodies of other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 or/and His435 are residues which also provide reduced Fc γ receptor binding if altered (Shields, R.L. et al, J.biol.chem.276(2001)6591 and 6604; Lund, J.et al, FASEB J.9(1995)115 and 119; Morgan, A. et al, Immunology 86(1995)319 and 324; EP 0307434). In one embodiment, with respect to Fc γ receptor binding, Fc belongs to the IgG4 subclass, or to the IgG1 or IgG2 subclass, has mutations in L234, L235, and/or D265 and/or comprises PVA236 mutations. In one embodiment, the FC comprises one or more of the mutations S228P, L234A, L235A, L235E and/or PVA236(PVA236 means the amino acid sequence ELLG (given in the single letter amino acid code) at amino acid positions 233 to 236 of IgG1 or the EFLG of IgG4 is substituted by PVA). In one embodiment, the mutations are S228P for IgG4 and L234A and L235A for IgG 1.

Fc region binding sites are known in the art and are described, for example, by Lukas, T.J., et al, J.Immunol.127(1981) 2555-2560; brunhouse, r. and Cebra, j.j., mol. immunol.16(1979) 907-; burton, D.R. et al, Nature 288(1980) 338-344; thommesen, J.E., et al, mol.Immunol.37(2000) 995-1004; idusogene, E.E., et al, J.Immunol.164(2000) 4178-; hezareh, M.et al, J.Virol.75(2001) 12161-; morgan, A. et al, Immunology 86(1995) 319-324; and EP 0307434. The Fc region binding site is characterized, for example, by amino acids L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat).

Fc regions with reduced effector function include those having substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DA" Fc mutants having a substitution of residue 265 to alanine and so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Certain Fc region variants having improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; and Shields, R.L. et al, J.biol.chem.276(2001) 6591-6604).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. nos. 6,194,551; WO 99/51642; and Idusogene, E.E., et al, J.Immunol.164(2000) 4178-.

For further examples of Fc region variants, see also Duncan, a.r. and Winter, g., Nature322(1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351.

In one embodiment, the heavy chain Fc region polypeptide has an amino acid mutation at one or more of positions 234, 235, 236, 237, 238, 239, 253, 254, 265, 266, 267, 268, 269, 270, 288, 297, 298, 299, 307, 311, 327, 328, 329, 330, 331, 332, 434 and 435. In one embodiment, one or more of the Fc receptors are fey receptors.

In one embodiment, the human IgG1 heavy chain polypeptide has the amino acid mutations L234A and L235A, and one or more of E233P, L235E, Δ G236, D265A, N297A, N297D, P329A, P329G, and P331S.

In one embodiment, the heavy chain Fc region polypeptide has an amino acid mutation at one or more of positions 248, 250, 251, 252, 253, 254, 255, 256, 257, 272, 285, 288, 290, 291, 308, 309, 310, 311, 314, 385, 386, 387, 428, 433, 434, 435, and 436. In one embodiment, one or more of the Fc receptors is FcRn.

The fusion polypeptide may comprise a linker polypeptide between the Fc receptor and the Fc region. This linker polypeptide can be used to modulate the distance between the Fc receptor and the Fc region to allow the two regions to function in the intended manner.

In one embodiment, the linker polypeptide is selected from the group comprising (G3S)3, (G3S)4, (G3S)5, (G3S)6, (G4S)3, (G4S)4, (G4S)5, (G5S)2, (G5S)3, and (G5S)4, and any combination thereof.

Furthermore, the fusion polypeptide may comprise a label between the Fc receptor and the Fc region, such as a label suitable for affinity purification or immobilization.

In one embodiment, the tag is selected from the group comprising an Arg tag, an Avi tag, a His-Avi tag, a His tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin binding peptide, a cellulose binding domain, a chitin binding domain, a GST tag, an MBP tag, streptavidin or avidin, biotin, a lectin, a polysaccharide, a steroid binding protein, a hormone, and a hormone receptor.

The linker polypeptide and the label may be combined in an intervening amino acid sequence located between the Fc receptor and the Fc region.

In one embodiment, the intervening amino acid sequence is selected from a first group comprising (G3S)3, (G3S)4, (G3S)5, (G3S)6, (G4S)3, (G4S)4, (G4S)5, (G5S)2, (G5S)3, and (G5S)4, or from a second group comprising an Arg tag, an Avi tag, a His-Avi tag, a His tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin binding peptide, a cellulose binding domain, a chitin binding domain, a GST tag, an MBP tag, or a combination of two elements selected from these groups.

The intervening amino acid sequence can be positioned before or after the cleavage site in the fusion polypeptide.

In one embodiment, the cleavage site is an enzymatic cleavage site. In one embodiment, the enzyme cleavage site is selected from the group comprising an IgA protease cleavage site, a granzyme B protease cleavage site, a Tev protease cleavage site, a PreScission protease cleavage site, a thrombin cleavage site, a Faktor10a protease site, an Ides protease cleavage site, a SUMO protease cleavage site and an enterokinase cleavage site. In one embodiment, the cleavage site is selected from the group of IgA protease cleavage site, PreScission protease cleavage site, granzyme B cleavage site and Ides protease cleavage site.

In one embodiment, the fusion polypeptide comprises an intrinsic cleavage site for papain, or pepsin, or Ides protease.

Since the fusion polypeptide as reported herein comprises an Fc-region, which in turn comprises an immunoglobulin hinge region, the dimeric fusion polypeptide comprises one or more disulfide bridges covalently linking the first fusion polypeptide to the second fusion polypeptide.

