HK1227040A1

HK1227040A1 - Interleukine-10 immunoconjugates

Info

Publication number: HK1227040A1
Application number: HK17100548.8A
Authority: HK
Inventors: Thomas Emrich; Pablo Umana; Ekkehard Moessner; Ralf Hosse; Jens Fischer; Lydia Jasmin Hanisch; Daigen Xu
Original assignee: 豪夫迈．罗氏有限公司
Priority date: 2014-02-06
Filing date: 2015-02-03
Publication date: 2017-10-13

Description

Interleukin-10 immunoconjugates

Technical Field

The present invention relates generally to fusion proteins of an antibody and interleukin-10 (IL-10). More particularly, the invention concerns fusion proteins of an antibody and a mutant IL-10 exhibiting improved properties for use as therapeutic agents, e.g. in the treatment of inflammatory diseases. In addition, the invention relates to polynucleotides encoding such fusion proteins, as well as vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the fusion proteins of the invention, and methods of using them in the treatment of disease.

Background

Biological function of IL-10

IL-10 is an alpha-helical cytokine expressed as a non-covalently linked homodimer of approximately 37 kDa. It plays a key role in tolerance induction and maintenance. Its dominant anti-inflammatory properties have been known for a long time. IL-10 suppresses the secretion of pro-inflammatory cytokines like TNF α, IL-1, IL-6, IL-12, and Th1 cytokines such as IL-2 and INF γ, and controls the differentiation and proliferation of macrophages, B cells and T cells (Glocker, E.O.et. al, Ann.N.Y.Acad.Sci.1246,102-107 (2011); Moore, K.W.et. al., Annu.Rev.Immunol.19,683-765 (2001); de Waal Malefyt, R.et. al., J.exp.Med.174,915-924 (1991); Williams, L.M.et. al., Immunology 113,281- (2004)). In addition, it is a potent inhibitor of antigen presentation, inhibiting MHC II expression and the upregulation of the costimulatory molecules CD80 and CD86 (Mosser, D.M. & Yhang, x., immunologicals reviews 226, 205-.

However, immunostimulatory properties have also been reported. IL-10 is able to co-stimulate B cell activation, prolong B cell survival, and aid in class switching in B cells. In addition, it can co-stimulate Natural Killer (NK) cell proliferation and cytokine production and act as a growth factor stimulating certain CD8⁺T cell subsets proliferate (Mosser, D.M.&Yhang, x., ImmunologicalReviews 226, 205-; cai, g.et al, eur.j.immunol.29,2658-2665 (1999); santin, a.d.et al, j.virol.74,4729-4737 (2000); rowbottom, a.w.et al, Immunology98,80-89 (1999); group, H.et al, J.Immunol.160,3188-3193 (1998)). Importantly, high doses of IL-10 (20 and 25. mu.g/kg, respectively) cause increased INF γ production in humans (Lauw, F.N.et al., J.Immunol.165,2783-2789 (2000); Tilg, H.et al., Gut 50,191-195 (2002)). The immunostimulatory activity of IL-10 was reported to be determined by the single amino acid isoleucine at position 87 in cellular IL-10 (Ding, Y.et., J.Exp.Med.191(2),213-223 (2000)).

IL-10 signals via a two-receptor complex consisting of two copies each of IL-10 receptor 1(IL-10R1) and IL-10R 2. IL-10R1 binds IL-10 with relatively high affinity (approximately 35-200pM) (Moore, K.W.et al, Annu.Rev.Immunol.19,683-765(2001)), and recruitment of IL-10R2 to the receptor complex contributes only marginally to ligand binding. However, the engagement of this second receptor to the complex enables signal transduction upon ligand binding. Thus, the functional receptor is composed of a dimer of IL-10R1 and IL-10R2 heterodimers. Most hematopoietic cells constitutively express low levels of IL-10R1, and receptor expression can often be significantly upregulated by a variety of stimuli. Non-hematopoietic cells (such as fibroblasts and epithelial cells) can also respond to stimuli by upregulating IL-10R 1. In contrast, IL-10R2 is expressed on most cells. Binding of IL-10 to the receptor complex activates Janus tyrosine kinases, JAK1 and Tyk2, which are associated with IL-10R1 and IL-10R2, respectively, phosphorylate the cytoplasmic tail of the receptor. This results in recruitment of STAT3 to IL-10R 1. Homodimerization of STAT3 results in its release from the receptor and translocation of the phosphorylated STAT homodimer into the nucleus, where it binds to STAT3 binding elements in the promoters of various genes. One of these genes is IL-10 itself, which is positively regulated by STAT 3. STAT3 also activates suppressors of cytokine signaling 3(SOCS3), and SOCS3 controls the quality and quantity of STAT activation. SOCS3 is induced by IL-10 and exerts negative regulatory effects on a variety of cytokine genes (Mosser, D.M. & Yhang, x., immunologicals reviews 226,205.218 (2008)).

Genetic linkage analysis and candidate gene sequencing revealed a direct link between mutations in IL-10R1 and IL-10R2 and early onset enterocolitis, a form of Inflammatory Bowel Disease (IBD) (Glocker, e.o.et al., n.engl.j.med.361(21),2033-2045 (2009)). Recent data suggest that early onset IBD may even be monogenic. Mutations in the IL-10 cytokine or its receptor result in loss of IL-10 function and cause severe enterocolitis in infants and young children (Glocker, e.o.et al., ann.n.y.acad.sci.1246,102-107 (2011)). Moreover, patients with severe forms of crohn's disease have defective IL-10 production in whole blood cell cultures and monocyte-derived dendritic cells (corea, i.et al, j.leukoc.biol.85(5),896-903 (2009)). IBD affects about 140 million people in the united states and about 220 million people in europe (Carter, m.j.et al, Gut 53(suppl.5), V1-V16 (2004); Engel, M.A. & Neurath, m.f., j.gastroenterol.45,571-583 (2010)).

Therapeutic approaches using IL-10

Recombinant IL-10 has been evaluated for therapeutic benefit in inflammatory and autoimmune disorders in phase I and II clinical trials investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, immunological and hematological effects of single or multiple doses administered intravenously or subcutaneously in a variety of settings in healthy volunteers, as well as in specific patient populations (Moore, K.W.et al, Annu.Rev.Immunol.19,683-765 (2001); Chernoff, A.E.et al, J.Immunol.154,5492-5499 (1995); Huhn, R.D.et al, Blood 87,699 Pharmacol.705 (1996); Huhn, R.D.et al, Clin.Therma.62, 171-180 (1997)). IL-10 is well tolerated at doses up to 25. mu.g/kg without serious side effects, and mild to moderate flu-like symptoms are observed in only a fraction of recipients at doses up to 100. mu.g/kg (Moore, K.W.et al, Annu. Rev. Immunol.19,683-765 (2001); Chernoff, A.E.et al, J.Immunol.154,5492-5499 (1995)). Psoriasis (the compilation of clinical studies can be seen in Mosser, D.M. & Yong, X., Immunological Reviews 226,205-218(2008)), Crohn's disease (Van Deventer S.J.et al, Gastroenterology 113,383-389 (1997)), Fedorak, R.N.et al, Gastroenterology 119,1473-1482 (2000); Schreiber, S.et al, gastroenterolotgy119,1461-1472 (2000); Colombel J.F.et al, Gut 49,42-46(2001)) and rheumatoid arthritis (Keystone, E.et al, Rheum.Dis.Imin.N.629.24, 629-639 (1998); Mosser, D.M. Yong, 2008, Y.226, 205-226) are frequently improving in clinical studies.

In conclusion, the clinical results were not satisfactory and the clinical development of recombinant human IL-10 (ilodebakin, TENOVIL, Schering-Plough Research Institute, Kenilworth, NJ), identical to endogenous human IL-10 except for the methionine residue at the amino terminus, was interrupted by the lack of efficacy. One systemic review on the efficacy and tolerability of recombinant human IL-10 for inducing regression in crohn's disease did not reveal statistically significant differences between IL-10 and placebo for complete or clinical regression, and stated that patients treated with IL-10 are significantly more likely to withdraw from the study due to adverse events relative to placebo (Buruiana, f.e.et al, Cochrane Database syst.rev.11, CD005109 (2010)). Several reasons for these unsatisfactory results are discussed in relation to crohn's disease (Herfarth, H.&J., Gut 50,146-147 (2002)): 1) local cytokine concentrations in the intestine are too low to mediate a sustained anti-inflammatory effect, 2) dose escalation with systemic administration of IL-10 is limited by side effects3) IL-10 on B cells and on CD4⁺、CD8⁺And/or the immunostimulatory properties of natural killer cells to produce INF γ counteract its immunosuppressive properties (Asadullah, K.et al., Pharmacol. Rev.55,241-269 (2003); Tilg, H.et al., Gut 50,191-195 (2002); Lauw, F.N.et al., J.Immunol.165,2783-2789 (2000)).

IL-10 exhibits a short plasma half-life due to its small size of about 37kDa, which leads to rapid renal clearance. In fact, its half-life in the system compartment is 2.5 hours, which limits mucosal bioavailability (Braat, h.et al, Expert opin. biol. ther.3(5), 725-. To improve cycle time, exposure, efficacy and reduce kidney uptake, several publications report pegylation of this cytokine (Mattos, a.et al, j.control Release162,84-91 (2012); mummm, j.b.et al, Cancer Cell 20(6), 781-. However, the longer systemic half-life of pegylated non-targeted IL-10 can exacerbate known adverse events of this molecule.

It has become clear that systemic treatment with recombinant human IL-10 is not sufficiently effective and the focus must be on local delivery of cytokines. There are several ways to achieve this goal: 1) IL-10 gene therapy of immune cells, 2) genetically modified, non-pathogenic IL-10 expressing bacteria, and 3) antibody-IL-10 fusion proteins for targeting and accumulating the cytokine in inflamed tissue.

IL-10 gene therapy of immune cells has proven effective in experimental colitis, but concerns about the safety of this approach to non-fatal disease have hampered clinical trials (Bract, h.et al, expetpopin, biol. ther.3(5),725-731 (2003)). IL-10 expressing transgenic bacteria (Lactococcus lactis) represent an alternative delivery route and the results of phase I trials in Crohn's disease have been published, requiring avoidance of systemic side effects from local delivery into mucosal compartments and biological inclusion (Braat, H.et al, gastroenterol. hepatol.4,754-759 (2006); Steidler, L.et al, Science 289,1352-1355 (2000)). A phase IIa randomization, placebo-controlled, double-blind, multi-center, dose escalation study to assess the safety, tolerability, pharmacodynamics, and efficacy of genetically modified, human IL-10 secreting lactococcus lactis (AG011, ActoGeniX) in patients with moderately active ulcerative colitis was well tolerated and safe. However, there was no significant improvement in mucosal inflammation (as measured by modified Baron scores) or clinical symptoms in patients receiving AG011 compared to placebo (Vermeire, s.et al, abstract 46presented at the diagnostic Disease Week annual meeting in New orans 02May 2010).

Antibody-cytokine fusion proteins (also known as immunocytokines) offer several advantages in terms of drug delivery and the modality of the drug itself. Local delivery of cytokines (e.g., IL-10) is achieved by fusion with antibodies or fragments thereof specific for appropriate disease markers. In this way, systemic side effects can be reduced/mitigated and local accumulation and retention of compounds at the site of inflammation can be achieved. In addition, depending on the fusion form and the antibody or antibody fragment used, properties such as plasma half-life, stability and developability can be improved. Although an established approach in oncology has long been, it has only recently been adapted for use in the treatment of inflammatory conditions and autoimmunity. Several cytokines, IL-10 among others, and photosensitizers are targeted to the psoriatic lesion by fusion with scFv antibody fragments specific for fibronectin ectodomain B (Trachsel, e.et al, j.invest.dermatol.127(4),881-886 (2007)). Furthermore, antibody fragments specific for fibronectin domain a (F8, DEKAVIL, philigen SpA) -IL-10 fusion proteins were used preclinically to inhibit the progression of established, collagen-induced Arthritis (Trachsel, e.et al, Arthritis res.ther.9(1), R9 (2007); Schwager, k.et al, Arthritis res.ther.11(5), R142(2009)) and were advanced into clinical trials. Recently, the same F8-IL-10 fusion protein was used to target endometritis lesions in syngeneic mouse models and to reduce the average lesion size compared to saline control groups (Schwager, k.et al, hum. reprod.26(9), 2344-.

The IgG-IL-10 fusion proteins of the invention have several advantages over IL-10 fusion proteins based on known antibody fragments (scFv, diabody, Fab), including improved producibility, stability, serum half-life, and surprisingly a significantly increased biological activity upon binding to a target antigen. Furthermore, the fusion proteins of the invention exhibit improved targeting efficiency via reduced affinity for the IL-10 receptor and reduced side effects caused by the immunostimulatory properties of IL-10.

Summary of The Invention

In one aspect, the invention provides a fusion protein of an IgG class antibody and a mutant IL-10 molecule, wherein the fusion protein comprises two identical heavy chain polypeptides and two identical light chain polypeptides, and wherein the mutant IL-10 molecule comprises an amino acid mutation that reduces the binding affinity of the mutant IL-10 molecule to the IL-10 receptor as compared to the wild-type IL-10 molecule. In one embodiment, the amino acid mutation reduces the binding affinity of the mutant IL-10 molecule to the IL-10 receptor by at least 2-fold, at least 5-fold, or at least 10-fold as compared to the wild-type IL-10 molecule. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the mutant IL-10 molecule comprises an amino acid substitution at a position corresponding to residue 87 of human IL-10(SEQ ID NO: 1). In a specific embodiment, the amino acid substitution is I87A. In one embodiment, the mutant IL-10 molecule is a human IL-10 molecule. In one embodiment, the mutant IL-10 molecule is a homodimer of two mutant IL-10 monomers. In one embodiment, the IL-10 receptor is IL-10R1, in particular human IL-10R 1.

In one embodiment, each of the heavy chain polypeptides comprises an IgG class antibody heavy chain and a mutant IL-10 monomer. In a more specific embodiment, the mutant IL-10 monomer is fused at its N-terminus to the C-terminus of the IgG class antibody heavy chain, optionally via a peptide linker. In one embodiment, the heavy chain polypeptides each consist essentially of an IgG class antibody heavy chain, a mutant IL-10 monomer, and an optional peptide linker. In one embodiment, each of the light chain polypeptides comprises an IgG class antibody light chain. In one embodiment, the light chain polypeptides each consist essentially of an IgG class antibody light chain.

In one embodiment, the mutant IL-10 monomer is a human IL-10 monomer. In one embodiment, the mutant IL-10 monomer comprises an amino acid substitution. In one embodiment, the mutant IL-10 monomer comprises an amino acid substitution at a position corresponding to residue 87 of human IL-10(SEQ ID NO: 1). In a specific embodiment, the amino acid substitution is I87A. In a specific embodiment, the mutant IL-10 monomer comprises SEQ ID NO: 98. In one embodiment, the mutant IL-10 monomer comprised in the heavy chain polypeptide forms a functional homodimeric mutant IL-10 molecule.

In one embodiment, the IgG class antibody comprises a modification that reduces the binding affinity of the antibody to an Fc receptor as compared to a corresponding IgG class antibody without the modification. In a particular embodiment, the Fc receptor is an fey receptor, in particular a human fey receptor. In one embodiment, the Fc receptor is an activating Fc receptor, particularly an activating fey receptor. In a specific embodiment, the Fc receptor is selected from the group consisting of: fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32) and Fc α RI (CD 89). In an even more specific embodiment, the Fc receptor is Fc γ IIIa, in particular human Fc γ IIIa. In one embodiment, the modification reduces effector function of an IgG class antibody. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the modification is in the Fc region of the IgG class antibody, in particular in the CH2 region. In one embodiment, the IgG-class antibody comprises an amino acid substitution at position 329(EU numbering) of the heavy chain of the antibody. In a specific embodiment, the amino acid substitution is P329G. In one embodiment, the IgG class antibody comprises amino acid substitutions at positions 234 and 235(EU numbering) of the heavy chain of the antibody. In a specific embodiment, the amino acid substitutions are L234A and L235A (LALA). In a particular embodiment, the IgG class antibody comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in the antibody heavy chain.

In one embodiment, the IgG class antibody is IgG₁Subclass antibody. In one embodiment, the IgG class antibody is a full length antibody. In one embodiment, the IgG class antibody is a human antibody. In one embodiment, the IgG class antibody is a monoclonal antibody.