The dimeric fusion polypeptide may be a homodimeric fusion polypeptide or a heterodimeric fusion polypeptide.

Additionally the dimeric fusion polypeptide may comprise a polypeptide according to formula I

R1-FC-R2 (formula I)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor, and

FC denotes a heavy chain Fc region polypeptide,

wherein R1 or R2 or both are present,

wherein FC does not substantially bind R1 and/or R2,

or a first fusion polypeptide according to formula II

R1-CS1-L1-CS2-FC-CS3-L2-CS4-R2 (formula II)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor,

FC denotes a heavy chain Fc region polypeptide,

CS1 denotes the first cleavage site,

CS2 denotes a second cleavage site which,

CS3 denotes a third cleavage site,

CS4 denotes a fourth cleavage site,

l1 denotes a first intervening amino acid sequence, and

l2 denotes a second intervening amino acid sequence,

wherein R1 or R2 or both are present,

wherein L1 and L2 may be present or absent independently of each other,

wherein FC does not substantially bind R1 and/or R2,

wherein the first and second fusion polypeptides of the dimeric fusion polypeptide may be selected independently from each other from formula I and formula II.

If the dimeric fusion polypeptide may comprise two different fusion polypeptides, a mechanism ensuring heterodimerization must be used.

In one embodiment the fusion polypeptide is characterized by

a) R1 and R2 of the first and second polypeptides are identical,

Use of the fusion polypeptides reported herein

One aspect as reported herein is the use of an immobilized fusion polypeptide as reported herein as affinity chromatography ligand.

In one embodiment, the fusion polypeptide is bound to a solid phase. In one embodiment, the solid phase is a chromatography material. In one embodiment, the fusion polypeptide is biotinylated and the solid phase is derivatized with streptavidin.

In one embodiment, the fusion polypeptide comprises a cleavage site between the Fc receptor and the Fc region. In one embodiment, the fusion polypeptide is cleaved prior to biotinylation.

In one embodiment, the fusion polypeptide comprises an immobilized label between the Fc receptor and the cleavage site. In one embodiment, the immobilization tag is a His-Avi tag.

Also reported is an affinity chromatography column comprising a matrix and a matrix-bound chromatography functionality, characterized in that the matrix-bound chromatography functionality comprises a fusion polypeptide as reported herein.

In one embodiment, the fusion polypeptide comprises an immobilized label between the Fc receptor and the cleavage site. In one embodiment, the immobilized tag is an Avi tag.

One aspect as reported herein is the use of an immobilized fusion polypeptide as reported herein for determining Fc receptor binding of an antibody.

In one embodiment the antibody is a low affinity antibody.

In one embodiment the determination is by surface plasmon resonance. In one embodiment the antibody is captured by a monomeric Fc receptor. In one embodiment the antibody is captured by a dimeric antibody.

Recombination method

One aspect as reported herein is a method for the production of soluble Fc receptors comprising the following steps

b) recovering the fusion polypeptide from the cell or the culture medium,

c) optionally cleaving the fusion polypeptide with a protease,

thereby producing soluble Fc receptors.

Methods and techniques known to those skilled in the art for practicing the present invention are described, for example, in Ausubel, f.m. editions, Current Protocols in Molecular Biology, volumes I to III (1997), Wiley and Sons; sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Such methods are, for example, affinity chromatography using microbially-derived proteins (e.g., protein A or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin), and mixed mode exchange chromatography), thiophilic adsorption (e.g., with β -mercaptoethanol and other SH ligands), hydrophobic interaction, or aromatic adsorption chromatography (e.g., with a hydrophobic interaction, or with other SH ligands)Using phenyl-sepharose, aza-arenophilic resins or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. using ni (ii) -and cu (ii) -affinity materials), size exclusion chromatography and preparative electrophoretic methods (e.g. gel electrophoresis, capillary electrophoresis) (vijayalaks hmi, m.a., appl.biochem.biotech.75(1998) 93-102). The fusion polypeptide can be enzymatically cleaved to release the Fc receptor, either before or after chromatographic purification. The expression cassette comprises a promoter, a DNA segment encoding a secretion signal sequence, a structural gene and a terminator/polyadenylation signal. The elements are assembled in an operably linked form on one plasmid encoding all of the desired different fusion polypeptides, or on two or more plasmids each encoding one fusion polypeptide. For expression of the structural gene, one or more plasmids are introduced into a suitable host cell. In mammalian cells such as CHO cell, NS0 cell, Sp2/0 cell, COS cell, HEK cell, K562 cell, BHK cell,Cells, etc., produce proteins. In one embodiment, the fusion polypeptide is expressed in CHO cells, or BHK cells, or HEK cells. The regulatory elements of the plasmids must be selected in such a way that they are functional in the host cell of choice. The expressed fusion polypeptide is functionally assembled.

An "expression plasmid" is a nucleic acid that provides all of the elements necessary for expression of one or more structural genes contained in a host cell. Typically, the expression plasmid comprises a prokaryotic plasmid propagation unit, for example, in the case of E.coli, an origin of replication, a selection marker, a eukaryotic selection marker, one or more expression cassettes for expressing one or more structural genes of interest, which each comprise a promoter, a structural gene and a transcription terminator (including polyadenylation signals). Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked" to the promoter. Similarly, a regulatory element is operably linked to a core promoter if the regulatory element modulates the activity of the core promoter.

Antibodies can be produced using recombinant methods and compositions such as those described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding a fusion polypeptide antibody described herein is provided. In one embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a first fusion polypeptide and an amino acid sequence comprising a second fusion polypeptide; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a first fusion polypeptide, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a second fusion polypeptide. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of producing a fusion polypeptide is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a fusion polypeptide provided above under conditions suitable for expression of the fusion polypeptide, and optionally recovering the fusion polypeptide from the host cell (or host cell culture medium).

For recombinant production of the fusion polypeptides as reported herein, the nucleic acid encoding the fusion polypeptide as described above is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to the gene encoding the fusion polypeptide).

Suitable host cells for cloning or expressing a vector encoding a fusion polypeptide include prokaryotic or eukaryotic cells as described herein. For example, fusion polypeptides can be produced in bacteria, particularly where glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides In bacteria see, for example, U.S. Pat. No. 5,648,237, U.S. Pat. No. 5,789,199 and U.S. Pat. No. 5,840,523 (see also Charlton, K.A., In: Methods In Molecular Biology, volume 248, Lo, B.K.C. (eds.), Humana Press, Totowa, NJ (2003), page 245-254, which describes expression of antibody fragments In E.coli). After expression, the fusion polypeptide can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion polypeptides, including fungi and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of fusion polypeptides having a partially or fully human glycosylation pattern (see Gerngross, T.U., nat. Biotech.22(2004) 1409-.

Host cells suitable for expression of glycosylated fusion polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures can also be used as hosts (see, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants)^TMTechnique)).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be used. Other examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham, f.l. et al, j.gen virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse support cells (TM 4 cells as described, for example, in Mather, J.P., biol. reprod.23(1980)243- & 252); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described, for example, in Mather, J.P., et al, Annals N.Y.Acad.Sci.383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian cell lines include Chinese Hamster Ovary (CHO) cells, packageInclude DHFR^-CHO cells (Urlaub, G., et al, Proc. Natl. Acad. Sci. USA 77(1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki, p. and Wu, a.m., Methods in Molecular Biology, volume 248, Lo, b.k.c. (eds.), Humana Press, Totowa, NJ (2004), page 255-268.

Methods and compositions for diagnosis and detection

In certain embodiments, any of the fusion polypeptides provided herein can be used to detect the presence of a molecule comprising an Fc region in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection.

In one embodiment, a method of diagnosing or detecting the use of the fusion polypeptide as reported herein is provided. In another aspect, methods are provided for detecting the presence of a molecule comprising an Fc region in a biological sample. In certain embodiments, the method comprises contacting a biological sample with a fusion polypeptide as reported herein under conditions allowing binding of the fusion polypeptide to a molecule comprising an Fc region, and detecting whether a complex is formed between the fusion polypeptide and the molecule comprising an Fc region. Such methods may be in vitro or in vivo.

In certain embodiments, a labeled fusion polypeptide is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent, colored, electron dense, chemiluminescent, and radioactive labels), as well as moieties that are indirectly detected (e.g., via an enzymatic reaction or molecular interaction), such as an enzyme or ligand. Exemplary labels include, but are not limited to, radioisotopes³²P、¹⁴C、¹²⁵I、³H and¹³¹I. fluorophores such as rare earth element chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases, e.g. firefly and bacterial luciferases (U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydronaphthyridinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g. glucoseCarbohydrate oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

Pharmaceutical composition

A pharmaceutical composition of a fusion polypeptide as reported herein in a lyophilized formulation or in an aqueous solution is prepared by: such fusion polypeptides having the desired purity are admixed with one or more optional pharmaceutically acceptable carriers (Remington's pharmaceutical Sciences 16 th edition, Osol, a. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citric acid and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzalkonium bromide; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly (vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannose, trehalose or sorbose; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rhuPH20 (r: (r))Baxter International, Inc.). Are described in U.S. patent publication Nos. 2005/0260186 and 2006/0104968Certain exemplary shasegps and methods of use are described, including rhuPH 20. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulation comprising histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in an amount effective for the intended purpose.

The active ingredient may be embedded in, for example, microcapsules prepared by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly (methylmethacylate) microcapsules), respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. (eds.) (1980).

Sustained release articles can be prepared. Suitable examples of sustained-release articles include semipermeable matrices of solid hydrophobic polymers containing the fusion polypeptide, which matrices are in the form of shaped articles (e.g., films or microcapsules).

The formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

Therapeutic methods and compositions

Any of the fusion polypeptides reported herein may be used in a method of treatment.

In one aspect, fusion polypeptides for use as a medicament are provided. In a further aspect, fusion polypeptides for use in treating diseases characterized by elevated antibody levels are provided. In one embodiment, the disease is an autoimmune disease. In certain embodiments, fusion polypeptides for use in methods of treatment are provided. In certain embodiments, the invention provides a fusion polypeptide for use in a method of treating an individual having a disease characterized by elevated antibody levels, the method comprising administering to the individual an effective amount of the fusion polypeptide. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In additional embodiments, the invention provides fusion polypeptides for reducing antibody levels. In certain embodiments, the present invention provides a fusion polypeptide for use in a method of reducing antibody levels in an individual, the method comprising administering to the individual an effective amount of the fusion polypeptide to reduce antibody levels. An "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides the use of a fusion polypeptide in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for treating a disease characterized by elevated antibody levels. In one embodiment the disease is an autoimmune disease. In another embodiment, the medicament is for use in a method of treating elevated antibody levels, the method comprising administering to an individual having a disease characterized by elevated antibody levels an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In another embodiment, the medicament is for reducing antibody levels. In another embodiment, the medicament is for use in a method of reducing antibody levels in an individual, the method comprising administering to the individual an effective amount of the medicament to reduce antibody levels. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides methods of treating diseases characterized by elevated antibody levels. In one embodiment the disease is an autoimmune disease. In one embodiment, the method comprises administering to an individual having such a disease characterized by elevated antibody levels an effective amount of a fusion polypeptide. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides methods for reducing the level of an antibody in an individual. In one embodiment, the method comprises administering to the individual an effective amount of the fusion protein to reduce antibody levels. In one embodiment, the "individual" is a human.