In one embodiment, the IgG-class antibody is capable of specifically binding to Fibroblast Activation Protein (FAP). In a specific embodiment, the fusion protein is capable of exhibiting an affinity constant (K) of less than 1nM, in particular less than 100pM, when measured by Surface Plasmon Resonance (SPR) at 25 ℃_D) Combining FAP. In one embodiment, the FAP is human, mouse, and/or cynomolgus FAP. In a specific embodiment, the IgG-class antibody comprises SEQ ID NO: 37, cdr (hcdr)1 of heavy chain, SEQ ID NO: HCDR2 of 41, SEQ ID NO: 49 HCDR3 of SEQ ID NO: 53 light chain cdr (lcdr)1, SEQ ID NO: LCDR 2 of 57 and SEQ ID NO: LCDR 3 of 61. In an even more specific embodiment, the IgG-class antibody comprises SEQ ID NO: 63 and the heavy chain variable region (VH) of SEQ ID NO: 65 (VL) in the light chain. In another specific embodiment, the IgG-class antibody comprises SEQ ID NO: 37 HCDR1, SEQ ID NO: 43, HCDR2 of SEQ ID NO: 47 HCDR3, SEQ ID NO: 51, LCDR 1 of SEQ ID NO: LCDR 2 of 55 and SEQ ID NO: LCDR 3 of 59. In an even more specific embodiment, the IgG-class antibody comprises SEQ ID NO: 67 and the VH of SEQ ID NO: 69.

In one embodiment, the fusion protein is capable of having an affinity constant (K) of about 100pM to about 10nM, particularly about 200pM to about 5nM, or about 500pM to about 2nM, when measured by SPR at 25 ℃_D) Binds to IL-10 receptor-1 (IL-10R 1). In a specific embodiment, the IL-10R1 is human IL-10R 1. In one embodiment, the affinity that binds IL-10R1 is measured by SPR at 25 ℃And constant (K)_D) Greater than the affinity constant (K) for said binding FAP_D). In a specific embodiment, the K that binds IL-10R1_DK greater than the combined FAP_DAbout 1.5 times, about 2 times, about 3 times, or about 5 times. In one embodiment, the fusion protein has at least a 2-fold, at least a 5-fold, or at least a 10-fold reduction in binding affinity for the IL-10 receptor as compared to a corresponding fusion protein comprising a wild-type IL-10 molecule.

In a particular embodiment, the invention provides a fusion protein of an IgG class antibody and a mutant IL-10 molecule, wherein the fusion protein comprises two identical heavy chain polypeptides and two identical light chain polypeptides; and wherein

(i) The IgG class antibody comprises SEQ ID NO: 37, cdr (hcdr)1 of heavy chain, SEQ ID NO: 43, HCDR2 of SEQ ID NO: 47 HCDR3, SEQ ID NO: 51, light chain cdr (lcdr)1, SEQ ID NO: LCDR 2 of 55 and seq id NO: 59, or an LCDR 3 comprising SEQ ID NO: 67 and the heavy chain variable region (VH) of SEQ ID NO: 69 (VL);

(ii) the IgG class antibody comprises the amino acid substitutions L234A, L235A and P329G in the antibody heavy chain (EU numbering);

(iii) each of the heavy chain polypeptides comprises an IgG class antibody heavy chain and a mutant IL-10 monomer fused at its N-terminus to the C-terminus of the IgG class antibody heavy chain via a peptide linker; and is

(iv) The mutant IL-10 monomer comprises SEQ ID NO: 98, respectively.

In another embodiment, the invention provides a fusion protein of an IgG class antibody and a mutant IL-10 molecule, wherein the fusion protein comprises two identical heavy chain polypeptides and two identical light chain polypeptides; and wherein

(i) The IgG class antibody comprises SEQ ID NO: 37, cdr (hcdr)1 of heavy chain, SEQ ID NO: HCDR2 of 41, SEQ ID NO: 49 HCDR3 of SEQ ID NO: 53 light chain cdr (lcdr)1, SEQ ID NO: LCDR 2 of 57 and seq id NO: 61, or an LCDR 3 comprising SEQ ID NO: 63 and the heavy chain variable region (VH) of SEQ ID NO: 65 light chain variable region (VL);

(iv) The mutant IL-10 monomer comprises SEQ ID NO: 98, respectively.

The invention further provides a polynucleotide encoding the fusion protein of the invention. Further provided is a vector, particularly an expression vector, comprising a polynucleotide of the invention. In another aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. The present invention also provides a method for producing a fusion protein of the invention, comprising the steps of: (i) culturing the host cell of the invention under conditions suitable for expression of the fusion protein, and (ii) recovering the fusion protein. Also provided are fusion proteins of an IgG class antibody and a mutant IL-10 molecule, which fusion proteins are produced by the methods.

In one aspect, the invention provides a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier. Also provided is a fusion protein or a pharmaceutical composition of the invention for use as a medicament and for the treatment or prevention of an inflammatory disease, in particular inflammatory bowel disease or rheumatoid arthritis, most particularly inflammatory bowel disease. Further provided are the use of a fusion protein of the invention for the preparation of a medicament for treating a disease in a subject in need thereof and a method of treating a disease in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a fusion protein of the invention in a pharmaceutically acceptable form. In one embodiment, the disease is an inflammatory disease. In a more specific embodiment, the inflammatory disease is inflammatory bowel disease, rheumatoid arthritis or idiopathic pulmonary fibrosis. In an even more specific embodiment, the inflammatory disease is inflammatory bowel disease. In one embodiment, the subject is a mammal, particularly a human.

Brief Description of Drawings

Fig. 1. Schematic representation of various antibody-IL-10 fusion versions. Panels (A) to (D) show IgG antibody-based profiles, and panels (E) to (G) show Fab fragment-based profiles. (A) "IgG-IL-10", a human IgG fused to the C-terminus of each IgG heavy chain (with an engineered Fc region to avoid effector functions, e.g., by amino acid substitutions L234A L235A (LALA) P329G) is an IL-10 molecule (wild-type human IL-10 cytokine sequence) (the IL-10 molecules on both heavy chains dimerize within the same IgG molecule). Linker between heavy chain and IL-10: for example (G)₄S)₄20 Polymer. (B) "IgG-Single chain (sc) IL-10", there is a single chain IL-10 dimer (scIL-10) fused to human IgG at the C-terminus of one of the IgG heavy chains (with an engineered Fc region to avoid effector functions and combining one "node" heavy chain and one "hole" heavy chain to drive both heterodimerization). The linker between heavy and single chain IL-10: for example (G)₄S)₃15 polymer. (C) "IgG-IL-10M 1", there is an engineered monomeric IL-10 molecule fused to human IgG at the C-terminus of one of the IgG heavy chains (with an engineered Fc portion to avoid effector function and combining one "node" heavy chain and one "hole" heavy chain to drive heterodimerization of the two). Linker between heavy chain and monomeric IL-10: for example (G)₄S)₃15 polymer. (D) "IgG- (IL-10M1)₂", a human IgG with one IL-10 monomer fused to the C-terminus of each IgG heavy chain (with an engineered Fc portion to avoid effector function) (monomeric IL-10 molecules on both heavy chains are not dimerized). Linker between heavy chain and IL-10: for example (G)₄S)₃15 polymer linker. (E) "Fab-IL-10", a Fab fragment with an IL-10 molecule (wild-type human IL-10 cytokine sequence) fused to the C-terminus of the Fab heavy chain (two of these fusions form a homodimeric active molecule via the IL-10 moiety by dimerization). Linker between heavy chain and IL-10: for example (G)₄S)₃15 polymer. (F)) "Fab-scIL-10-Fab", a tandem Fab fragment interrupted by a single chain IL-10 dimer (i.e., two IL-10 molecules passing through, for example (G)₄S)₄A 20-mer linker was attached and inserted between the C-terminus of the first Fab heavy chain (HC1) and the N-terminus of the second Fab heavy chain (HC2), resulting in a single peptide chain comprising HC 1-IL-10-HC 2). Two light chains (which may be identical to those used for other constructs) are paired with these two heavy chains. (G) "Fab-IL-10M 1-Fab", a tandem Fab fragment interrupted by an engineered monomeric IL-10 molecule. This pattern is identical to (F) except for the monomeric IL-10 moiety.

Fig. 2. Purification of an IgG-IL-10 construct based on FAP targeting 4B9 (see SEQ ID NO 25 and 27). (A) Elution profile of protein a purification step. (B) Elution profile of size exclusion chromatography step. (C) Analytical SDS-PAGE of the final product (reduced (R): NuPAGE Novex Bis-Tris Mini gel, Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-acetate, Invitrogen, Tris-acetate running buffer). M: and (4) marking the size. (D) Analytical size exclusion chromatography of the final product on a Superdex200 column. The monomer content was 99.8%.

Fig. 3. Purification of an IgG-scIL-10 construct (see SEQ ID NO 7, 11 and 13) based on FAP targeting 4G 8. (A) Elution profile of protein a purification step. (B) Elution profile of the size exclusion chromatography step (expected product is indicated by dashed box). (C) Analytical SDS-PAGE of the final product (reduced (R): NuPAGE Novex Bis-Tris Mini gel, Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-acetate, Invitrogen, Tris-acetate running buffer); the extra lower MW band on the non-reducing gel probably represents a half molecule consisting of one heavy and light chain. (D) Analytical size exclusion chromatography of the final product on a TSKgel G3000SW XL column. The monomer content was 80.6%.

Fig. 4. Purification of an IgG-IL-10M1 construct (see SEQ ID NO 7, 13 and 15) based on FAP targeting 4G 8. (A) Elution profile of protein a purification step. (B) Elution profile of size exclusion chromatography step. (C) Analytical SDS-PAGE of the final product (reduced (R): NuPAGE Novex Bis-Tris Mini gel, Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-acetate, Invitrogen, Tris-acetate running buffer). (D) Analytical size exclusion chromatography of the final product on a Superdex200 column. The monomer content was 98.2%.

Fig. 5. IgG- (IL-10M1) based on FAP targeting 4B9₂Purification of the constructs (see SEQ ID NO 25 and 29). (A) Elution profile of protein a purification step. (B) Elution profile of size exclusion chromatography step. (C) LabChipGX (Caliper) analysis of the final product. (D) Analytical size exclusion chromatography of the final product on a TKSgel G3000SW XL column. The monomer content is 100%.

Fig. 6. Purification of Fab-IL-10 constructs (see SEQ ID NO 25 and 31) based on FAP targeting 4B 9. (A) Elution profile of protein a purification step. (B) Elution profile of size exclusion chromatography step. (C) Analytical SDS-PAGE of the final product (reduced (R): NuPAGE Novex Bis-Tris Mini gel, Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-acetate, Invitrogen, Tris-acetate running buffer). (D) Analytical size exclusion chromatography of the final product on a Superdex200 column. The monomer content was 92.9%.

Fig. 7. Purification of FAP targeting 4G8 based Fab-scIL-10-Fab constructs (see SEQ ID NOs 7 and 21). (A) Elution profile of protein a purification step. (B) Elution profile of size exclusion chromatography step. (C) Analytical SDS-PAGE of the final product (reduced (R): NuPAGE Novex Bis-Tris Mini gel, Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-acetate, Invitrogen, Tris-acetate running buffer). (D) Analytical size exclusion chromatography of the final product on a Superdex200 column. The monomer content is 100%.

Fig. 8. Purification of FAP targeting 4G 8-based Fab-IL-10M1-Fab fusions (see SEQ ID NOs 7 and 23). (A) Elution profile of protein a purification step. (B) Elution profile of size exclusion chromatography step. (C) Analytical SDS-PAGE of the final product (reduced (R): NuPAGE Novex Bis-Tris Mini gel, Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-acetate, Invitrogen, Tris-acetate running buffer). (D) Analytical size exclusion chromatography of the final product on a Superdex200 column. The monomer content is 100%.

Fig. 9. SPR assay setup on ProteOn XPR 36. (A) Anti-pentahis IgG (capture agent) was covalently immobilized on GLM chip by amine coupling followed by capture of FAP (ligand) followed by injection of anti-FAP antibody-IL-10 fusion construct (analyte). (B) Biotinylated human IL-10R1 (ligand) was immobilized on a neutravidin derivatized sensor chip (NLC), followed by injection of an anti-FAP antibody-IL-10 fusion construct (analyte).

Fig. 10. Suppression of monocyte IL-6 production by different antibody-IL-10 fusion proteins. 4G8Fab-IL-10(B) or 4G8IgG-IL-10(A) was immobilized on cell culture plates coated with different concentrations of recombinant human FAP, after which monocytes and 100ng/ml LPS as stimulator were added for 24 hours. The concentration of IL-6 in the supernatant was then measured. The same data in table 8 are plotted, but in a different comparison.

Fig. 11. Comparison of Size Exclusion Chromatography (SEC) profiles of Fab-IL-10 and IgG-IL-10 versions. The arrows indicate the desired dimer product, aggregates are indicated by dashed circles, and monomers are indicated by solid circles. In contrast to the Fab-IL-10 format, the IgG-IL-10 format does not generate monomers or "half molecules" due to the covalent homodimerization of the disulfide linkages of its heavy chains.

Fig. 12. Biochemical characterization of IL-10-his wild-type cytokine (SEQ ID NO: 90). A) Analytical SDS-PAGE of the final product (NuPAGE Novex Bis-Tris Mini gel (Invitrogen), TRIS-glycine sample buffer, MOPS running buffer; reduced (R) and non-reduced (NR) SDS-PAGE; B) the final product was subjected to analytical size exclusion chromatography on a Superdex 75,10/300GL column.

FIG. 13. Biochemical characterization of IL-10(I87A) -his mutant cytokine (SEQ ID NO: 92). A) Analytical SDS-PAGE of the final product (NuPAGE Novex Bis-Tris Mini gel (Invitrogen), TRIS-Glycine sample buffer, MES running buffer; reduced (R) and non-reduced (NR) SDS-PAGE; B) the final product was subjected to analytical size exclusion chromatography on a Superdex200, 10/300GL column.

Fig. 14. Biochemical characterization of IL-10(R24A) -his mutant cytokine (SEQ ID NO: 94). A) Analytical SDS-PAGE of the final product (NuPAGE Novex Bis-Tris Mini gel (Invitrogen), TRIS-Glycine sample buffer, MES running buffer; reduced (R) and non-reduced (NR) SDS-PAGE; B) the final product was subjected to analytical size exclusion chromatography on a Superdex200, 10/300GL column.

Fig. 15. SPR assay setup on ProteOn XPR 36. Biotinylated IL-10R1-Fc (ligand) was immobilized on the NLC chip by neutravidin capture followed by injection of IL-10-his cytokine (analyte).

Detailed Description

Definition of

Unless otherwise defined below, terms are used herein as generally used in the art.

"fibroblast activation protein" (abbreviated FAP, also known as Seprase (EC 3.4.21)) refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed FAP, as well as any form of FAP that results from processing in a cell. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. In one embodiment, the antibody of the invention is capable of specifically binding to human, mouse and/or cynomolgus FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 128) or NCBI (www.ncbi.nlm.nih.gov /) RefSeq NP _ 004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid and nucleotide sequences of His-tagged human FAP ECD are shown in SEQ ID NOs 81 and 82, respectively. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 107) or NCBI RefSeq NP _ 032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NOs 83 and 84 show the amino acid and nucleotide sequences of the His-tagged mouse FAP ECD, respectively. SEQ ID NOs 85 and 86 show the amino acid and nucleotide sequences of the His-tagged cynomolgus FAP ECD, respectively.

The "IL-10 receptor" (abbreviated IL-10R) is a natural transmembrane receptor for IL-10 and is composed of IL-10R1 (or IL-10R α) and IL-10R2 (or IL-10R β) subunits.

By "human IL-10R 1" (sometimes also referred to as IL-10 receptor subunit α) is meant the protein described in UniProt accession No. Q13651 (version 115), in particular the extracellular domain of said protein extending from full sequence amino acid position 22 to amino acid position 235. SEQ ID NO 87 and 88 show the amino acid and nucleotide sequences, respectively, of human IL-10R1ECD fused to a human Fc region.

As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising an antibody and an IL-10 molecule, wherein the components of the fusion protein are linked to each other by peptide bonds, either directly or via a peptide linker. For clarity, the individual peptide chains of the antibody building blocks of the fusion proteins may be non-covalently linked, for example by disulfide bonds.

By "fusion" is meant that the components are linked by peptide bonds, either directly or via one or more peptide linkers.

By "specific binding" is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a particular antigen can be measured via enzyme-linked immunosorbent assays (ELISAs) or other techniques well known to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljebelad et al, Glyco J17, 323-containing 329(2000)), as well as conventional binding assays (Heeley, Endocr Res 28, 217-containing 229 (2002)). In one embodiment, the degree of binding of the antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen as measured, for example, by SPR. In certain embodiments, an antigen-binding antibody has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or moreSmall, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (K)_D)。

"affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be in terms of dissociation constant (K)_D) Expressed as dissociation and association rate constants (k, respectively)_DissociationAnd k_{Bonding of}) The ratio of (a) to (b). As such, equal affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. One particular method for measuring affinity is Surface Plasmon Resonance (SPR).