In another aspect, the invention provides a pharmaceutical composition comprising any of the fusion polypeptides as reported herein, e.g. for use in any of the above methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the fusion polypeptides as reported herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the fusion polypeptides reported herein and at least one additional therapeutic agent.

The fusion polypeptides of the invention may be used alone or in combination with other active agents in therapy. For example, a fusion polypeptide of the invention can be co-administered with at least one additional therapeutic agent.

Such combination therapies as indicated above encompass combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the fusion polypeptide of the invention may be performed prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants.

The fusion polypeptides of the invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal and, if required for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at multiple time points, bolus administrations, and pulse infusions.

The fusion polypeptides of the invention will be formulated, administered and administered in a manner that meets good medical criteria. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the active agent, the method of administration, the administration schedule and other factors known to the medical practitioner. The fusion polypeptide need not be, but is optionally formulated with one or more active agents currently used to prevent or treat the condition in question. The effective amount of such other active agents will depend on the amount of fusion polypeptide present in the formulation, the type of disease or therapy, and other factors discussed above. These are typically used at the same dosages and using the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of the fusion polypeptide of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of fusion polypeptide, the severity and course of the disease, whether the fusion polypeptide is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the fusion polypeptide, and the discretion of the attending physician. The fusion polypeptide is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.5mg/kg-10mg/kg) of the fusion polypeptide can be an initial candidate dose for administration to a patient, whether administered, for example, by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a common daily dose may be from about 1. mu.g/kg to 100mg/kg or more. For repeated administration over a range of days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the fusion polypeptide will range from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses (or any combination thereof) of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or10 mg/kg may be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every 3 weeks (e.g., such that the patient receives from about 2 to about 20 or, e.g., about 6 doses of the fusion polypeptide). However, other dosage regimens may be used. The progress of such therapy is readily monitored by conventional techniques and assays.

It will be appreciated that any of the above formulations or methods of treatment may be performed with the immunoconjugates of the invention in place of or in addition to the fusion polypeptide.

Article of manufacture

In another aspect of the invention, there is provided an article of manufacture comprising a substance as described above which is useful in the treatment, prevention and/or diagnosis of a condition. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective, by itself or in combination with another composition, in the treatment, prevention and/or diagnosis of a condition and may have a sterile access port (e.g., the container may be an intravenous bag or vial having a stopper penetrable by a hypodermic needle). At least one active agent in the composition is a fusion polypeptide of the invention. The label or package insert suggests that the composition is for use in treating the selected condition. Additionally, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises a fusion polypeptide of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic agent or therapeutic agent. The article of manufacture in this embodiment of the invention may also comprise package inserts indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials that are popular from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

It is understood that any of the above articles of manufacture may comprise an immunoconjugate of the invention in place of or in addition to a fusion polypeptide.

The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made in the illustrated methods without departing from the spirit of the invention.

Examples

Example 1

Generation of expression plasmids

a) Generation of expression plasmid for Fc γ RIIIaV158-Avi-IgA protease-Fc LALA P239G fusion polypeptide

The Fc γ RIIIaV158-Avi-IgA protease-Fc LALA P239G fusion polypeptide encoding gene was assembled by fusing chemically synthesized DNA fragments encoding: i) the murine immunoglobulin heavy chain signal sequence (MGWSCIILFLVATATGVHS: SEQ ID NO: 55); ii) the human Fc γ receptor IIIaV158 from amino acid residues 2-193 (i.e. excluding the initial methionine); and iii) a human Fc- γ -1-heavy chain constant region (hinge-CH 2-CH3) with mutations L234A, L235A, and P329G.

In addition to the Fc γ RIIIaV158-Avi-IgA protease-Fc LALA P239G fusion polypeptide expression cassette, the expression plasmid for transient expression of Fc γ RIIIaV158-Avi-IgA protease-FcLALA P239G fusion polypeptide in HEK293 cells contained an origin of replication from the vector pUC18 that allows this plasmid to replicate in e.coli, and a β -lactamase gene that confers ampicillin resistance in e.coli. In detail, the transcriptional unit of the Fc γ RIIIaV158-Avi-IgA protease-Fc LALALAP 239G fusion polypeptide coding gene comprises the following functional elements:

immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A,

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence,

-a soluble human Fc gamma receptor III V158 polypeptide from amino acid positions 2-193 of the wild-type human Fc gamma receptor III V158 protein,

human Fc-. gamma. -1-heavy chain constant region (hinge-CH 2-CH3, LALA P329G), and

bovine growth hormone polyadenylation sequence (BGH poly A signal sequence).