"reduced binding", e.g. reduced binding to an IL-10 receptor or an Fc receptor, refers to a reduction in affinity of the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes a decrease in affinity to 0 (or below the detection limit of the analytical method), i.e. complete elimination of the interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the corresponding interaction.

As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule in vitro to an intact antibody that comprises a portion of the intact antibody that binds to an antigen that is bound to the intact antibody. Examples of antibody fragments includeBut are not limited to Fv, Fab '-SH, F (ab')₂For reviews of certain antibody fragments see Hudson et al, Nat Med 9,129-134(2003) for reviews of scFv fragments see, e.g., Pl ü ckthun, in the Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315(1994), see also WO 93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458 for Fab and F (ab') comprising salvage receptor binding epitope residues and having an extended in vivo half-life₂See U.S. Pat. No.5,869,046 for a discussion of fragments. Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Tri-and tetrabodies are also described in Hudson et al, Nat Med 9, 129-. Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1). Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and generation by recombinant host cells (e.g., e.coli or phage), as described herein.

The terms "full-length antibody," "intact antibody," and "full antibody" are used interchangeably herein to refer to an antibody having a structure that is substantially similar to a native antibody structure.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has one variable region (VH), also known as variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as heavy chain constant regions. Similarly, from N-terminal to C-terminalEach light chain has a variable region (VL), also known as the variable light or light chain variable domain, followed by a light chain constant domain (CL), also known as the light chain constant region, the heavy chains of antibodies can be classified into one of five types, some of which can be further divided into subtypes, e.g., gamma (ig), called α (IgA), (IgD), (IgE), gamma (IgG), or mu (IgM)₁(IgG₁)、γ₂(IgG₂)、γ₃(IgG₃)、γ₄(IgG₄)、α₁(IgA₁) And α₂(IgA₂). Based on the amino acid sequence of its constant domains, the light chains of antibodies can be classified into one of two types called kappa (κ) and lambda (λ).

As used herein, a "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising the VL domain of a light chain and a constant domain (CL) and the VH domain of a heavy chain and a first constant domain (CH 1).

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

"IgG class antibody" refers to an antibody having the structure of a naturally occurring immunoglobulin G (IgG) molecule. The antibody heavy chain of an IgG class antibody has the domain structure VH-CH1-CH2-CH 3. The antibody light chain of an IgG class antibody has the domain structure VL-CL. An IgG class antibody consists essentially of two Fab fragments and one Fc domain connected via an immunoglobulin hinge region.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology,6^thed., W.H.Freeman Co., page 91 (2007). Is singleThe VH or VL domain may be sufficient to confer antigen binding specificity.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or which forms structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody comprises 6 HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from Complementarity Determining Regions (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196,901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 50-65 of 31-35B, H2 of H1, and 95-102 of H3 (Kabat et al, Sequences of Proteins of immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md (1991)). In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDR is contained within a CDR region called the shortened-CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) exist at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1, and 95-102 of H3 (see Almagro and Fransson, Front. biosci.13,1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra (referred to as "Kabat numbering").

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of 4 FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an antibody heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises IgG CH2 and IgG CH3 domains. The "CH 2 domain" of the human IgG Fc region typically extends from approximately the amino acid residue at position 231 to approximately the amino acid residue at position 340. In one embodiment, the carbohydrate chain is attached to a CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH 3 domain" comprises a stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about the amino acid residue at position 341 to about the amino acid residue at position 447 in an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "bump" ("knob") introduced in one of its strands and a corresponding "cavity" ("hole") introduced in its other strand; see U.S. Pat. No.5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains, as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or 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 specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as EU index, as described in Kabat et al, Sequences of Proteins of immunological interest,5th ed.

The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, cell surface receptor (e.g., B cell receptor) downregulation, and B cell activation.

An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc region of an antibody, initiates a signaling event that stimulates cells bearing the receptor to perform effector function. Activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89). One specific activating Fc receptor is human Fc γ RIIIa (see UniProt accession number P08637 (version 141)).

"native IL-10" (also referred to as "wild-type IL-10") means a naturally occurring IL-10, as opposed to a "modified" or "mutant IL-10" which has been modified from the naturally occurring IL-10, for example, in order to alter one or more of its characteristics, such as stability or receptor binding affinity. Modified or mutant IL-10 molecules may, for example, comprise modifications in the amino acid sequence, such as amino acid substitutions, deletions, or insertions. For example, a modified IL-10 molecule with increased stability in monomeric form has been described (Josephson et al, J Biol Chem 275,13552-13557 (2000)).

Native IL-10 is a homodimer consisting of two alpha-helical monomer domains. The sequence of the native human IL-10 monomer domain is shown in SEQ ID NO: 1. thus, an "IL-10 monomer" is a protein that is substantially similar in sequence and/or structure to the monomer domain of native IL-10.

"stable" or "stability" when used in reference to a protein means that the structural integrity (e.g., its secondary structure) of the protein is retained.

"functional" when used in reference to a protein means that the protein is capable of mediating a biological function, particularly a biological function that would be mediated by a corresponding protein found in nature (e.g., native IL-10). In the case of IL-10, the biological functions may include activation of IL-10 receptor signaling in cells expressing the IL-10 receptor (e.g., monocytes), suppression of proinflammatory cytokine(s) (such as TNF α, IL-1, IL-6, IL-12, IL-2, and/or INF γ) secretion, inhibition of MHC II expression, and upregulation of costimulatory molecules (such as CD80 and/or CD 86).

The term "peptide linker" is a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable, non-immunogenic linker peptides include, for example, (G)₄S)_n、(SG₄)_nOr G₄(SG₄)_nA peptide linker. "n" is generally a number between 1 and 10, typically between 2 and 4.

"node-in-pocket modification" refers to a modification within the interface between two antibody heavy chains in the CH3 domain, wherein i) in the CH3 domain of one heavy chain one amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance ("segment") within the interface in the CH3 domain of one heavy chain, which can be placed in a cavity ("pocket") within the interface in the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain one amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity ("pocket") within the interface of the second CH3 domain, wherein the protuberance ("segment") within the interface in the first CH3 domain can be placed. In one embodiment, a "node-into-hole modification" comprises the amino acid substitution T366W and optionally amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitution T366S, L368A, Y407V and optionally Y349C in the other of the antibody heavy chains. Techniques for entering the acupoints are described, for example, in US 5,731,168; US 7,695,936; ridgway et al, Prot Eng9,617-621(1996) and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") in the interface of the second polypeptide such that the protuberance can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of the same or similar size as the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine). The introduction of two cysteine residues at positions S354 and Y349, respectively, results in the formation of a disulfide bridge between the two antibody heavy chains in the Fc region, further stabilizing the dimer (Carter, J immunological Methods 248,7-15 (2001)).

Amino acid "substitution" refers to the replacement of one amino acid with another amino acid in a polypeptide. In one embodiment, the amino acid is replaced with another amino acid having similar structural and/or chemical properties, such as a conservative amino acid substitution. "conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acidAcids and glutamic acid. Non-conservative substitutions may require the exchange of a member of one of these classes for a member of another class. For example, an amino acid substitution can also result in the substitution of one amino acid with another amino acid having a different structural and/or chemical property, e.g., the substitution of an amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., basic). Amino acid substitutions can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may also be useful. Various nomenclature may be used herein to refer to the same amino acid substitution. For example, substitution of the heavy chain of an antibody at position 329 from proline to glycine may be at 329G, G329, G₃₂₉P329G, or Pro329 Gly.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared. For purposes herein, however, the sequence comparison computer program ALIGN-2 is used to generate% amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was authored by Genentech, inc, and the source code has been submitted with the user document to the us copyright Office (u.s.copyright Office), Washington d.c., 20559, which is registered under us copyright registration No. txu 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program should be compiled for use in a UNIX operating system, including the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were unchanged. In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a to/with/against a given amino acid sequence B (or which may be expressed in terms of phrases as a given amino acid sequence a having or comprising a particular% amino acid sequence identity to/with/against a given amino acid sequence B) is calculated as follows:

fraction X/Y X100

Wherein X is the number of amino acid residues scored as an identical match by the sequence alignment program ALIGN-2 in an alignment of said program pairs A and B, and wherein Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. Unless explicitly stated otherwise, all% amino acid sequence identity values used herein were obtained as described in the preceding paragraph using the ALIGN-2 computer program.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. The nucleotide sequence may be interrupted by non-nucleotide building blocks. The polynucleotide may comprise modifications made post-synthetically, such as conjugation to a label.

The term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification (e.g., glycosylation) of a polypeptide.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived therefrom (regardless of the number of passages). Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell. Host cells are any type of cell system that can be used to produce the fusion proteins of the invention. Host cells include cultured cells, for example, mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells, plant cells and the like, and also include cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues.

An "effective amount" of an agent refers to an amount necessary to effect a physiological change in the cell or tissue to which it is administered.

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount (in a necessary dose and for a necessary time) effective to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of the disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other ingredients that have unacceptable toxicity to the subject to whom the formulation will be administered.

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

As used herein, "treatment" refers to an attempt to alter the natural course of disease in the treated individual (and grammatical variations thereof), and may be a clinical intervention performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or delay the progression of a disorder.

Fusion proteins of the invention

The present invention provides novel antibody-IL-10 fusion proteins with particularly advantageous properties, such as producibility, stability, binding affinity, biological activity, targeting efficiency, and reduced toxicity.

In a first aspect, the invention provides a fusion protein of an IgG class antibody and a mutant IL-10 molecule, wherein the fusion protein comprises two identical heavy chain polypeptides and two identical light chain polypeptides, and wherein the mutant IL-10 molecule comprises an amino acid mutation that reduces the binding affinity of the mutant IL-10 molecule to the IL-10 receptor as compared to the wild-type IL-10 molecule. In one embodiment, the heavy chain polypeptides each comprise an IgG class antibody heavy chain and a mutant IL-10 monomer. In a more specific embodiment, the mutant IL-10 monomer is fused at its N-terminus to the C-terminus of the heavy chain of the IgG class antibody, optionally via a peptide linker. In one embodiment, the heavy chain polypeptides each consist essentially of an IgG class antibody heavy chain, a mutant IL-10 monomer, and an optional peptide linker. In one embodiment, the light chain polypeptides each comprise an IgG class antibody light chain. In one embodiment, the light chain polypeptides each consist essentially of an IgG class antibody light chain. The presence of antibodies of the IgG class confers advantageous pharmacokinetic properties to the fusion proteins of the invention compared to fusion proteins based on antibody fragments, including a prolonged serum half-life (due to recirculation via binding to FcRn and the molecular size well beyond the threshold of renal filtration). The presence of IgG class antibodies also allows for simple purification of the fusion protein by, for example, protein a affinity chromatography. Surprisingly, as shown in the examples comparing the IgG-based IgG-IL-10 fusion proteins of the invention with the corresponding fusion protein based on Fab fragments (Fab-IL-10), the presence of antibodies of the IgG class also improves the biological activity of the fusion protein when binding to its target antigen. The use of identical heavy (and light) chain polypeptides allows for simple generation of fusion proteins, avoids the formation of unwanted side products and circumvents the need for modifications, such as knob-to-hole modifications, that promote heterodimerization of non-identical heavy chains.

In one embodiment, the mutant IL-10 molecule is a human IL-10 molecule. In one embodiment, the mutant IL-10 molecule comprises an amino acid mutation that reduces the binding affinity of the mutant IL-10 molecule for the IL-10 receptor by at least 2-fold, at least 5-fold, or at least 10-fold as compared to the wild-type IL-10 molecule. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the mutant IL-10 molecule comprises an amino acid substitution at a position corresponding to residue 87 of human IL-10(SEQ ID NO: 1). In a specific embodiment, the amino acid substitution is I87A. As shown in the examples, this amino acid substitution reduces binding affinity for IL-10R1, but maintains substantial immunosuppressive activity of the mutant IL-10 molecule. In addition, it is expected to reduce the unwanted immunostimulation of IL-10.

In one embodiment, the mutant IL-10 molecule is a homodimer of two mutant IL-10 monomers. In one embodiment, the mutant IL-10 molecule comprises an amino acid mutation in each of the two mutant IL-10 monomers comprising it that reduces the binding affinity of the mutant IL-10 molecule for the IL-10 receptor as compared to the wild-type IL-10 molecule. In one embodiment, the mutant IL-10 molecule comprises only a single amino acid mutation in each of the two mutant IL-10 monomers comprising it, which amino acid mutation reduces the binding affinity of the mutant IL-10 molecule for the IL-10 receptor as compared to the wild-type IL-10 molecule.

In some embodiments, the mutant IL-10 monomer is a human IL-10 monomer. In one embodiment, the mutant IL-10 monomer comprises an amino acid substitution. In one embodiment, the mutant IL-10 monomer comprises an amino acid substitution at a position corresponding to residue 87 of human IL-10(SEQ ID NO: 1). In a specific embodiment, the amino acid substitution is I87A. In a specific embodiment, the mutant IL-10 monomer comprises SEQ ID NO: 98. In one embodiment, the mutant IL-10 monomer comprised in the heavy chain polypeptide forms a functional homodimeric mutant IL-10 molecule. This type of fusion protein is particularly advantageous in that the two IL-10 monomers form a fully functional, biologically active IL-10 dimer. In addition, in contrast to fusion proteins based on antibody fragments, dimerization occurs not only between IL-10 monomers in the fusion proteins of the invention, but also between the antibody heavy chains fused to the monomers. Thus, the fusion proteins of the invention comprise IL-10 dimers that have a reduced tendency to break down into two monomers compared to, for example, the Fab-IL-10 fusion proteins described herein (see FIG. 11). Importantly, this version of the fusion protein is also superior in biological activity to the other fusion protein versions described herein.

In one embodiment, the IgG class antibody is IgG₁Subclass antibody. In one embodiment, the IgG class antibody is a human antibody, i.e., it comprises human variable and constant regions. Exemplary human IgG₁The sequences of the heavy and light chain constant regions are shown in SEQ ID NOs 79 and 80, respectively. In one embodiment, theIgG class antibodies comprise a human Fc region, in particular a human IgG Fc region, more particularly a human IgG₁An Fc region. In one embodiment, the IgG class antibody is a full length antibody. In one embodiment, the IgG class antibody is a monoclonal antibody.

Although the Fc domain of IgG class antibodies confers the fusion protein with favorable pharmacokinetic properties, including a long serum half-life (contributing to better accumulation in the target tissue and favorable tissue-to-blood distribution ratio), it may at the same time cause unwanted targeting of the fusion protein to Fc receptor expressing cells, rather than to preferred antigen bearing cells. Furthermore, activation of the Fc receptor signaling pathway can cause cytokine release leading to (pro-inflammatory) cytokine receptor activation and serious side effects when administered systemically. Thus, in one embodiment, the IgG class antibody comprises a modification that reduces the binding affinity of the antibody to an Fc receptor as compared to a corresponding IgG class antibody without the modification. In a particular embodiment, the Fc receptor is an fey receptor, in particular a human fey receptor. Binding affinity to Fc receptors can be readily determined, for example by ELISA or by Surface Plasmon Resonance (SPR), using standard instrumentation such as BIAcore instrumentation (GE Healthcare) and Fc receptors, such as can be obtained by recombinant expression. A specific exemplary and illustrative embodiment for measuring binding affinity is described below. According to one embodiment, the ligand (Fc receptor) immobilized on a CM5 chip was used at 25 deg.CThe T100 instrument (GE Healthcare) measures binding affinity for Fc receptors by surface plasmon resonance. Briefly, carboxymethylated dextran biosensor chips (CM5, GEHealthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide hydrochloride (NHS) according to the supplier's instructions. The recombinant ligand was diluted to 0.5-30. mu.g/ml with 10mM sodium acetate pH 5.5, followed by injection at a flow rate of 10. mu.l/min to achieve approximately 100 and 5000 Response Units (RU) of conjugated protein. After injection of ligand, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, 3 to 5-fold serial dilutions (ranging between about 0.01nM and 300 nM) of antibody in HBS-EP + (GE Healthcare, 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) were injected at 25 ℃ at a flow rate of about 30-50. mu.l/min. Using a simple one-to-one Langmuir (Langmuir) binding model by simultaneous fitting of binding and dissociation sensorgrams(s) ((R))T100Evaluation software Evaluation 1.1.1) calculation of the binding Rate (k)_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D). See, e.g., Chen et al, J Mol Biol 293,865- & 881 (1999). Alternatively, cell lines known to express specific Fc receptors, such as NK cells expressing Fc γ IIIa receptors, can be used to assess the binding affinity of an antibody to an Fc receptor.

In one embodiment, the modification comprises one or more amino acid mutations that reduce the binding affinity of the antibody for an Fc receptor. In one embodiment, the amino acid mutation is an amino acid substitution. Typically, the same amino acid mutation or mutations are present in each of the two heavy chains of an antibody. In one embodiment, the amino acid mutation reduces the binding affinity of the antibody to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the antibody to the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the antibody to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, the IgG class antibody exhibits a binding affinity for an Fc receptor of less than 20%, particularly less than 10%, more particularly less than 5%, compared to a corresponding IgG class antibody without the modification.

In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is selected from the group consisting of: fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32) and Fc α RI (CD 89). In a specific embodiment, the Fc receptor is an Fc γ receptor, more specifically an Fc γ RIIIa, Fc γ RI or Fc γ RIIa receptor. Preferably, the binding affinity for each of these receptors is reduced. In an even more specific embodiment, the Fc receptor is Fc γ IIIa, in particular human Fc γ IIIa. In some embodiments, the binding affinity to complement components, particularly to C1q is also reduced. In one embodiment, the binding affinity for neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the antibody exhibits greater than about 70% of the binding affinity of the unmodified form of the antibody for FcRn, i.e., retains the binding affinity of the antibody for the receptor. IgG class antibodies comprised in the fusion proteins of the invention may exhibit such affinity of greater than about 80% and even greater than about 90%.

In one embodiment, the modification that reduces the binding affinity of the antibody to an Fc receptor is in the Fc region of an IgG class antibody, particularly the CH2 region. In one embodiment, the IgG-class antibody comprises an amino acid substitution at antibody heavy chain position 329(EU numbering). In a more particular embodiment, the amino acid substitution is P329A or P329G, in particular P329G. In one embodiment, the IgG class antibody comprises amino acid substitutions at antibody heavy chain positions 234 and 235(EU numbering). In a specific embodiment, the amino acid substitutions are L234A and L235A (LALA). In one embodiment, the IgG-class antibody comprises an amino acid substitution at antibody heavy chain position 329(EU numbering) and a further amino acid substitution at a position selected from antibody heavy chain positions 228, 233, 234, 235, 297 and 331. In a more specific embodiment, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the IgG-class antibody comprises amino acid substitutions at antibody heavy chain positions P329, L234 and L235(EU numbering). In a more specific embodiment, the IgG-class antibody comprises the amino acid substitutions L234A, L235A and P329G (LALA P329G) in the antibody heavy chain. This combination of amino acid substitutions is almost particularly effective in abolishing Fc γ receptor binding of human IgG class antibodies, as described in PCT publication No. wo 2012/130831, incorporated herein by reference in its entirety. PCT publication No. wo 2012/130831 also describes methods of making such modified antibodies and methods for determining properties thereof, such as Fc receptor binding or effector function.

Antibodies comprising modifications in the heavy chain of an antibody can be made by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide change can be verified by, for example, sequencing.

Antibodies comprising modifications that reduce Fc receptor binding typically have reduced effector function, particularly reduced ADCC, as compared to the corresponding unmodified antibody. Thus, in one embodiment, the modification that reduces the binding affinity of an IgG class antibody to an Fc receptor reduces the effector function of the IgG class antibody. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, ADCC is reduced to less than 20% of the ADCC induced by the corresponding IgG-class antibody without the modification. Effector function of an antibody can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83, 7059-; U.S. Pat. Nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assays (CellTechnology, inc., Mountain View, CA); and CytotoxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al.Proc NatlAcad Sci USA95, 652-. In some embodiments, the binding of IgG-class antibodies to complement components (particularly C1q) is also reduced. Thus, Complement Dependent Cytotoxicity (CDC) may also be reduced. A C1q binding assay may be performed to determine whether an antibody is capable of binding C1q and thus has CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J Immunol Methods 202,163 (1996); Cragg et al, Blood 101,1045-1052 (2003); and Cragg and Glennie, Blood 103,2738-2743 (2004)).

Antibodies with reduced Fc receptor binding and/or effector function in addition to the IgG class antibodies described herein above and in PCT publication No. wo 2012/130831 also include those that replace one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. patent No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acids 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

IgG₄Subclass antibody display and IgG₁Reduced binding affinity to Fc receptors and reduced effector function compared to antibodies. Thus, in some embodiments, the IgG class antibody comprised in the fusion protein of the invention is IgG₄Subclass antibodies, particularly human IgG₄Subclass antibody. In one embodiment, the IgG is₄Subclass antibodies comprise an amino acid substitution in the Fc region at position S228, specifically amino acid substitution S228P. To further reduce its binding affinity to Fc receptors and/or its effector function, in one embodiment, the IgG₄Subclass antibodies comprise amino acid substitutions at position L235, specifically amino acid substitution L235E. In another embodiment, the IgG is₄Subclass antibodies comprise amino acid substitutions at position P329, specifically amino acid substitution P329G. In a particular embodiment, the IgG is₄Subclass antibodyAmino acid substitutions are included at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G. Such modified IgG₄Subclass antibodies and their Fc γ receptor binding properties are described in PCT publication No. wo 2012/130831, which is incorporated herein by reference in its entirety.

The antibodies of the invention combine a number of particularly advantageous properties, for example for therapeutic applications.

In one embodiment, the IgG-class antibody is capable of specifically binding to Fibroblast Activation Protein (FAP). FAP has been identified as a suitable target for the treatment of inflammatory diseases using the fusion proteins of the invention. In a specific embodiment, the fusion protein is capable of exhibiting an affinity constant (K) of less than 1nM, in particular less than 100pM, when measured by Surface Plasmon Resonance (SPR) at 25 ℃_D) Combining FAP. Described herein is a method of measuring binding affinity for FAP by SPR. In one embodiment, affinity of the fusion protein was measured by SPR at 25 ℃ with His-tagged antigen immobilized by anti-His antibody covalently coupled to GLM chip using ProteOn XPR36 instrument (Biorad)_D). In one illustrative method, the target protein (FAP) was captured via its H6 tag by covalently immobilized anti-pentahis IgG (Qiagen #34660, mouse monoclonal antibody) immobilized at high levels (up to about 5.000RU) at 30 μ Ι/min onto separate vertical channels of the GLM chip by simultaneously activating all channels with a freshly prepared mixture of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (shhs) for 5 minutes, followed by injection of 15 μ g/ml anti-pentahis IgG in 10mM sodium acetate buffer pH 4.5 for 180 seconds. The channel was blocked by injection of ethanolamine for 5 minutes. The His 6-tagged FAP was captured from a 5. mu.g/ml dilution in running buffer along the vertical channel at 30. mu.l/min for 60s to achieve a ligand density of between about 250 and 600 RU. In a single click kinetics assay setup (OSK), the fusion protein was injected as analyte along the horizontal channel at 100. mu.l/min in 5-fold serial dilutions ranging from 50 to 0.08 nM. The association phase was recorded for 180s and the dissociation phase for 600 s. In interactions exhibiting very slow off-ratesIn this case, the dissociation rate record was extended to 1800s to observe dissociation of the complexes. Running buffer (PBST) was injected along the sixth channel to provide an "on-line" blank as a reference. Binding rates (k) were calculated using a simple 1:1 Langmuir (Langmuir) binding model (ProteOnManager software version2.1) by simultaneous fitting of binding and dissociation sensorgrams_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D)。

In one embodiment, the FAP is human, mouse, and/or cynomolgus FAP. Preferably, the IgG-like antibodies comprised in the fusion protein of the invention are cross-reactive to human and cynomolgus monkey and/or mouse FAP, which can be studied in vivo, e.g. in cynomolgus monkey and/or mouse, prior to human use.

In a specific embodiment, the IgG-class antibody comprises SEQ ID NO: 37, cdr (hcdr)1 of heavy chain, SEQ ID NO: HCDR2 of 41, SEQ ID NO: 49 HCDR3 of SEQ ID NO: 53 light chain CDR (LCDR)1, SEQ ID NO: LCDR 2 of 57 and SEQ ID NO: LCDR 3 of 61. In an even more specific embodiment, the IgG-class antibody comprises SEQ ID NO: 63 and the heavy chain variable region (VH) of SEQ ID NO: 65 (VL) in the light chain. In another specific embodiment, the IgG-class antibody comprises SEQ ID NO: 37 HCDR1, SEQ ID NO: 43 HCDR2, seq id NO: 47 HCDR3, SEQ ID NO: 51, LCDR 1 of SEQ ID NO: LCDR 2 of 55 and SEQ ID NO: LCDR 3 of 59. In an even more specific embodiment, the IgG-class antibody comprises SEQ ID NO: 67 and SEQ ID NO: 69. As shown in the examples, these antibodies showed particularly strong binding affinity/avidity for human, mouse and cynomolgus FAP

In further specific embodiments, the IgG-class antibody comprises SEQ ID NO: 39 HCDR1 of SEQ id no: 45 HCDR2, SEQ ID NO: 49 HCDR3 of SEQ ID NO: 53 light chain cdr (lcdr)1, SEQ ID NO: LCDR 2 of 57 and SEQ ID NO: LCDR 3 of 61. In an even more specific embodiment, the IgG-class antibody comprises SEQ ID NO: 71 and the VH of SEQ ID NO: 73 VL. In another specific embodiment, the IgG-class antibody comprises SEQ ID NO: 37 HCDR1, SEQ ID NO: HCDR2 of 41, SEQ ID NO: 47 HCDR3, SEQ ID NO: 51, LCDR 1 of SEQ ID NO: LCDR 2 of 55 and SEQ ID NO: LCDR 3 of 59. In an even more specific embodiment, the IgG-class antibody comprises SEQ ID NO: 75 and the VH of SEQ ID NO: VL of 77.

In one embodiment, the fusion protein is capable of having an affinity constant (K) of about 100pM to about 10nM, particularly about 200pM to about 5nM, or about 500pM to about 2nM, when measured by SPR at 25 ℃_D) Binds to IL-10 receptor-1 (IL-10R 1). Described herein is a method of measuring binding affinity to IL-10R1 by SPR. In one embodiment, the affinity of the fusion protein was measured by SPR at 25 ℃ using a ProteOn XPR36 instrument (Biorad) with biotinylated IL-10R1 immobilized on NLC chip by neutravidin capture (K)_D). In one exemplary method, 400 to 1600RU of IL-10R1 was captured on neutravidin derivatized chip substrates along a vertical channel at a concentration of 10 μ g/ml and a flow rate of 30 μ l/sec for different contact times. Binding to biotinylated IL-10R1 was measured by injection at 100. mu.l/min in a horizontal orientation at six different analyte concentrations (50, 10, 2, 0.4, 0.08, 0nM), with an on rate recorded for 180s and an off rate recorded for 600 s. Running buffer (PBST) was injected along the sixth channel to provide an "on-line" blank as a reference. Binding rates (k) were calculated using a simple 1:1 Langmuir (Langmuir) binding model (ProteOn Manager software evolution 2.1) by simultaneous fitting of binding and dissociation sensorgrams_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D)。

In a specific embodiment, the IL-10R1 is human IL-10R 1. In one embodiment, the affinity constant (K) for binding to IL-10R1, as measured by SPR at 25 ℃_D) Greater than the affinity constant (K) for said binding FAP_D). In a specific embodiment, the K that binds IL-10R1_DK greater than the combined FAP_DAbout 1.5 times, about 2 times, about 3 times, or about 5 times.The fusion protein of the invention binds to FAP and K of IL-10R1_DThe specific ratios of values make them particularly suitable for efficiently targeting IL-10 to FAP-expressing tissues. Without wishing to be bound by theory, because their binding affinity for FAP is higher than their binding affinity for IL-10R1, the fusion proteins of the invention are less likely to bind to cells expressing IL-10R1 outside of the target tissue (e.g., in circulation) before reaching the target tissue expressing FAP.

In a particular aspect, the invention provides a human IgG capable of specifically binding FAP and comprising a modification₁Fusion proteins of subclass antibodies and mutant IL-10 molecules comprising amino acid mutations, with corresponding human IgG without said modifications₁Subclass antibody, the modification reduces the binding affinity of the antibody to the Fc receptor, and the amino acid mutation reduces the binding affinity of the mutant IL-10 molecule to the IL-10 receptor as compared to the wild-type IL-10 molecule, wherein the fusion protein comprises two identical heavy chain polypeptides (each comprising a fusion to human IgG at its N-terminus₁Mutant IL-10 monomers C-terminal to the subclass antibody heavy chain) and two identical light chain polypeptides. In one embodiment, the heavy chain polypeptide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO 96. In one embodiment, the light chain polypeptide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO. 25.

In a specific embodiment, the fusion protein comprises a sequence identical to SEQ ID NO:96 and a heavy chain polypeptide at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO:25, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain polypeptide. In yet another specific embodiment, the fusion protein comprises a sequence identical to SEQ ID NO:96 and two heavy chain polypeptides that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO:25, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

Polynucleotide

The invention also provides a polynucleotide encoding a fusion protein as described herein or an antigen-binding fragment thereof.

Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in SEQ ID NOs 26,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,89,97, and 99, including functional fragments or variants thereof.

The polynucleotide encoding the fusion protein of the invention may be expressed as a single polynucleotide encoding the entire fusion protein, or as multiple (e.g., two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides may associate via, for example, disulfide bonds or other means to form a functional fusion protein. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide from the heavy chain portion of an antibody. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antibody.

In one embodiment, the invention relates to a polynucleotide encoding a fusion protein of an IgG-class antibody and a mutant IL-10 molecule, or an antigen-binding fragment thereof, wherein said polynucleotide comprises a sequence encoding a variable region sequence as set forth in SEQ ID NO63, 65, 67, 69, 71, 73, 75, or 77. In another embodiment, the invention relates to a polynucleotide encoding a fusion protein of an IgG class antibody and a mutant IL-10 molecule or a fragment thereof, wherein said polynucleotide comprises a sequence encoding a polypeptide sequence as set forth in SEQ id no 25 or 96. In another embodiment, the invention further relates to a polynucleotide encoding a fusion protein of an IgG-class antibody and a mutant IL-10 molecule or fragment thereof, wherein said polynucleotide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence set forth in SEQ id no 26,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78, or 89. In another embodiment, the invention relates to a polynucleotide encoding a fusion protein of an IgG-class antibody and a mutant IL-10 molecule or a fragment thereof, wherein said polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO 2,6, 8, 10, 18, 26, 28, 30, 38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,89,97, or 99. In another embodiment, the invention relates to a polynucleotide encoding a fusion protein of an IgG-class antibody and a mutant IL-10 molecule, or a fragment thereof, wherein said polynucleotide comprises a sequence encoding a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO63, 65, 67, 69, 71, 73, 75, or 77. In another embodiment, the invention relates to a polynucleotide encoding a fusion protein of an IgG-class antibody and a mutant IL-10 molecule, or a fragment thereof, wherein said polynucleotide comprises a sequence encoding a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO 25 or 96. The present invention encompasses polynucleotides encoding fusion proteins of an IgG-class antibody and a mutant IL-10 molecule, or fragments thereof, wherein said polynucleotides comprise a sequence encoding the variable region sequence of SEQ ID NO63, 65, 67, 69, 71, 73, 75, or 77 and conservative amino acid substitutions. The invention also encompasses polynucleotides encoding fusion proteins of IgG-class antibodies and mutant IL-10 molecules, or fragments thereof, wherein said polynucleotides comprise a sequence encoding the polypeptide sequence of SEQ ID NO 25 or 96 and conservative amino acid substitutions.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.

Recombination method

The fusion protein of the invention can be obtained, for example, by solid phase peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the fusion proteins (fragments) (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector (preferably an expression vector) comprising one or more polynucleotides of the invention is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the fusion protein (fragment) and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, e.g., those described in Maniatis et al, Molecular CLONING: A Laboratory Manual, Cold Spring Harbor LABORATORY, N.Y. (1989); and Ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, Greene publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be a plasmid, part of a virus or may be a nucleic acid fragment. Expression vectors comprise an expression cassette in which a polynucleotide encoding a fusion protein (fragment) (i.e., a coding region) is cloned in operable association with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region (if present), but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides which are separated post-translationally or co-translationally into the final protein via proteolytic cleavage. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode a heterologous coding region, fused or unfused with a polynucleotide encoding a fusion protein (fragment) of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, if a promoter is capable of effecting transcription of a nucleic acid encoding a polypeptide, the promoter region will be in operable association with the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements such as enhancers, operators, repressors, and transcriptional termination signals can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter, in combination with intron-a), simian virus 40 (e.g., early promoter), and retroviruses (e.g., Rous (Rous) sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit beta globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (specifically, internal ribosome entry sites or IRES, also known as CITE sequences). The expression cassette may also comprise other features, such as an origin of replication and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs) or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

The polynucleotide and nucleic acid coding regions of the invention may be associated with additional coding regions that encode secretion or signal peptides that direct the secretion of the polypeptide encoded by the polynucleotide of the invention. For example, if secretion of the fusion is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the fusion protein of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein upon initiation of export of the growing protein chain across the rough endoplasmic reticulum. One of ordinary skill in the art knows that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of such a sequence that retains the ability to direct secretion of the polypeptide with which it is operably associated, is used. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase. The amino acid and nucleotide sequences of exemplary secretory signal peptides are shown in SEQ ID NOs 35 and 36, respectively.