The amino acid sequence of the mature Fc gamma RIIIaV158-Avi-IgA protease-Fc LALA P239G fusion polypeptide is:

GMRTEDLPKA VVFLEPQWYR VLEKDSVTLK CQGAYSPEDN STQWFHNESLISSQASSYFIDAATVDDSGE YRCQTNLSTL SDPVQLEVHI GWLLLQAPRWVFKEEDPIHL RCHSWKNTAL HKVTYLQNGKGRKYFHHNSD FYIPKATLKDSGSYFCRGLV GSKNVSSETV NITITQGLAV STISSFFPPGYQGLNDIFEAQKIEWHELVVAPPAPEDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALGAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

(SEQ ID NO：56)。

the following fusion polypeptides can be obtained analogously:

-Fc γ RIIa-LR (H131) -Avi-IgA protease-Fc LALA P239G fusion polypeptide:

QAAAPPKAVL KLEPPWINVL QEDSVTLTCQ GARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYT CQTGQTSLSD PVHLTVLSEW LVLQTPHLEFQEGETIMLRCHSWKDKPLVK VTFFQNGKSQ KFSHLDPTFS IPQANHSHSGDYHCTGNIGY TLFSSKPVTI TVQVPSMGSSSPMGIGLNDI FEAQKIEWHELVVAPPAPED KTHTCPPCPA PEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISKAKG QPREPQVYTL PPSRDELTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

(SEQ ID NO：57)。

-Fc γ RIIb-Avi-IgA protease-Fc LALA P239G fusion polypeptide:

APPKAVLKLE PQWINVLQED SVTLTCRGTH SPESDSIQWF HNGNLIPTHTQPSYRFKANNNDSGEYTCQT GQTSLSDPVH LTVLSEWLVL QTPHLEFQEGETIVLRCHSW KDKPLVKVTF FQNGKSKKFSRSDPNFSIPQ ANHSHSGDYHCTGNIGYTLY SSKPVTITVQ APGLNDIFEA QKIEWHELVVAPPAPEDKTHTCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVSNKALGAPIEK TISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGK

(SEQ ID NO：58)。

-Fc γ RIIIb-Avi-IgA protease-Fc LALA P239G fusion polypeptide:

GMRTEDLPKA VVFLEPQWYS VLEKDSVTLK CQGAYSPEDN STQWFHNESLISSQASSYFIDAATVNDSGE YRCQTNLSTL SDPVQLEVHI GWLLLQAPRWVFKEEDPIHL RCHSWKNTAL HKVTYLQNGKDRKYFHHNSD FHIPKATLKDSGSYFCRGLV GSKNVSSETV NITITQGLAV STISSFSPPGYQGLNDIFEAQKIEWHELVV APPAPEDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALGAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

(SEQ ID NO：59)。

minimum Fc γ RIIIa-Avi-Fc LALA p239G fusion polypeptide (no protease cleavage site): GWLLLQAPRWVFKEEDPIHL RCHSWKNTAL HKVTYLQNGK GRKYFHHNSDFYIPKATLKD SGSYFCRGLV GSKNVSSETVNITITQGLAV STISSFFPPGYQGLNDIFEA QKIEWHELED KTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAP IEKTISKAKG QPREPQVYTLPPSRDELTKN QVSLTCLVKG FYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

(SEQ ID NO：60)。

c) Production of "binding-hole" expression plasmids for dimeric Fc receptor fusion polypeptides

The expression plasmids for transient expression of Fc receptor Fc region fusion polypeptides (wells) in HEK293 cells were derived from the expression vectors described under a) above. It differs from the DNA sequence encoding the Fc region with the hole mutations T366S, L368A, Y407V and Y349C in the human gamma-1 heavy chain constant region.

The expression plasmids for transient expression of Fc receptor Fc region fusion polypeptides (knots) in HEK293 cells were derived from the expression vectors described under a) above. It differs from the DNA sequence encoding the Fc region with the junction mutations T366W and S354C within the human gamma-1 heavy chain constant region.

In addition to the fusion polypeptide (knot/well) expression cassette, the expression plasmid for transient expression of the Fc receptor Fc region fusion polypeptide (knot/well) in HEK293 cells contains an origin of replication from the vector pUC18 that allows this plasmid to replicate in e.coli, and a beta-lactamase gene that confers ampicillin resistance in e.coli. In detail, the transcription unit of the gene encoding the fusion polypeptide (node/pore) comprises the following functional elements:

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence,

human Fc- γ -1 heavy chain constant region with the mutations T366S, L368A, Y407V and Y349C or the knot mutations T366W and S354C (hinge-CH 2-CH3) within the human γ -1 heavy chain constant region, and

bovine growth hormone polyadenylation sequence (BGH poly A signal sequence).

Example 2

Transient expression, purification and analytical characterization of the Fc γ RIIIaV158-Avi-IgA protease-Fc LALA P239G fusion polypeptide

Fusion polypeptides were obtained by transient transfection of HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). For transfection, "293-Free" transfection reagent (Novagen) was used. The pair of hole-binding fusion polypeptides was expressed from two different plasmids using an equimolar plasmid ratio at the time of transfection. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants containing the fusion polypeptides were harvested seven days post transfection. The supernatant was stored at reduced temperature until purification.

General information on the recombinant expression of human immunoglobulins in, for example, HEK293 cells is given in Meissner, P.et al, Biotechnol.Bioeng.75(2001) 197-203.