DNA encoding short protein sequences that can be used to facilitate later purification (e.g., histidine tag) or to aid in labeling of the fusion protein can be incorporated into or at the end of the fusion protein (fragment) encoding polynucleotide.

In a particular embodiment, host cells are provided that comprise one or more polynucleotides of the invention. In certain embodiments, host cells comprising one or more vectors of the invention are provided. Polynucleotides and vectors may be incorporated herein, individually or in combination, separatelyAny of the features described for the polynucleotide and vector. In one such embodiment, the host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide encoding (part of) a fusion protein of the invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce a fusion protein of the invention or a fragment thereof. Host cells suitable for replication and to support expression of the fusion protein are well known in the art. Such cells may be transfected or transduced with a particular expression vector as appropriate, and a large number of vector-containing cells may be cultured for seeding a large-scale fermentor to obtain a sufficient amount of the fusion protein for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese Hamster Ovary (CHO), insect cells, and the like. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in production of polypeptides having partially or fully human glycosylation patterns. See Gerngross, NatBiotech 22, 1409-. Host cells suitable for the expression (glycosylation) of 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 for use with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may 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 (PLANTIBODIIES described for the production of antibodies in transgenic plants^TMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Another example of a mammalian host cell line that may be used is the monkey kidney CV1 line transformed by SV40 (COS-7) (ii) a Human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al, J Gen Virol 36,59(1977)), baby hamster kidney cells (BHK), mouse Sertoli (Sertoli) cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23,243-251 (1980)), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), Kiwi kidney cells (MDCK), bovine and murine hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, for example, in Mather et al, Annals N.Y. Acad Sci 383,44-68 (1982)), C5 cells and MRFS 35 cells. Other mammalian host cell lines that may be used include Chinese Hamster Ovary (CHO) cells, including dhfr^-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells such as mammalian culture cells, yeast cells, insect cells, bacterial cells, plant cells, and the like, but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising either the heavy chain or the light chain of an antibody can be engineered such that the other antibody chain is also expressed, such that the product of expression is an antibody having both a heavy chain and a light chain.

In one embodiment, a method of producing a fusion protein according to the invention is provided, wherein the method comprises culturing a host cell (as provided herein) comprising a polynucleotide encoding the fusion protein under conditions suitable for expression of the fusion protein, and recovering the fusion protein from the host cell (or host cell culture medium).

In the fusion protein of the present invention, the components (IgG class antibody and IL-10 molecule) are genetically fused to each other. The fusion proteins can be designed such that their components are fused to each other directly or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and the efficacy can be tested. Additional sequences are included to incorporate cleavage sites to separate the various components of the fusion protein, if desired, such as endopeptidase recognition sequences.

In certain embodiments, the fusion proteins of the invention comprise at least an antibody variable region that binds an antigen, such as FAP. The variable regions may form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods for generating polyclonal and monoclonal Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, antigen manual," Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be generated using solid phase peptide synthesis constructs, can be generated recombinantly (e.g., as described in U.S. patent No.4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see, e.g., U.S. patent No.5,969,108 to McCafferty).

Antibodies of any animal species may be used in the present invention. Non-limiting antibodies useful in the invention can be of murine, primate, or human origin. If the antibody is intended for human use, a chimeric form of the antibody may be used in which the constant regions of the antibody are from a human. Humanized or fully human forms of antibodies can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No.5,565,332 to Winter). Humanization can be achieved by a variety of methods, including but not limited to (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions, with or without retention of critical framework residues (e.g., those important for retaining better antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (c) grafting intact non-human variable domains, but "cloak" them with human-like moieties by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front Biosci 13,1619-1633(2008), and also described, for example, in Riechmann et al, Nature 332,323-329 (1988); queen et al, Proc Natl Acad Sci USA 86, 10029-; U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; jones et al, Nature 321,522-525 (1986); morrison et al, Proc Natl Acad Sci 81,6851-6855 (1984); morrison and Oi, AdvImmunol 44,65-92 (1988); verhoeyen et al, Science 239, 1534-; padlan, Molec Immun 31(3),169-217 (1994); kashmiri et al, Methods36, 25-34(2005) (describing SDR (a-CDR) grafting); padlan, Mol Immunol 28,489-498(1991) (description "resurfacing"); dall' Acqua et al, Methods36, 43-60(2005) (description "FR shuffling"); and Osbourn et al, Methods36,61-68(2005) and Klimka et al, Br J Cancer 83,252-260(2000) (describing the "guided selection" approach to FR shuffling). Particular antibodies according to the invention are human antibodies. Human antibodies and human variable regions can be generated using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5,368-74(2001), and Lonberg, Curr Opin Immunol 20, 450-. The human variable region can form part of and be derived from human monoclonal antibodies produced by the hybridoma method (see, e.g., monoclonal antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)). Human antibodies and Human variable regions can also be prepared by administering immunogens to transgenic animals that have been modified to produce fully Human antibodies or fully antibodies with Human variable regions in response to antigen challenge (see, e.g., Lonberg, Nat Biotech 23,1117-1125 (2005). Human antibodies and Human variable regions can also be generated by isolating Fv clone variable region sequences selected from Human-derived phage display libraries (see, e.g., Hoogenboom et al. in methods in Molecular Biology 178,1-37 (O' Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352,624-628 (1991)). phage typically display antibody fragments, either as single chain Fv fragments (scFv) or as Fab fragments; see, for a detailed description of the preparation of antibodies by phage display in WO 2012/020006, are hereby incorporated by reference in their entirety

In certain embodiments, the antibodies comprised in the fusion proteins of the present invention are engineered to have enhanced binding affinity according to methods disclosed, for example, in PCT publication WO 2012/020006 (see examples relating to affinity maturation) or U.S. patent application publication No.2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of an antibody of the invention to bind a particular antigenic determinant may be measured via enzyme-linked immunosorbent assays (ELISAs) or other techniques well known to those skilled in the art, such as surface plasmon resonance techniques (Liljebelad, et., Glyco J17, 323-containing 329(2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-containing 229 (2002)). Competition assays can be used to identify antibodies that compete with a reference antibody for binding to a particular antigen, e.g., antibodies that compete with 4G8 antibody for binding to FAP. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by the reference antibody. A detailed exemplary method for locating epitopes to which antibodies bind is provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular biology vol.66(Humana Press, Totowa, NJ). In one exemplary competition assay, an immobilized antigen (e.g., FAP) is incubated in a solution comprising a first labeled antibody (e.g., 4G8 antibody) that binds the antigen and a second unlabeled antibody that is tested for its competition with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen was incubated in a solution comprising the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample relative to the control sample, this is an indication that the second antibody is competing with the first antibody for binding to the antigen. See Harlowand Lane (1988) Antibodies, laboratory Manual ch.14(Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y.).

Fusion proteins prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, fusion protein-bound antibodies, ligands, receptors or antigens can be used. For example, for affinity chromatography purification of the fusion protein of the present invention, a matrix having protein a or protein G may be used. Fusion proteins can be separated using sequential protein a or G affinity chromatography and size exclusion chromatography, substantially as described in the examples. The purity of the fusion protein can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, fusion proteins expressed as described in the examples appear to be intact and correctly assembled as evidenced by reducing and non-reducing SDS-PAGE (see e.g. fig. 2, 5).

Compositions, formulations and routes of administration

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

Also provided is a method of producing a fusion protein of the invention in a form suitable for in vivo administration, the method comprising: (a) obtaining a fusion protein according to the invention, and (b) formulating said fusion protein together with at least one pharmaceutically acceptable carrier, thereby formulating a fusion protein preparation for in vivo administration.

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of one or more fusion proteins dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce an adverse, allergic, or other untoward reaction when administered to an animal such as, for example, a human, as appropriate. In accordance with the present disclosure, the preparation of Pharmaceutical compositions containing at least one fusion protein and optionally additional active ingredients will be known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences,18th ed. mack Printing Company,1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it will be understood that the formulations should meet sterility, pyrogenicity, general safety and purity Standards as required by the FDA Office of Biological Standards or corresponding agencies in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, such similar materials, and combinations thereof, as will be known to those of ordinary skill in the art (see, e.g., Remington's pharmaceutical Sciences,18th ed. machine Printing Company,1990, pp.1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

The composition may contain different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. Can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intracapsular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical (topocally), topical (locally), administration of the fusion protein of the invention (and any additional therapeutic agent) by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, local perfusion directly bathing target cells, via catheter, via lavage, in emulsion, in liquid compositions (e.g., liposomes), or by other methods or any combination of the foregoing, as will be appreciated by one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences,18th ed. mack Printing Company,1990, incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used for administering polypeptide molecules such as the fusion proteins of the present invention.

Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the fusion proteins of the invention may be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as Hanks 'solution, Ringer's solution or physiological saline buffer. The solution may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the fusion protein may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the fusion protein of the invention in the required amount in the appropriate solvent with various other ingredients enumerated below, as required. Sterility can be readily achieved, for example, by filtration through a sterile filtration membrane. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first made isotonic before injection with sufficient saline or glucose. The compositions must be stable under the conditions of manufacture and storage and provide protection against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at safe levels, for example below 0.5ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphoric acid, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; chlorhexidine di-ammonium; benzalkonium chloride; benzethonium chloride; 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 polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl-lipids or triglycerides or liposomes.

The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly- (methacrylate) microcapsules, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions (macroemulsions). Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack printing company, 1990). Sustained release formulations can be prepared. Examples of suitable sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In particular embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of absorption delaying agents, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

In addition to the previously described compositions, the fusion proteins can also be formulated as depot (depot) formulations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fusion protein may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

Pharmaceutical compositions comprising the fusion proteins of the invention may be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants which facilitate processing of the protein into pharmaceutically acceptable preparations. Suitable formulations depend on the route of administration chosen.

The fusion protein may be formulated into a composition in free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, such as those formed with the free amino groups of the proteinaceous composition, or with inorganic acids, such as for example hydrochloric or phosphoric acids, or with organic acids, such as acetic, oxalic, tartaric or mandelic acid. The salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxide; or an organic base such as isopropylamine, trimethylamine, histidine or procaine (procaine). Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

Therapeutic methods and compositions

Any of the fusion proteins provided herein can be used in a method of treatment.

For use in a method of treatment, the fusion proteins of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors 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 agent, the method of administration, the timing of administration, and other factors known to medical practitioners.

In one aspect, a fusion protein of the invention is provided for use as a medicament. In other aspects, fusion proteins of the invention are provided for use in treating a disease. In certain embodiments, fusion proteins of the invention are provided for use in a method of treatment. In one embodiment, the invention provides a fusion protein as described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the invention provides a fusion protein for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the fusion protein. In certain embodiments, the disease to be treated is an inflammatory disease. Exemplary inflammatory diseases include inflammatory bowel disease (e.g., crohn's disease or ulcerative colitis) and rheumatoid arthritis. In a particular embodiment, the disease is inflammatory bowel disease or rheumatoid arthritis, particularly inflammatory bowel disease, more particularly crohn's disease or ulcerative colitis. In another specific embodiment, the disease is idiopathic pulmonary fibrosis. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-inflammatory agent (if the disease to be treated is an inflammatory disease). An "individual" according to any of the embodiments above is a mammal, preferably a human.

In a further aspect, the invention provides the use of a fusion protein of the invention in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having a disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is an inflammatory disease. In a particular embodiment, the disease is inflammatory bowel disease or rheumatoid arthritis, particularly inflammatory bowel disease, more particularly crohn's disease or ulcerative colitis. In another specific embodiment, the disease is idiopathic pulmonary fibrosis. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-inflammatory agent (if the disease to be treated is an inflammatory disease). An "individual" according to any of the embodiments above is a mammal, preferably a human.

In a further aspect, the invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a fusion protein of the invention. In one embodiment, the individual is administered a composition comprising a fusion protein of the invention in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is an inflammatory disease. In a particular embodiment, the disease is inflammatory bowel disease or rheumatoid arthritis, particularly inflammatory bowel disease, more particularly crohn's disease or ulcerative colitis. In another specific embodiment, the disease is idiopathic pulmonary fibrosis. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-inflammatory agent (if the disease to be treated is an inflammatory disease). An "individual" according to any of the embodiments above may be a mammal, preferably a human.

The fusion proteins of the present invention are also useful as diagnostic reagents. The binding of the fusion protein to the antigenic determinant can be easily detected, for example by means of a label attached to the fusion protein or by using a labeled secondary antibody specific for the fusion protein of the invention.

In some embodiments, an effective amount of a fusion protein of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a fusion protein of the invention is administered to an individual to treat a disease.

For the prevention or treatment of disease, the appropriate dosage of the fusion protein 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 route of administration, the weight of the patient, the type of fusion protein, the severity and course of the disease, whether the fusion protein is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic intervention, the patient's clinical history and response to the fusion protein, and the judgment of the attending physician. The practitioner responsible for administration will determine at any event the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

The fusion protein is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the fusion protein may be a starting candidate dose for administration to a patient, whether, for example, by one or more divided administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the fusion protein will be in the range of about 0.005mg/kg to about 10 mg/kg. In other non-limiting examples, the dosage may further include from about 1 μ g/kg body weight, about 5 μ g/kg body weight, about 10 μ g/kg body weight, about 50 μ g/kg body weight, about 100 μ g/kg body weight, about 200 μ g/kg body weight, about 350 μ g/kg body weight, about 500 μ g/kg body weight, about 1mg/kg body weight, about 5mg/kg body weight, about 10mg/kg body weight, about 50mg/kg body weight, about 100mg/kg body weight, about 200mg/kg body weight, about 350mg/kg body weight, about 500mg/kg body weight, to about 1000mg/kg body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the amounts listed herein, based on the numbers described above, a range of about 5mg/kg body weight to about 100mg/kg body weight, a range of about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, and the like may be administered. Thus, about 0.5mg/kg, 2.0mg/kg, 5.0mg/kg, or 10mg/kg (or any combination thereof) of one or more doses 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 protein). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of the therapy is readily monitored by conventional techniques and assays.

The fusion proteins of the invention will generally be used in an amount effective for the purpose intended. For use in treating or preventing a disease condition, the fusion protein of the invention, or a pharmaceutical composition thereof, is administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The dosage can then be formulated in animal models to achieve inclusion of the IC as determined in cell culture₅₀The circulating concentration range of (c). Such information can be used to more accurately determine the dosage available in a human.

Initial doses can also be estimated from in vitro data, such as animal models, using techniques well known in the art. One of ordinary skill in the art can readily optimize administration to humans based on animal data.

The dosage amount and time interval can be adjusted individually to provide plasma levels of the fusion protein sufficient to maintain a therapeutic effect. Typical patient doses for administration by injection range from about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

In the case of local administration or selective uptake, the effective local concentration of the fusion protein may not be related to the plasma concentration. One skilled in the art would be able to optimize therapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the fusion proteins described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the fusion protein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. LD can be determined using cell culture assays and animal studies₅₀(lethal dose for 50% of the population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD₅₀/ED₅₀And (4) the ratio. Fusion proteins exhibiting a large therapeutic index are preferred. In one embodiment, the fusion protein according to the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage suitable for use in humans. Preferably, the dose is at a circulating concentration (including ED) with little or no toxicity₅₀) Within the range of (1). The dosage within this range may vary depending on a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be selected by The individual physician in view of The condition of The patient (see, e.g., Fingl et al, 1975, in: The pharmacological basis of Therapeutics, Ch.1, p.1, herein incorporated by reference in its entirety).

The attending physician of a patient treated with a fusion protein of the invention will know how and when to terminate, discontinue or adjust administration (due to toxicity, organ dysfunction, etc.). Conversely, if the clinical response is not adequate (excluding toxicity), the attending physician will also know how to adjust the treatment to higher levels. The magnitude of the administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, the route of administration, and the like. The severity of the condition can be assessed, for example, in part, by standard prognostic assessment methods. In addition, the dosage and possibly the frequency of administration will also vary with the age, weight and response of the individual patient.

Other Agents and treatments

The fusion proteins of the invention may be administered in combination with one or more other agents in therapy. For example, the fusion protein of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise any active ingredient suitable for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an anti-inflammatory agent.

Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents will depend on the amount of fusion protein used, the type of disorder or treatment, and other factors described above. The fusion proteins are generally used at the same dosages and administration routes as described herein, or at about 1 to 99% of the dosages described herein, or by any dosage and any route empirically/clinically determined to be appropriate.