The culture supernatant containing the fusion polypeptide was filtered and purified by two chromatographic steps. PBS (1 mMKH) was used₂PO₄、10mM Na₂HPO₄137mM NaCl, 2.7mM KCl), HiTrap MabSelectSuRe (GEHealthcare) balanced at pH 7.4, and the fusion polypeptide was captured by affinity chromatography. Unbound protein was removed by washing with equilibration buffer and the fusion polypeptide was recovered with 0.05M citrate buffer pH 3 and neutralized to pH6.5 with 1M Tris base pH 9.0 immediately after elution. Using Superdex 200^TMSize exclusion chromatography on (GE Healthcare) was used as the second purification step. Size exclusion chromatography was performed in 2mM MOPS buffer, 0.125M NaCl, pH 7.2. The eluted fusion polypeptides were concentrated using an Ultrafree-CL centrifugal filtration unit (Millipore, Billerica, Mass.) equipped with a Biomax-SK membrane and stored at-80 ℃.

Four different Fc γ RIIIa-Fc fusion polypeptides were purified according to this procedure:

a) fc gamma RIIIaV158-Avi-Fc LALA P239G (without cleavage site)

b) Minimum Fc gamma RIIIaV158-Avi-Fc LALA P239G (no cleavage site)

c) Fc gamma RIIIaV158-Avi-PreScission Protease (PP) -Fc LALA P239G

d) Fc gamma RIIIaV158-Avi-IgA protease-Fc LALA P239G

The protein concentration of the fusion polypeptide was determined by measuring the 280nm Optical Density (OD) using the molar extinction coefficient calculated from the amino acid sequence. The purity of the fusion polypeptides and the correct dimer formation were analyzed by SDS-PAGE and Coomassie blue staining in the presence and absence of reducing agent (5mM 1.4-dithiothreitol). By using Superdex 200^TMHigh performance SEC of analytical size exclusion column (GEHealthcare) was used to determine the aggregate content of the fusion polypeptide preparation. After removal of N-glycans by treatment with a combination of neuraminidase, O-glycanase and peptide N-glycosidase f (roche Applied science), the integrity of the amino acid backbone of the reduced fusion polypeptide was verified by Nano electrophoresis QTOF mass spectrometry.

Example 3

Cleavage by papain

Fc γ RIIIa-Fc fusion polypeptides that do not contain an enzymatic cleavage site can be cleaved by papain. The Fc γ RIIIa-Fc fusion polypeptide was cleaved at 37 ℃ for 1 hour by addition of cysteine and 0.1mU/mg of fusion polypeptide papain (from Carica papaya, Roche diagnostics GmbH). The subsequent purification was carried out as described in example 2. An analytical SDS-PAGE gel is shown in FIG. 2.

Example 4

Cleavage by Ides protease

Cleavage of the Fc γ RIIIaV158-Avi-Fc LALA P239G fusion polypeptide with the Ides protease is very inefficient and therefore not useful in this case.

Example 5

Cleavage by PreScission protease

After dialysis against 50mM Tris, 150NaCl, 1mM EDTA, 1mM DTT pH 7.4, the Fc γ RIIIa- (PP) -Fc fusion polypeptide was cleaved overnight at room temperature by addition of between 1-15U of PreScission protease (GE Healthcare)/100 μ g of fusion polypeptide. Only a portion of the protein may be cleaved. On the other hand, non-specific cleavage of the receptor without PP cleavage site by PreScission protease was observed.

Example 6

Cleavage by IgA protease

After dialysis against 50mM Tris pH 8 with Slide-a-lyzer dialysis cassette, the Fc γ RIIIa-Fc fusion polypeptide was cleaved overnight at 21 ℃ by addition of IgA protease (Roche Diagnostics GmbH) in a ratio w (protease)/w (fusion polypeptide) of 1: 100. Cleavage was controlled by analytical size exclusion chromatography (SEC, Superdex 75; GE Healthcare). After cleavage, the Fc γ RIIIa receptor was passed through Superdex 75^TMPreparative size exclusion chromatography on (GE Healthcare) was separated from IgA protease and from Fc-Tag by HiTrap MabSelectSuRe (GE Healthcare) column. An analytical SDS-PAGE gel is shown in FIG. 3.

Table (b): yield of fermentation and purification of fusion polypeptides comprising different Fc γ RIIIa V158

Example 7

Preparation of Fc gamma RIIIa V158 affinity column

An affinity column with Fc γ RIIIa V158 was prepared by biotinylating the Avi label in vitro and then coupling to streptavidin agarose. This can be done with the fusion polypeptide intact and with the receptor after the Fc region has been cleaved off. It is a very fast and efficient method for the preparation of affinity columns for analytical and preparative purposes.

Biotinylation of the receptor

The soluble extracellular domain of Fc γ RIIIaV158 with Avi tag expressed in HEK293 cells was biotinylated following the following protocol after purification: biotinylated biotinylation kit from Avidity was used to biotinylate between 1.2 and 12mg of labeled Fc γ RIIIaV158 or between 2.4 and 24mg of labeled Fc γ RIIIaV158Fc region fusion polypeptides in 3ml PBS, 125mM NaCl pH 7.2, 0.02% Tween and 1 pan of holoprotease inhibitor (Roche) in PBS according to the manufacturer's instructions. The biotinylation reaction was carried out at room temperature overnight. The modified polypeptide was dialyzed against 20mM sodium phosphate buffer, 150mM NaCl pH 7.5 at 4 ℃ overnight to remove excess biotin.

Coupling to streptavidin Sepharose

1g of streptavidin agarose (GE Healthcare) was added to biotinylated and dialyzed receptors, incubated with shaking for 2 hours, and finally loaded onto a 1ml XK column (GE Healthcare).

Example 8

Chromatography method

General conditions:

equilibration buffer a: 20mM citric acid/150 mM NaCl pH 6.0

Elution buffer B: 20mM citric acid/150 mM NaCl pH 3.0

And (3) elution: the reaction time of the reaction mixture is 5 minutes and 100 percent of A,

within 60 minutes to 100% of B,

0.1 minute 100% of B,

6 min 100% A

Sample amount: 50 μ g or more

Separation of fucosylated and afucosylated (afucosylated) antibodies

Chromatography of the antibodies on an FcgRIIIa column allows quantification of fully fucosylated and afucosylated antibody fractions. The afucosylated antibody fraction is related to ADCC of the antibody preparation.

In fig. 4, the separation and quantification of different glycosylated forms of anti-Her antibody (wild type, top) and glycoengineered anti-Her antibody on the Fc-FcgRIIIa column is shown. Analysis time can be shortened by changing the gradient while maintaining resolution.

Comparison of affinity columns using Fc γ RIIIaV158 and Fc-labeled Fc γ RIIIaV158

After equimolar coupling of the two receptor constructs, the affinity column performed equally on the fully fucosylated and afucosylated antibodies (FIG. 5: black: Fc γ RIIIaV 158; blue: Fc γ RIIIaV 158-Fc).

Example 9

Fc gamma RIIIaV158-Avi-IgA protease-Fc LALA P329G-IgG interaction measurement

The system is well established for the study of molecular interactions. It allows continuous real-time monitoring of ligand/analyte binding, thereby determining the association rate constant (k)_a) Dissociation rate constant (k)_d) And equilibrium constant (K)_D). The change in refractive index indicates a mass change on the surface caused by the interaction of the immobilized ligand with the analyte injected in solution. If the molecule binds to the immobilized ligand on the surface, the mass increases and in case of dissociation, the mass decreases.

For the activity assay of the Fc γ RIIIaV158-Fc LALA P329G fusion polypeptide, a direct binding assay was used.

An approximately 400 Resonance Unit (RU) capture system (20. mu.g/ml human Fab capture kit GE Healthcare,28-9583-25) was coupled to a CM5 chip (GE Healthcare BR1005-30) at pH 5.0 using an amine coupling kit supplied by GE. The sample and system buffer was HBS-P + pH 7.4(10mM HEPES, pH 7.4, 150mM NaCl, 0.05% (v/v) surfactant P20). The flow cell was set at 25 ℃ and the sample zone at 12 ℃. The antibody was captured by injecting 50nM solution at a flow rate of 10. mu.l/min for 360 seconds. Binding was measured by association with 50nM of Fc γ RIIIa fusion polypeptide injected at a flow rate of 50 μ l/min for 180 seconds and dissociation for 360 seconds. The surface was regenerated by washing with glycine pH 2.1 solution at a flow rate of 20 μ l/min for 60 seconds. For activity evaluation of the constructs, signal height and dissociation behavior were compared.

As shown in figure 6, the response of Fc γ RIIIaV158-FcLALA P329G fusion polypeptide showed more than 100 response units compared to 40RU of Fc γ RIIIaV 158.

Example 10

Measurement of the Pre-and post-cleavage IgG kinetic interaction of the Fc γ RIIIaV158-Avi-IgA protease-Fc LALA P329G fusion polypeptide

For the activity assay of the cleaved Fc γ RIIIaV158-Fc LALA P329G fusion polypeptide, a direct binding assay was used.

An approximately 400 Resonance Unit (RU) capture system (20. mu.g/ml human Fab capture kit GE Healthcare,28-9583-25) was coupled to a CM5 chip (GE Healthcare BR1005-30) at pH 5.0 using an amine coupling kit supplied by GE. The sample and system buffer was HBS-P + pH 7.4(10mM HEPES, pH 7.4, 150mM NaCl, 0.05% (v/v) surfactant P20). The flow cell was set at 25 ℃ and the sample zone at 12 ℃. The antibody was captured by injecting 50nM solution at a flow rate of 10. mu.l/min for 80 seconds.

Binding was measured at 25 ℃ for 120 seconds by passing different concentrations of antibody ranging from 0 to 250nM (1:2 dilution) through the flow cell at a flow rate of 30. mu.l/min. The dissociation period was monitored by switching from the sample solution to running buffer for 420 seconds. The surface was regenerated by washing with glycine pH 2.1 solution at a flow rate of 20 μ l/min for 60 seconds.

The bulk refractive index difference was corrected by subtracting the response obtained from the surface of the RIIIaV158 without capture. Blank buffer injections (double reference) were also subtracted.

The determination of k, defined as k, was carried out by analyzing sensorgram curves obtained with several different concentrations using the BIAevaluation software package_a/k_dEquilibrium dissociation constant (K) of_D). The fitting of the data follows a suitable binding model. In FIG. 7, a sensorgram of the Fc γ receptor V158-Fc LALA P329G fusion polypeptide (FIG. 7a), Fc γ receptor V158 (FIG. 7b), cleaved Fc γ receptor V158-FcLALA P329G fusion polypeptide (FIG. 7c) is shown.

Claims

1. A fusion polypeptide according to formula I:

R1-FC-R2 (formula I)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor, and

FC denotes a heavy chain Fc region polypeptide,

wherein R1 or R2 is present, or both R1 and R2 are present,

wherein FC does not bind R1 and/or R2,

wherein R1 and R2 are independently of each other selected from the group of human Fc γ -receptor, human neonatal Fc receptor, murine Fc receptor, and rabbit neonatal Fc receptor, and

wherein FC is a mutated human IgG1 heavy chain Fc region polypeptide,

wherein the mutations are L234A, L235A, P329G, and the pore mutations T366S, L368A, Y407V and Y349C, or

The mutations were L234A, L235A, P329G, and junction mutations T366W and S354C.