Such combination therapies recited above encompass both combined administration (where two or more therapeutic agents are included in the same composition or separate compositions) and separate administration, in which case administration of the fusion protein of the invention can occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants.

Article of manufacture

In another aspect of the invention, articles of manufacture containing materials useful in the treatment, prevention and/or diagnosis of the disorders described above are provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition, which by itself or in combination with other compositions is effective for treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper penetrable by a hypodermic needle). At least one active ingredient in the composition is a fusion protein of the invention. The label or package insert indicates that the composition is for use in treating the selected condition. In addition, the article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises a fusion protein of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional therapeutic agent. The article of manufacture in this embodiment of the invention may also comprise a package insert indicating that the composition is useful for treating 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 further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced in view of the general description provided above.

Recombinant DNA technology

DNA is manipulated using standard methods, such as Sambrook et al, Molecular cloning: A laboratory and; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions. The DNA sequence was determined by double-strand sequencing. For general information on the nucleotide sequences of the light and heavy chains of human immunoglobulins see: kabat, E.A.et., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242.

Gene synthesis

The desired gene segments were generated by PCR using appropriate templates or synthesized from synthetic oligonucleotides and PCR products by automated gene synthesis in Geneart AG (Regensburg, Germany). The gene segments flanked by single restriction endonuclease cleavage sites were cloned into standard cloning/sequencing vectors. Plasmid DNA was purified from transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments are designed with appropriate restriction sites to allow subcloning into the corresponding expression vector. All constructs were designed with a 5' DNA sequence encoding a leader peptide (MGWSCIILFLVATATGVHS) that targets secretion of the protein in eukaryotic cells.

Cloning of antibody-IL-10 fusion constructs

In-frame insertion of amplified DNA fragments of heavy and light chain V domains into a vector containing human IgG₁Or a mammalian expression vector for the corresponding receptor for either the Fab constant heavy chain or the human constant light chain. The heavy and light chains are always encoded on separate plasmids. While the light chain-encoding plasmids are the same for both IgG-based and Fab-based IL-10 fusion constructs, for the Fab-based constructs, the heavy chain-encoding plasmids contain one or two VH-CH1 domains along with the corresponding IL-10 portion, depending on the version. In the case where the Fab heavy chain plasmid contains two VH-CH1 domains (tandem Fab interrupted by single chain IL-10 dimer or engineered monomeric IL-10(Josephsonet al, J Biol Chem 275,13552-7(2000)), the two V domains must be inserted in a two-step cloning procedure using a different combination of restriction sites for each of them. The IL-10 portion of these constructs was always cloned in-frame with the heavy chains of these antibodies, used between the C-terminus of the Fab or IgG heavy chains and the N-terminus of the cytokine, respectively (G)₄S)₃15 polymer linker. Only the IgG-IL-10 version (FIG. 1A) is at the C-terminus of the IgG heavy chain andthe cytokine comprises (G) between N-termini₄S)₄A 20 mer linker. The C-terminal lysine residue of IgG heavy chain is removed upon addition of the linker. For single chain IL-10, an insertion (G) is made between the two IL-10 chains₄S)₄A 20 mer linker. In the case where only one of the two different IgG heavy chains is fused to IL-10, two heavy chain plasmids need to be constructed and transfected to drive heterodimerization through knob-to-hole modification in the IgG CH3 domain. The "hole" heavy chain linked to the IL-10 moiety carries the Y349C, T366S, L368A and Y407V mutations in the CH3 domain, while the non-fused "segment" heavy chain carries the S354C and T366W mutations in the CH3 domain (EU numbering). To eliminate Fc γ R binding/effector function and prevent FcR co-activation, the following mutations were introduced into the CH2 domain of each IgG heavy chain: L234A, L235A and P329G (EU numbering). Expression of the antibody-IL-10 fusion construct was driven by the MPSV promoter and transcription was terminated by a synthetic polyA signal sequence located downstream of the CDS. In addition to the expression cassettes, each vector contains an EBV oriP sequence for autonomous replication in EBV-EBNA expressing cell lines.

Preparation of antibody-IL-10 fusion protein

Details of the generation, affinity maturation and characterization of antigen binding modules for FAP can be found in the examples of PCT publication No. wo 2012/020006, particularly examples 2-6 (preparations) and 7-13 (characterizations), which are incorporated herein by reference in their entirety. As described herein, a variety of antigen binding domains for FAP were generated by phage display, including those referred to as 4G8 and 4B9 used in the examples below.

Calcium phosphate transfection was used to co-transfect exponentially growing HEK293-EBNA cells with mammalian expression vectors to generate the antibody-IL-10 fusion constructs used in the examples. Alternatively, HEK293EBNA cells in suspension growth were transfected with expression vectors via Polyethyleneimine (PEI). All FAP targeting antibody-IL-10 fusion constructs based on clones 4G8 and 4B9 can be purified by affinity chromatography using a protein a matrix.

Briefly, FAP targeting constructs fused to IL-10 (single chain (sc) IL-10 or IL-10M1) were purified by a procedure consisting of one affinity chromatography step (protein A) followed by size exclusion chromatography (Superdex 200, GEHealthcare). The protein a column was equilibrated in 20mM sodium phosphate, 20mM sodium citrate pH 7.5, the supernatant was loaded, and the column was washed with 20mM sodium phosphate, 20mM sodium citrate (optionally with or without 500mM sodium chloride), pH 7.5, followed by 13.3mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH 5.45 (in the case where FBS was present in the supernatant). Optionally, a third wash is performed with 10mM MES, 50mM NaCl pH 5. The fusion construct was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine pH 3. The eluted fractions were pooled and refined by size exclusion chromatography in the final formulation buffer (25 mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH6.7 or 20mM histidine, 140mM NaCl pH 6.0).

Protein concentration of the purified antibody-IL-10 fusion construct was determined by measuring the Optical Density (OD) at 280nm using a molar extinction coefficient calculated based on the amino acid sequence. By SDS-PAGE in the presence and absence of reducing agent (5mM 1, 4-dithiothreitol) and with Coomassie blue (SimpleBlue)^TMSafeStain, Invitrogen) and analyzed for purity, integrity and monomer status of the fusion constructs. Use according to the manufacturer's instructions (4-20% Tris-glycine gel or 3-12% Bis-Tris)Pre-gel systems (Invitrogen). Alternatively, the reduced and non-reduced antibody-IL-10 fusion constructs were analyzed using LabChip GX (Caliper) according to the manufacturer's instructions. Analytical size exclusion column (GE Healthcare) was used at 25 ℃ using Superdex 20010/300 GL (using 2mM MOPS, 150mM NaCl, 0.02% NaN₃pH 7.3 running buffer) or TSKgel G3000SW XL column (at 25mM K)₂HPO₄125mM NaCl, 200mM arginine, 0.02% (w/v) NaN₃pH6.7 running buffer) samples of the immunoconjugates were analyzed for aggregate content.

The results of the purification and subsequent analysis of the different constructs are shown in FIGS. 2-8. IgG-IL-10 constructs exhibit several production advantages over other IL-10 fusion formats. First, in comparison to Fab-IL-10 versions, the IL-10 homodimer was anchored within the same antibody molecule. Thus, upon production, the monomeric IL-10 molecule seen for the Fab-IL-10 version does not occur, wherein after affinity chromatography of the Fab-IL-10 version, monomeric and dimeric protein species are observed, of which only the dimer is the desired active product (compare FIG. 2B and FIG. 6B). Second, in contrast to heterodimeric IgG-based versions containing knob-into-hole modifications (e.g., IgG-scIL-10 and IgG-IL-10M1), the IgG-IL-10 construct contains two identical heavy chains. This avoids unwanted by-products like pocket-pocket or node-node homodimers.

Determination of affinity by SPR

Kinetic rate constants (k) of antibody-IL-10 fusion constructs for FAP from three different species (human, murine and cynomolgus) and human IL-10R1 (k) were measured by Surface Plasmon Resonance (SPR) using a Proteon XPR36(BioRad) instrument with PBST running buffer (10mM phosphate, 150mM sodium chloride pH 7.4, 0.005% Tween 20) at 25 deg.C_onAnd k_off) And affinity (K)_D). To determine affinity for FAP, the target protein was captured via its H6 tag by a covalently immobilized anti-H6 antibody (fig. 9A). Briefly, anti-pentahis IgG (Qiagen #34660, mouse monoclonal antibody) was immobilized at high levels (up to about 5.000RU) at 30 μ l/min onto separate vertical channels of the GLM chip by simultaneous activation of all channels with a freshly prepared mixture of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (shhs) for 5min followed by injection of 15 μ g/ml anti-pentahis IgG in 10mM sodium acetate buffer pH 4.5 for 180 sec. The channel was blocked by injection of ethanolamine for 5 minutes. FAP from different species (see SEQ ID NOs 81, 83 and 85) with H6 tag captured from 5 μ g/ml dilution in running buffer along the vertical channel at 30 μ l/min for 60s to achieve a ligand density between about 250 and 600 RU. In a single click kinetics assay setup (OSK), the antibody-IL-10 fusion construct was injected as an analyte along the horizontal channel at 100. mu.l/min in 5-fold serial dilutions ranging from 50 to 0.08 nM. The association phase was recorded for 180s and the dissociation phase for 600 s. In thatIn the case of interactions that exhibit very slow dissociation rates, the dissociation rate recordings were extended to 1800s to observe dissociation of the complexes. However, in some cases, it is still not possible to fit these off-rates, so the estimate 1x 10 is used^-51/s to calculate K_D. Running buffer (PBST) was injected along the sixth channel to provide an "on-line" blank as a reference. Binding rates (k) were calculated using a simple 1:1 Langmuir (Langmuir) binding model (ProteOn Manager software version2.1) by simultaneous fitting of binding and dissociation sensorgrams_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D). Regeneration was performed in a horizontal orientation at 100 μ l/min by two 10mM glycine pH 1.5 and 50mM NaOH pulses for 30s, dissociating anti-pentaHisIgG from the captured FAP and bound antibody-IL-10 fusion construct.

To measure the interaction between the antibody-IL-10 fusion construct and human IL-10R1, a NLC chip was used to immobilize biotinylated receptors (FIG. 9B). For different contact times, 400 to 1600RU of human IL-10R1 fused to an IgG Fc region was captured on a neutravidin-derivatized chip substrate at a concentration of 10. mu.g/ml and a flow rate of 30. mu.l/sec along the vertical channel (see SEQ ID NO: 87). Binding to biotinylated human IL10R1 was measured by injection at 100. mu.l/min in a horizontal orientation at six different analyte concentrations (50, 10, 2, 0.4, 0.08, 0nM), with an on rate recorded for 180s and an off rate recorded for 600 s. Running buffer (PBST) was injected along the sixth channel to provide an "on-line" blank as a reference. Binding rates (k) were calculated using a simple 1:1 Langmuir (Langmuir) binding model (ProteOn Manager software version2.1) by simultaneous fitting of binding and dissociation sensorgrams_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D). Since human IL-10R1 could not be regenerated without loss of activity, two subsequent steps (ligand capture and analyte injection) were performed channel by channel using a freshly immobilized sensor chip surface for each interaction.

Tables 1 and 2 show a summary of kinetic rates and equilibrium constants for binding to FAP and human IL-10R1 from different species based on antibody-IL-10 fusion constructs against FAP clone 4G8 or 4B9, respectively.

Table 1: summary of kinetic rates and equilibrium constants based on antibody fusions against FAP clone 4G 8. Binding to FAP from different species and to human IL-10R 1.

Table 2: summary of kinetic rates and equilibrium constants based on antibody fusions against FAP clone 4B 9. Binding to FAP from different species and to human IL-10R 1.

We did not fuse with antibody at hand but C-terminally tagged with H6 wild-type (wt) IL-10 cytokine showed K of 52pM for human IL-10R1_D(k_on2.5x 10⁶1/Ms，k_off1.3x10^-41/s). For the dimeric cytokine based antibody-IL-10 fusion construct, the affinity for IL-10R1 was comparable to that of the unfused cytokine, as well as two-digit pM (ranging from 18 to 73 pM). This shows that this cytokine is resistant to fusion with antibodies or fragments thereof at the N-terminus without significant loss of affinity for human IL-10R 1. In contrast, monomeric cytokine-based antibody-IL-10 fusion constructs did not show an affinity effect for dimeric IL-10 fusions, and therefore their affinity for the receptor was in the three-digit pM or one-digit nM range (815 pM and 1.1nM, respectively). Binding to FAP was dependent on the corresponding antibody, with clone 4B9 showing higher affinity/avidity for human and cynomolgus FAP, while clone 4G8 showed higher affinity/avidity for murine FAPForce. In fact, the affinity of the 4G8 antibody for murine FAP was so strong that it was not possible to determine the dissociation rate of the complex under the conditions applied.

The interaction between IL-10 and IL-10R1 is high affinity (avidity) ranging from about 35-200pM (Moore, K.W.et al, Annu.Rev.Immunol.19,683-765 (2001)). For constructs containing a dimeric IL-10 portion or two independent monomers, fusion to the antibody appeared to have no significant change in affinity (approximately 19-73 pM). However, for the monomeric IL-10 fusion construct, this binding strength is significantly reduced, most likely because there is no affinity effect that occurs in the case of a dimeric cytokine or two monomers fused to the same IgG. Ideally, the affinity of the antibody-IL-10 fusion construct for the target FAP should be higher than the affinity for the high affinity cytokine receptor IL-10R1, thereby achieving effective targeting of FAP-expressing tissues. Despite the high affinity between IL-10 and IL-10R1, the affinity exhibited by molecules based on the IgG-IL-10 pattern for the target FAP is still higher: clone 4B9IgG-IL-10 (48 pM for IL-10R1 versus 11pM for human FAP) and clone 4G8 (25 pM for IL-10R1 versus 6pM for murine FAP), respectively. These affinities for IL-10R1 and for FAP appear to be appropriate for achieving effective targeting to tissues overexpressing FAP, and IL-10R1 does not appear to represent a sink for these molecules.

Suppression of LPS-induced production of primary monocyte proinflammatory cytokines

For functional characterization and differentiation between FAP-targeting IL-10 constructs based on IgG or Fab, the efficacy of these molecules was evaluated in different in vitro assays. For example, the efficacy of suppressing LPS-induced production of primary monocyte proinflammatory cytokines was measured. For this experiment, 200ml of heparinized peripheral blood (obtained from healthy volunteers, Medical Services department, Roche Diagnostics GmbH, Penzberg, Germany) was separated by Ficoll Hypaque density gradient followed by negative separation of monocytes (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, # 130-. In culture medium (supplemented with 10% human serum, 2mM L-glutamine [ Gibco, # 25030%]And Pen/Strep RPMI1640 [ Gibco/Invitrogen，Darmstadt，Germany，cat.no.#10509-24]) In the scale of 5x10⁴Individual cells/well purified monocytes were seeded in 96-well F cell culture plates (Costar/Corning Life Sciences, Amsterdam, the Netherlands; # 3596).

All antibody-IL-10 fusion proteins were tested in (a) solution and (b) experimental setup, in which recombinant human FAP (C) was incubated overnight at 4 ℃_{Final (a Chinese character of 'gan')}1 μ g/ml) were coated onto plates (or 60-90min at room temperature) and the antibody-IL-10 fusion protein was immobilized by binding to coated FAP.

For setting (a), cells were stimulated directly with 100ng/ml LPS (Sigma-Aldrich/Nunc, Taufkirchen, Germany, # L3129) in the presence or absence of titrated amounts (normally 0-500nM) of the indicated antibody-fusion construct or recombinant wild-type human IL-10 as a positive control after vaccination. For set-up (b), unbound FAP was removed after coating and the plate was blocked with medium (see above) for 1 hour at room temperature before incubation with the IL-10 construct for an additional 1 hour. Thereafter, the plates were washed with medium, after which monocytes were added to the culture together with a suitable stimulus (100ng/ml LPS).

For all experiments, cells were incubated at 37 ℃ and 5% CO₂Incubation for 24 hours supernatant was collected (optionally stored at-20/-80 ℃) and tested for cytokine production using the CBA Flex kit for IL-1 β, IL-6, G-CSF, and/or TNF α (BD Biosciences, Heidelberg, Germany, #558279, #558276, #558326, and #558299), plates were measured with FACS arrays (from BD) and analyzed using FCAP software (from BD).

As shown in Table 3, the in vitro potency of 4G8Fab-IL-10 (see SEQ ID NO 7 and 19) and IgG-IL-10 (see SEQ ID NO 7 and 9) in suppressing the proinflammatory cytokines IL-1 β, IL-6, and TNF α was comparable in setting (a). In contrast, in setting (b), the IgG-based format exhibited superior efficacy compared to Fab-IL-10. The EC50 value for the IgG-IL-10 construct in set (b) was similar to the EC50 value for recombinant wt human IL-10 (which could only be tested in set (a)).

Table 3: 4G8-IgG-IL-10 and 4G8-Fab-IL-10 suppressed the EC50 value for monocyte cytokine production (donor 1).

This result was reproduced in a separate experiment using two different blood donors (tables 4 and 5). In this experiment, the IgG-based targeting IL-10 construct was again significantly superior to the Fab-based molecule in suppressing all three cytokines tested, as indicated by the EC50 values obtained in setting (b). In configuration (a), all molecules are equivalent.

Table 4: 4G8-IgG-IL-10 and 4G8-Fab-IL-10 suppressed the EC50 value for monocyte cytokine production (donor 2).

Table 5: 4G8-IgG-IL-10 and 4G8-Fab-IL-10 suppressed the EC50 value for monocyte cytokine production (donor 3).

In yet another experiment, Fab and IgG based IL-10 constructs were again evaluated for their efficacy in suppressing monocyte IL-6 production and compared to wt IL-10 and non-targeting Fab-IL-10 and IgG-IL-10 constructs that did not bind FAP (Table 6). Again, 4G8-IgG-IL-10 was found to be more effective at suppressing IL-6 production in experimental setting (b) than 4G8-Fab-IL-10, whereas the non-targeting construct caused suppression only at the highest concentration. In contrast, in setting (a), the potency of all constructs was comparable.

Table 6: 4G8-IgG-IL-10 and 4G8-Fab-IL-10 suppressed the EC50 value for monocyte IL-6 production (donor 4).

Titration of FAP coating amount (c) because the concentration of recombinant human FAP used for coating in previous assays may reflect artificial or non-physiological conditions_finBetween 0.25 and 5 μ g/ml) and its effect on EC50 values was evaluated in experimental setup (b).

As shown in tables 7 and 8, in summary, there was no significant difference in the ratio of EC50 values for IgG and Fab based constructs. At all concentrations, the IgG-IL-10 construct was more potent in inhibiting IL-6 induction (tables 7 and 8). However, the FAP coating concentration did affect the experimental results, with a general increase in EC50 values as the concentration decreased, which may reflect the amount of immobilized construct on the plate (Table 8; for Fab-based constructs, a decrease in cytokine was observed at the lowest FAP concentration, but EC50 could not be calculated). Interestingly, at high FAP concentrations (5. mu.g/ml), an increase in the total amount of IL-6 secretion was detected (FIG. 10).

Table 7: 4G8-IgG-IL-10 and human wild-type IL-10 (in solution) suppressed the EC50 value for monocyte IL-6 production (donor 5).

Table 8: 4G8-IgG-IL-10 and 4G8-Fab-IL-10 immobilized on coated FAP at different concentrations suppressed the EC50 value for monocyte IL-6 production (donor 5).

In yet another experiment, IL-10 fusion constructs comprising different FAP targeting domains, i.e., affinity matured anti-FAP antibody variant 4B9, were tested. Again, the in vitro efficacy of the constructs in suppressing LPS-induced monocyte IL-6 production was evaluated in experimental settings (a) and (b).

Table 9 shows that for the 4B 9-based constructs, IgG-IL-10 molecules (see SEQ ID NO 25 and 27) were superior to Fab-IL-10 constructs (see SEQ ID NO 25 and 31) in suppressing IL-6 production in experimental set-up (a) (and comparable in set-up (B)). In general, the 4B9 and 4G8 constructs exhibited similar potency.

Table 9: IgG-IL-10 and Fab-IL-10 based on 4G8 and 4B9 suppressed the EC50 value for monocyte IL-6 production (donor 7).

In a further series of experiments, 4G 8-based IgG-IL-10, Fab-IL-10M1-Fab and IgG-IL-10M1 constructs were compared. The suppression of LPS-induced production of the monocyte proinflammatory cytokines IL-6, IL-1. beta. and TNF. alpha. was evaluated in experimental settings (a) and (b). The results of these experiments are shown in tables 10-12 (three different donors). As in previous experiments, IgG-IL-10 was the most powerful construct, especially in experimental setting (b).

Table 10: the 4G8IgG-IL-10, 4G8Fab-IL-10M1-Fab and 4G8IgG-IL-10M1 fusion proteins suppressed the EC50 value for monocyte cytokine production (donor 1).

Table 11: the 4G8IgG-IL-10, 4G8Fab-IL-10M1-Fab and 4G8IgG-IL-10M1 fusion proteins suppressed the EC50 value for monocyte cytokine production (donor 2).

Table 12: the 4G8IgG-IL-10, 4G8Fab-IL-10M1-Fab and 4G8IgG-IL-10M1 fusion proteins suppressed the EC50 value for monocyte cytokine production (donor 3).

In yet another series of experiments, Fab-IL-10, Fab-scIL-10-Fab and Fab-IL-10M1-Fab constructs based on 4G8 were compared. The suppression of LPS-induced monocyte IL-6, IL-1 β, TNF α and G-CSF production was evaluated in experimental settings (a) and (b). The results of these experiments are shown in tables 13-17 (six different donors). The results show that constructs comprising dimeric IL-10 molecules are more potent than constructs with either scIL-10 or monomeric IL-10M1 molecules.

Table 13: the 4G8Fab-IL-10, 4G8Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins suppressed the EC50 values for monocyte cytokine production.

Table 14: the 4G8Fab-IL-10, 4G8Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins suppressed the EC50 values for monocyte IL-6 production.

Table 15: the 4G8Fab-IL-10, 4G8Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins suppressed the EC50 values for monocyte IL-1 β production.

Table 16: the 4G8Fab-IL-10, 4G8Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins suppressed the EC50 values for monocyte G-CSF production.

Table 17: the 4G8Fab-IL-10, 4G8Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins suppressed the EC50 values for monocyte TNF α production.

Finally, Fab-IL-10 and IgG- (IL-10M1) based on 4B9 and 4G8 were compared₂And (3) constructing the structure. The suppression of LPS-induced monocyte IL-6 production was assessed in experimental settings (a) and (b). The results of this experiment are shown in table 19. The results showed that IgG- (IL-10M1) was included₂All constructs within perform better in setting (b) than in setting (a).

Table 18: 4B9IgG-IL-10, 4G8IgG-IL-10 and 4G8IgG- (IL-10M1)₂EC50 values for the suppression of monocyte IL-6 production by fusion proteins.

Repressing IFN gamma-induced upregulation of MHC-II molecules on primary monocytes

For functional characterization and differentiation between FAP-targeting IL-10 constructs based on IgG and Fab, their ability to suppress IFN γ -induced MHC-II expression in monocytes was evaluated. This experiment was carried out with the construct, similarly to the cytokine repression assay, either in solution (experimental setup (a); see above) or immobilized by binding to FAP coated on cell culture plates (experimental setup (b); see above). In principle, monocytes were isolated and cultured as described above, but stimulated with 250U/ml IFN γ (BD, #554616) for 24 hours. Prior to stimulation, the cells are optionally treated with recombinant wild-type (wt) IL-10 or a different antibody-IL-10 fusion construct. After incubation, cells were dissociated by Accutase treatment (PAA, # L11-007) and stained with anti-HLA-DR antibody (BD, #559866) in PBS containing 3% human serum (Sigma, #4522) to avoid any non-specific Fc γ R binding prior to final FACS analysis.

The results of this experiment are shown in Table 19, demonstrating that 4B 9-based construct IgG-IL-10 molecules were superior to the Fab-IL-10 construct in experimental setting (B) (comparable in setting (a)) in downregulating IFN γ -induced MHC-II expression on primary monocytes.

Table 19: EC50 values for down-regulation of IFN γ -induced MHC-II expression on primary monocytes for 4B9IgG-IL-10 and 4B9 Fab-IL-10.

Sample (I)	EC50[nM]Setting (a) (solution)	EC50[nM]Setting (b) (immobilization)
			Fab-IL-10	0.072	Is not calculable
IgG-IL-10	0.064	0.018
			hu wt IL-10	0.004	Not tested

IL-10 induced STAT3 phosphorylation in isolated primary monocytes

Because IL-10R signaling causes STAT3 phosphorylation, the function of several targeted IL-10 constructs and patterns was evaluated in the pSTAT3 assay using freshly isolated blood mononuclear cells (Finbloom)&Winestock,J.Immunol.1995；Moore et al.,Annu.Rev.Immunol.2001；Mosser&Zhang, ImmunologicalReviews 2008). Briefly, CD14 was isolated touchless from Ficoll-isolated PBMCs of healthy donors as described above⁺A monocyte. Typically, 3-10X 10 in 300. mu.l medium (RPMI 1640/10% FCS/L-glutamine/pen/strep)⁵Individual cells were transferred into FACS tubes and were typically incubated at 37 ℃ in 5% CO₂Incubation with 0-200/500nM wt human IL-10 or the antibody-IL-10 fusion protein shown for 30 minutes. Then, 300. mu.l of pre-warmed fixation buffer I (BD Biosciences, #557870) was added to each tube, vortexed, and incubated at 37 ℃ for 10 minutes, after which the cells were washed once with 2ml PBS/2% FCS and centrifuged at 250x g for 10 minutes. Subsequently, 300. mu.l of ice-cooled permeabilization buffer III (BD Biosciences, #558050) was added per tube for cell permeabilization and incubated on ice for 30 minutes, after which the cells were washed again as described above. Finally, the cells were resuspended in 100. mu.l antibody dilution (anti Stat-3. A647; BD Biosciences, #557815) and incubated at 4 ℃ for 30 minutes, after which the cells were washed and processed for FACS analysis.

The EC50 values obtained for the different constructs in this experiment are shown in tables 20 and 21. The results show that constructs comprising dimeric IL-10 molecules (based on Fab or IgG) are more active than constructs comprising either scIL-10 molecules or monomeric IL-10M1 molecules.

Table 20: EC50 values for IL-10 induced STAT3 phosphorylation of 4G 8-based antibody-IL-10 fusion proteins in isolated primary monocytes.

Table 21: EC50 values for IL-10 induced STAT3 phosphorylation in isolated primary monocytes based on antibody-IL-10 fusion proteins of 4B 9.

Sample (I)	EC50[nM]pSTAT3 Induction
		Human wt IL-10	0.017
IgG-IL-10	0.130
		IgG-(IL-10M1)₂	0.435

Biodistribution of FAP-targeted and non-targeted antibody-IL-10 fusion proteins

Tissue biodistribution of FAP-targeted, In-111 labeled 4B9IgG-IL-10, 4G8IgG-IL-10 and non-targeted DP47GS IgG-IL 10 was determined at 50 μ G per mouse In DBA/1J mice with collagen-induced arthritis that achieved a predetermined arthritis score >3 (28 days after first immunization). Biodistribution was performed 72 hours after i.v. injection of radiolabeled conjugate in 5 mice per group.

The results of this experiment are shown in table 22. The uptake of the non-targeting antibody-IL-10 fusion protein DP47GS IgG-IL-10 in inflamed joints was significantly (p <0.0001) lower than that of the targeting IgG-IL-10 fusion protein, indicating that the uptake of 4B9IgG-IL-10 and 4G8IgG-IL-10 was mediated by FAP. Spleen uptake was most likely mediated by IL-10, since all three constructs showed similar levels of spleen accumulation.

Table 22: uptake of antibody construct (% injected dose/g tissue).

Tissue of	4B9IgG-IL-10	4G8IgG-IL-10	DP47GS IgG-IL-10
				Inflamed joint	20.7±1.1	19.6±1.0	8.6±1.0
Spleen	6.3±0.4	7.3±0.3	6.7±0.5
				Blood, blood-enriching agent and method for producing the same	4.2±0.5	1.1±0.1	7.3±1.0

To investigate the effect of IL-10 on IgG-IL-10 biodistribution, In a second experiment, the biodistribution of In-111 labeled 4G8IgG-IL-10 was compared to that of In-111 labeled 4G8 IgG.

The results of this experiment are shown in table 23. There was no significant difference in accumulation in inflamed joints between 4G8IgG and 4G8IgG-IL-10, indicating that IL-10 had no significant effect on targeting 4G8IgG to inflamed sites. Splenic uptake of 4G8IgGI-IL-10 was significantly higher than that of 4G8IgG (p <0.0001), indicating that uptake in the spleen is mediated in part by IL-10.

Table 23: uptake of antibody construct (% injected dose/g tissue).

Tissue of	4G8IgG	4G8IgG-IL-10
			Inflamed joint	18.1±1.0	19.6±1.0
Spleen	2.9±0.2	7.3±0.3
			Blood, blood-enriching agent and method for producing the same	3.9±0.8	1.1±0.1

Preparation of mutant IL-10 molecules and antibody fusion proteins thereof

To improve targeting of the corresponding antibody fusion protein to the antibody target expression site rather than to the IL-10 receptor expression site, several mutant IL-10 molecules were designed based on known or expected reduced affinity for human IL-10R 1. Two of these mutant IL-10 molecules, IL-10I87A and IL-10R24A molecules, were used in the examples below.

Cloning of IL-10 wild-type and mutant cytokines

Inserting a DNA fragment encoding the IL-10 wild-type cytokine in-frame into a recipient mammalian expression vector. IL-10 mutants were generated by site-directed mutagenesis based on the IL-10 wild-type DNA sequence. All IL-10 cytokine constructs were fused C-terminally to a hexahistidine tag, enabling affinity purification of the recombinant protein. Cytokine expression is driven by the P-MPSV promoter and transcription is terminated by a synthetic polyA signal sequence located downstream of the CDS. In addition to the expression cassettes, each vector contains an EBV oriP sequence for autonomous replication in EBV-EBNA expressing cell lines.

Production and purification of IL-10 cytokines

Calcium phosphate transfection was used to transiently transfect exponentially growing adherent HEK293-EBNA cells with mammalian expression vectors to generate IL-10 cytokines for use in the examples below. All IL-10 cytokines were purified from culture supernatants by immobilized metal ion affinity chromatography (IMAC) via a C-terminal hexahistidine tag.

Briefly, IL-10 cytokines were purified by a procedure consisting of an affinity step (NiNTA Superflow card, Qiagen) followed by size exclusion chromatography (HiLoad 16/60Superdex 200, GE Healthcare).

NiNTA Superflow cartridges pre-filled with 5ml Ni-NTA resin were equilibrated with 10 column volumes of TRIS 25mM, NaCl 500mM, imidazole 20mM pH 8.0. 200ml culture supernatant was loaded and the column was washed with TRIS 25mM, NaCl 500mM, imidazole 20mM pH 8.0. The his-tagged IL-10 cytokine was eluted into TRIS 25mM, NaCl 500mM, imidazole 500mM pH 8.0 on 5 column volumes with a shallow linear gradient at 5ml/min and 1ml fractions were collected. Fractions containing the dimeric cytokine peak were spin concentrated in Millipore Amicon MWCO 10k at 2500rpm gently spun for 15 minutes at 4 ℃. The concentrated eluate was purified by size exclusion chromatography on a HiLoad 16/60Superdex 200 column in final formulation buffer 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH6.7 at a flow rate of 1 ml/min. Fractions were collected and gently spun at 2500rpm to a final concentration of 0.5-1mg/ml in Millipore Amicon MWCO 10k to concentrate those fractions containing dimeric IL-10 cytokine (10 fold) before they were snap frozen in liquid nitrogen and stored at-80 ℃.

IL-10 cytokine purity and integrity was analyzed by SDS-PAGE in the presence or absence of reducing agent (5mM 1, 4-dithiothreitol) and Coomassie blue (SimpleBlue)^TMSafesain, Invitrogen). Use according to the manufacturer's instructions (4-16% Bis-Tris Mini gel)Pre-glue system (Invitrogen). Analytical size exclusion column (GEHealthcare) using Superdex 7510/300 GL or Superdex 20010/300 GL at 25 ℃ with 2mM MOPS, 150mM NaCl, 0.02% NaN₃The running buffer was assayed for aggregate content as well as monomer content of IL-10 cytokine at pH 7.3 (FIGS. 12-14).

Determination of affinity by Surface Plasmon Resonance (SPR)

Kinetic rate constants (k) for human IL-10R1 for IL-10 wild-type and mutant cytokines were measured by Surface Plasmon Resonance (SPR) using a ProteOn XPR36(BioRad) instrument with PBST running buffer (10mM phosphate, 150mM sodium chloride pH 7.4, 0.005% Tween 20) at 25 ℃. (k_onAnd k_off) And affinity (K)_D)。

The SPR assay setup is depicted in figure 15. Neutravidin derivatized on NLC chip at concentration 30. mu.g/ml and flow rate 30. mu.l/min along vertical channel for contact time of 240sApproximately 770RU of biotinylated human IL-10R1 fused to an IgG Fc region was captured on the chip substrate (see SEQ ID NO: 87). Binding to huIL10R1 was measured by injection at 50. mu.l/min in a horizontal orientation at 5 different analyte concentrations (50, 10, 2, 0.4, 0.08nM), with an on-rate recording of 180s and an off-rate recording of 600 s. Running buffer (PBST) was injected along the sixth channel to provide an "on-line" blank as a reference. Binding rates (k) were calculated using a simple 1:1 Langmuir (Langmuir) binding model (ProteOn managersofware version2.1) by simultaneous fitting of binding and dissociation sensorgrams_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D). Since human IL-10R1 cannot be regenerated without loss of activity, subsequent ligand capture and analyte injection steps were performed per channel using freshly immobilized sensor chip surface channels for each interaction.