2. The fusion polypeptide according to claim 1, wherein the fusion polypeptide has formula II

R1-CS1-L1-CS2-FC-CS3-L2-CS4-R2 (formula II)

Wherein

R1 represents a first Fc receptor,

r2 represents a second Fc receptor,

FC denotes a heavy chain Fc region polypeptide,

CS1 denotes the first cleavage site,

CS2 denotes a second cleavage site which,

CS3 denotes a third cleavage site,

CS4 denotes a fourth cleavage site,

l1 denotes a first intervening amino acid sequence, and

l2 denotes a second intervening amino acid sequence,

wherein R1 or R2 is present, or both R1 and R2 are present,

wherein L1 and L2 may be present or absent independently of each other,

wherein FC does not bind R1 and/or R2.

3. The fusion polypeptide according to claim 1, wherein the human Fc γ receptor is selected from the group consisting of human Fc γ RI (CD64), human Fc γ RII (CD32), human Fc γ RIIA, human Fc γ RIIB, human Fc γ RIIC, human Fc γ RIII (CD16), human Fc γ RIIIA, and human Fc γ RIIIB.

4. A fusion polypeptide according to claim 3, wherein the human neonatal Fc receptor is human FcRn.

5. The fusion polypeptide according to claim 1, wherein the murine Fc receptor is selected from the group consisting of murine fcyri (CD64), murine fcyrii (CD32), murine fcyriib, murine fcyriii (CD16), murine fcyriii-2 (CD16-2), and murine fcyriv.

6. The fusion polypeptide according to claim 1, wherein one or more of the Fc receptors is an fey receptor.

7. The fusion polypeptide according to claim 1, wherein one or more of the Fc receptors is FcRn.

8. The fusion polypeptide according to claim 2, wherein the intervening amino acid sequence is selected from a first group comprising (G3S)3, (G3S)4, (G3S)5, (G3S)6, (G4S)3, (G4S)4, (G4S)5, (G5S)2, (G5S)3, and (G5S)4, or from a second group comprising an Arg tag, an Avi tag, a His-Avi tag, a His tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a GST tag, or an MBP tag, or from a combination of two elements selected from these groups.

9. The fusion polypeptide according to claim 2, wherein the cleavage site is selected from the group consisting of an IgA protease cleavage site, a granzyme B protease cleavage site, a Tev protease cleavage site, a Precision protease cleavage site, a thrombin cleavage site, a Faktor10a protease site, an Ides protease cleavage site, an enterokinase cleavage site or a SUMO protease cleavage site.

10. The fusion polypeptide according to claim 2, wherein the fusion polypeptide does not comprise an additional protease cleavage site but comprises an intrinsic protease cleavage site.

11. The fusion polypeptide according to claim 10, wherein the intrinsic protease cleavage site is a papain cleavage site, a pepsin cleavage site or an Ides protease cleavage site.

12. A dimeric fusion polypeptide comprising two fusion polypeptides according to any one of claims 1 to 11.

13. The fusion polypeptide according to claim 12, wherein,

FC of the first fusion polypeptide is a mutated human IgG1 heavy chain FC region polypeptide, wherein the mutations are L234A, L235A, P329G, and junction mutations T366W and S354C; and is

The FC of the second fusion polypeptide was a mutated human IgG1 heavy chain FC region polypeptide, wherein the mutations were L234A, L235A, P329G, and the pore mutations T366S, L368A, Y407V, and Y349C.

14. The fusion polypeptide of claim 12, wherein the fusion polypeptide is characterized by

a) R1 and R2 of the first and second fusion polypeptides are identical,

c) r1 of the first and second fusion polypeptides are the same and R2 of the first and second fusion polypeptides are the same but different from R1,

h) r1 of the first fusion polypeptide and R2 of the second fusion polypeptide are different and R2 of the first fusion polypeptide is absent and R1 of the second fusion polypeptide is absent.

15. A method of producing a soluble Fc receptor, the method comprising the steps of

a) Culturing a cell comprising a nucleic acid encoding the fusion polypeptide according to any one of claims 1 to 14,

b) recovering the fusion polypeptide from the cell or the culture medium,

c) optionally cleaving the fusion polypeptide with a protease,

and thereby produce soluble Fc receptors.

16. Use of an immobilized fusion polypeptide or an immobilized dimeric fusion polypeptide according to any one of claims 1 to 14 as affinity chromatography ligand.

17. Use of an immobilized fusion polypeptide or an immobilized dimeric fusion polypeptide according to any one of claims 1 to 14 for determining Fc receptor binding of an antibody.

18. Use according to claim 17, wherein the fusion polypeptide is bound to a solid phase.

19. A pharmaceutical composition comprising a fusion polypeptide according to any one of claims 1 to 14.

20. Use of a fusion polypeptide according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of a disease.

21. The use according to claim 20, wherein the medicament is for the treatment of an inflammatory disease.

22. Use according to any one of claims 20 to 21, wherein the disease is a disease characterized by increased antibody levels.

23. Use according to any one of claims 20 to 21 wherein the disease is an autoimmune disease.

24. Use according to any one of claims 20 to 21 wherein the disease is rheumatoid arthritis.