IL-10 wild-type cytokine exhibits a K of about 39pM for human IL-10R1_D(k_on2.76x 10⁶1/Ms，k_off1.08x 10^-41/s). As expected, the two IL-10 cytokine mutants, IL-10I87A and IL-10R24A, exhibited reduced affinity for human IL-10R1, at about 476pM and about 81pM, respectively (Table 24). Reduced affinity for IL-10R1 may represent a unique advantage when IL-10 is targeted to inflamed tissue via fusion to an antibody. Ideally, the affinity of a targeting antibody fused to an IL-10 cytokine should be significantly higher for inflammatory targets than for cytokines for their receptors, to achieve effective targeting and avoid off-target effects. In this regard, a more than 10-fold decrease in the affinity of the IL-10I87A cytokine compared to the IL-10 wild-type should result in excellent targeting of the IgG-IL-10I87A fusion molecule to the site of inflammation. In contrast, IL-10R24A only exhibited a 2-fold decrease in affinity for IL-10R 1. This mutation is described in Yoon, S.II, et al, Journal of Biological Chemistry 281(46),35088-35096 (2006).

Table 24: summary of kinetic rates and equilibrium constants for IL-10 cytokine. Binding of IL-10 wild type or IL-10 mutant to human IL-10R 1.

IL-10 mutants	k_on[1/Ms]	k_off[1/s]	K_D[pM]
				IL-10wt	2.76x 10⁶	1.08x 10^-4	38.9
IL-10I87A	4.61x 10⁵	2.19x 10^-4	476
				IL-10R24A	2.28x 10⁶	1.84x 10^-4	80.7

Suppression of proinflammatory cytokine production by monocytes by different human IL-10 mutants

The efficacy of these molecules was evaluated in different in vitro assays for functional characterization and differentiation between interleukin-10 (IL-10) mutants. For example, the efficacy of suppressing LPS-induced production of primary monocyte proinflammatory cytokines was measured.For this experiment, 200ml heparinized peripheral blood (obtained from healthy volunteers, Medical Services department, Roche Diagnostics GmbH, Penzberg, Germany) was separated by Ficoll Hypaque density gradient followed by negative separation of monocytes (Miltenyi Biotec, # 130-. In culture medium (supplemented with 10% human serum, 2mM L-glutamine [ Gibco, # 25030%]And Pen/Strep RPMI1640 [ Gibco/Invitrogen, #10509-24]) Medium by 5x10⁴Individual cells/well purified monocytes were seeded in 96-well F cell culture plates (Costar/Corning Life Sciences, # 3596).

Isolated monocytes were stimulated directly with 100ng/ml LPS (Sigma-Aldrich/Nunc, # L3129) in the presence of the indicated IL-10 mutant (mutation I87A or R24A) after inoculation, in comparison with wild-type (wt) human IL-10 as a positive control. The cells were then incubated at 37 ℃ and 5% CO₂After 24 hours incubation, supernatants were collected (optionally stored at-20/-80 ℃) and tested for cytokine production using the CBAFlex kit for IL-1 β, IL-6, G-CSF, and/or TNF α (BD Biosciences, #558279, #558276, #558326, and # 558299). the plates were measured with FACS arrays and analyzed using FCAP software (all from BD).

As shown in Table 25, the different IL-10 mutants had the highest in vitro potency in suppressing the proinflammatory cytokines IL-1 β and TNF α, with the wt IL-10 mutant being the weakest (as indicated by the highest EC) in the R24A mutant₅₀Value).

Table 25: inhibition of monocyte-derived cytokine production following LPS stimulation. Comparison of different IL-10 proteins and mutants.

Suppression of proinflammatory cytokine production by monocytes by different human antibody-mutant IL-10 fusion proteins

Next, the efficacy of the I87A IL-10 variant was compared to wt IL-10 with a human IgG fusion pattern targeting FAP. Briefly, untargeted wt IL-10 was tested in two different in vitro assays, in comparison to two 4B9IgG-IL-10 constructs comprising wtIL-10 (see SEQ ID NOS 25 and 27) or IL-10I87A (see SEQ ID NOS 25 and 96).

For the first setting ("in solution"), cells were stimulated directly with 100ng/ml LPS after inoculation in the presence or absence of titrated amounts (normally 0-500nM) of the indicated antibody-fusion construct or recombinant wild-type human IL-10 as a positive control. In another setup ("FAP-coated"), recombinant human FAP (C) will be used at 4 ℃_{Final (a Chinese character of 'gan')}1 μ g/ml) were coated onto plates overnight (or 60-90 minutes at room temperature). Unbound FAP was removed after coating and the plate was blocked with medium (see above) for 1 hour at room temperature before incubation with the IL-10 construct for another 1 hour. Thereafter, the plates were washed with medium before adding monocytes to the culture along with LPS stimulators as described above.

In the solution assay format, wt IL-10 elicited the highest potency in suppressing IL-6 and TNF α, followed by 4B9IgG-hIL-10wt (Table 26). In this assay, 4B9IgG-hIL-10I87A showed significantly reduced efficacy. In FAP targeting assay settings, the difference between 4B9IgG-hIL-10wt and 4B9IgG-hIL-10I87A was less pronounced.

Table 26: inhibition of monocyte-derived cytokine production following LPS stimulation. Comparison of different 4B9 IgG-mutant human IL-10 fusion proteins.

Similar to the human IL-10 molecule, further experiments were performed, and murine RAW cell lines (mouse macrophage cell lines) were also used to test murine IL-10 variants and fusion constructs. Briefly, the efficacy of the I87A IL-10 variant was compared to wt IL-10 in a human IgG fusion model targeting FAP. Briefly, untargeted wt IL-10 was tested in two different in vitro assays, compared to two 4G8IgG-IL-10 constructs containing murine wt IL-10 or IL-10I87A and untargeted IgG-IL-10wt constructs.

Briefly, 3x 10 seeded in 96-well F cell culture plates in medium (DMEM supplemented with 10% FCS and 4mM L-glutamine)⁵RAW 264.7 cells/well. Two assay variants were performed (solution-and human FAP-coated as described above). All constructs were titrated at 0-150nM, either directly on monocytes (in solution assay) or at 37 ℃ and 5% CO₂Incubation with FAP-coated wells for 1 hour followed by addition of monocytes equal for all test conditions followed by addition of 100ng/ml LPS (Sigma # L3129) and evaluation of mouse TNF α in the supernatant after 48 hours the results of these experiments are shown in table 27.

Table 27: inhibition of murine RAW cell-derived TNF α production following LPS stimulation. Comparison of different 4G8 IgG-mutant murine IL-10 fusion proteins.

In addition to efficient targeting of inflamed tissues and sufficient immunosuppressive activity, IL-10 fusion proteins should ideally not exert immunostimulatory properties. It is known that viral (e.g., epstein-barr virus) IL-10 lacks several immunostimulatory effects on certain cell types (like thymocytes and mast cells) in contrast to human IL-10, while retaining immunosuppressive activity that inhibits interferon gamma production. Ding and colleagues showed that the single amino acid isoleucine at position 87 in cellular IL-10 (human IL-10, murine IL-10) is required for its immunostimulatory function (Ding, y.et., j.exp. med.191(2), 213-. Thus, replacement of isoleucine 87(I87A) with alanine not only reduced the affinity of the cytokine for IL-10R1, but also abolished its several immunostimulatory activities, potentially leading to an improved therapeutic window compared to human wild-type IL-10. Although less potent than the IL-10 wild-type cytokine in several in vitro assays, IgG-IL-10I87A may be superior to fusion proteins comprising wild-type IL-10 in clinical benefit by more efficiently targeting inflamed tissues and reducing side effects caused by immunostimulatory properties.

In addition to the IL-10 wild-type cytokine and the single amino acid mutants I87A and R24A, several other single amino acid mutants as well as double mutants (i.e., a combination of two single amino acid mutations) of human IL-10 cytokine were investigated by SPR-based binding assays and in vitro potency assays. These additional mutants were similarly selected due to a known or expected decrease in affinity for human IL-10R 1. Interestingly, binding affinity to IL-10R1 is not always associated with in vitro potency. Importantly, human IL-10I87A had the lowest affinity (476pM) for human IL-10R1, but was not necessarily the least potent mutant tested in several cellular assays. The relatively low affinity for human IL-10R1, the retention of in vitro potency levels and the elimination of stimulatory properties similar to viral IL-10 may represent a unique advantage over other IL-10 mutants and could make IgG-IL-10I87A a promising candidate for therapeutic agents.

***

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

A fusion protein of an IgG-class antibody and a mutant IL-10 molecule, wherein the fusion protein comprises two identical heavy chain polypeptides and two identical light chain polypeptides, and wherein the mutant IL-10 molecule comprises an amino acid mutation that reduces the binding affinity of the mutant IL-10 molecule to the IL-10 receptor as compared to the wild-type IL-10 molecule.
2. The fusion protein of claim 1, wherein the mutant IL-10 molecule comprises an amino acid substitution at a position corresponding to residue 87 of human IL-10(SEQ id no: 1).
3. The fusion protein of claim 2, wherein the amino acid substitution is I87A.
4. The fusion protein of any one of the preceding claims, wherein the mutant IL-10 molecule is a homodimer of two mutant IL-10 monomers.
5. The fusion protein of any one of the preceding claims, wherein the mutant IL-10 molecule is a human IL-10 molecule.
6. The fusion protein of any preceding claim, wherein each of the heavy chain polypeptides comprises an IgG class antibody heavy chain and a mutant IL-10 monomer.
7. The fusion protein of claim 6, wherein the mutant IL-10 monomer is fused at its N-terminus to the C-terminus of the IgG class antibody heavy chain, optionally via a peptide linker.
8. The fusion protein of any one of the preceding claims, wherein the heavy chain polypeptides each consist essentially of an IgG class antibody heavy chain, a mutant IL-10 monomer, and optionally a peptide linker.
9. The fusion protein of any one of claims 6 to 8, wherein the mutant IL-10 monomer comprised in the heavy chain polypeptide forms a functional homodimeric mutant IL-10 molecule.
10. The fusion protein of any one of the preceding claims, wherein the IgG class antibody comprises a modification that reduces the binding affinity of the antibody to an Fc receptor as compared to a corresponding IgG class antibody without the modification.
11. The fusion protein of claim 10, wherein the Fc receptor is an fey receptor, in particular a human fey receptor.
12. The fusion protein of claim 10 or 11, wherein the Fc receptor is an activating Fc receptor.
13. The fusion protein of any one of claims 10 to 12, wherein the Fc receptor is selected from the group consisting of: fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32) and Fc α RI (CD 89).
14. The fusion protein of any one of claims 10 to 13, wherein the Fc receptor is fcyiiia, in particular human fcyiiia.
15. The fusion protein of any one of claims 10 to 14, wherein the IgG-class antibody comprises an amino acid substitution at position 329 of the antibody heavy chain (EU numbering)
16. The fusion protein of claim 15, wherein the amino acid substitution is P329G.
17. The fusion protein of any one of claims 10 to 16, wherein the IgG-class antibody comprises amino acid substitutions at positions 234 and 235 of the heavy chain of the antibody (EU numbering).
18. The fusion protein of claim 17, wherein the amino acid substitutions are L234A and L235A (LALA).
19. The fusion protein of any one of claims 10 to 18, wherein the IgG-class antibody comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in the antibody heavy chain.
20. The fusion protein of any one of the preceding claims, wherein the IgG-class antibody is IgG₁Subclass antibody.
21. The fusion protein of any one of the preceding claims, wherein the IgG-class antibody is a full-length antibody.
22. The fusion protein of any one of the preceding claims, wherein the IgG-class antibody is a human antibody.
23. The fusion protein of any one of the preceding claims, wherein the IgG-class antibody is capable of specifically binding to Fibroblast Activation Protein (FAP).
24. The fusion protein of claim 23, wherein the fusion protein is capable of having an affinity constant (K) of less than 1nM, in particular less than 100pM, when measured by Surface Plasmon Resonance (SPR) at 25 ℃_D) Combining FAP.
25. The fusion protein of claim 23 or 24, wherein the FAP is human, mouse, and/or cynomolgus FAP.
26. The fusion protein of any one of claims 23 to 25, wherein the IgG-class antibody comprises the amino acid sequence of SEQ ID NO: 37, cdr (hcdr)1 of heavy chain, SEQ ID NO: HCDR2 of 41, SEQ ID NO: 49 HCDR3 of SEQ ID NO: 53 light chain cdr (lcdr)1, SEQ ID NO: LCDR 2 of 57 and SEQ ID NO: LCDR 3 of 61.
27. The fusion protein of claim 26, wherein the IgG-class antibody comprises SEQ ID NO: 63 and the heavy chain variable region (VH) of SEQ ID NO: 65 (VL) in the light chain.
28. The fusion protein of any one of claims 23 to 25, wherein the IgG-class antibody comprises the amino acid sequence of SEQ ID NO: HCDR1 of 37, SEQ ID NO: 43, HCDR2 of SEQ ID NO: HCDR3 of SEQ ID NO: 51, LCDR 1 of SEQ ID NO: LCDR 2 of 55 and SEQ ID NO: LCDR 3 of 59.
29. The fusion protein of claim 28, wherein the IgG-class antibody comprises SEQ ID NO: 67 and the VH of SEQ ID NO: 69.
30. The fusion protein of any one of the preceding claims, wherein the fusion protein is capable of having an affinity constant (K) of about 100pM to about 10nM, particularly about 200pM to about 5nM, or about 500pM to about 2nM, when measured by SPR at 25 ℃_D) Binds to IL-10 receptor-1 (IL-10R 1).
31. The fusion protein of claim 30, wherein the IL-10R1 is human IL-10R 1.
32. The fusion protein of claim 30 when dependent on claim 23, wherein said affinity constant (K) for binding IL-10R1 is measured by SPR at 25 ℃_D) Greater than the affinity constant (K) for said binding FAP_D)。
33. The fusion polypeptide of any one of claims 1 to 25 or 28 to 32, wherein the heavy chain polypeptide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 96.
34. The fusion polypeptide of any one of claims 1 to 25 or 28 to 33, wherein the light chain polypeptide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 25.
35. A polynucleotide encoding the fusion protein of any one of the preceding claims.
36. A vector, particularly an expression vector, comprising the polynucleotide of claim 35.
37. A host cell comprising the polynucleotide of claim 35 or the vector of claim 36.
38. A method of producing a fusion protein of an IgG-class antibody and a mutant IL-10 molecule, comprising the steps of: (i) culturing the host cell of claim 37 under conditions suitable for expression of the fusion protein, and (ii) recovering the fusion protein.
A fusion protein of an IgG class antibody and a mutant IL-10 molecule produced by the method of claim 38.
40. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 34 or 39 and a pharmaceutically acceptable carrier.
41. The fusion protein of any one of claims 1 to 34 or 39 or the pharmaceutical composition of claim 40 for use as a medicament.
42. The fusion protein of any one of claims 1 to 34 or 39 or the pharmaceutical composition of claim 40 for use in the treatment or prevention of an inflammatory disease.
43. The fusion protein or pharmaceutical composition of claim 42, wherein the inflammatory disease is inflammatory bowel disease, rheumatoid arthritis, or idiopathic pulmonary fibrosis.
44. Use of the fusion protein of any one of claims 1-34 or 39 for the preparation of a medicament for treating a disease in an individual in need thereof.
45. The use of claim 44, wherein the disease is an inflammatory disease.
46. The use of claim 45, wherein the inflammatory disease is inflammatory bowel disease, rheumatoid arthritis or idiopathic pulmonary fibrosis.
47. The use of any one of claims 44 to 46, wherein the individual is a mammal, in particular a human.
48. A method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of a composition comprising the fusion protein of any one of claims 1 to 34 or 39 in a pharmaceutically acceptable form.
49. The method of claim 48, wherein the disease is an inflammatory disease.
50. The method of claim 49, wherein the inflammatory disease is inflammatory bowel disease, rheumatoid arthritis, or idiopathic pulmonary fibrosis.
51. The method of any one of claims 48 to 50, wherein the individual is a mammal, in particular a human.
52. The invention as described in the specification.