HK1187929B - Antibody molecules which bind il-17a and il-17f - Google Patents

Description

Antibody molecules that bind IL-17A and IL-17F

The present invention relates to antibody molecules specific for antigenic determinants of IL-17A and IL-17F. The invention also relates to the therapeutic use of said antibody molecules and to methods for producing said antibody molecules.

IL-17A (originally designated CTLA-8 and also known as IL-17) is a cytokine that promotes inflammation and is a founder member of the IL-17 family (Rouvier et al, 1993, J.Immunol.150: 5445-5456). Subsequently, 5 additional family members (IL-17B-IL-17F) were identified, including the most related IL-17F (ML-1) sharing about 55% amino acid sequence homology with IL-17A (Moseley et al, 2003, CytokineGrowth Factor Rev.14: 155-174). The recently defined subset Th17 of autoimmune-related helper T cells expresses IL-17A and IL-17F, and also IL-21 and IL-22 characteristic cytokines (Korn et al, 2009, Annu. Rev. Immunol.27: 485-) -517). IL-17A and IL-17F are expressed as homodimers, but can also be expressed as IL-17A/F heterodimers (Wright et al 2008, J.Immunol.181: 2799-2805). IL-17A and F signal through the receptors IL-17R, IL-17RC or the IL-17RA/RC receptor complex (Gaffen2008, cytokine.43: 402-. Both IL-17A and IL-17F are associated with a variety of autoimmune diseases.

Thus, bivalent antagonists of IL-17A and IL-17F would be more effective than single antagonists for treating IL-17 mediated diseases. Antibodies that bind to IL-17A and IL-17F have been described in WO2007/106769, WO2008/047134, WO2009/136286, and WO 2010/025400.

The present invention provides improved neutralizing antibodies that bind both IL-17A and IL-17F with high affinity. In particular, the antibodies of the invention are capable of specifically binding to both IL-17A and IL-17F, i.e., the antibodies do not bind to other subtypes of IL-17. Preferably, the antibodies of the invention also bind to the IL-17A/IL-17F heterodimer. Preferably, the antibodies of the invention neutralize the activity of IL-17A and IL-17F. In one embodiment, the antibodies of the invention also neutralize the activity of the IL-17A/IL-17F heterodimer. The antibodies of the invention thus have the advantageous property that they can inhibit the biological activity of both IL-17A and IL-17F. Accordingly, the invention also provides the use of such antibodies in the treatment and/or prophylaxis of diseases mediated by either or both IL-17A or IL-17F, such as autoimmune or inflammatory diseases or cancer.

The term "neutralizing antibody" as used herein describes an antibody capable of neutralizing both IL-17A and IL-17F biological signaling activity, for example by blocking the binding of IL-17A and IL-17F to its receptor(s) and by blocking the binding of IL-17A/IL-17F heterodimer to its receptor(s). It will be appreciated that the term "neutralization" as used herein refers to a reduction in biological signaling activity, which may be partial or complete. Further, it will be appreciated that the degree of neutralization of the activity of the antibody against IL-17A and IL-17F may be the same or different. In one embodiment, the degree of neutralization of IL-17A/IL-17F activity may be the same or different from the degree of neutralization of IL-17A or IL-17F activity.

In one embodiment, the antibodies of the invention specifically bind to IL-17A and IL-17F. By specifically binding is meant that the antibody has a greater affinity for IL-17A and IL-17F polypeptides (including IL-17A/IL-17F heterodimers) than for other polypeptides. Preferably, the IL-17A and IL-17F polypeptides are of human origin. In one embodiment, the antibody also binds to cynomolgus IL-17A and IL-17F.

IL-17A and IL-17F polypeptides or mixtures of both or cells expressing one or both of the polypeptides can be used to produce antibodies that specifically recognize both polypeptides. The IL-17 polypeptides (IL-17A and IL-17F) may be "mature" polypeptides or biologically active fragments or derivatives thereof, which preferably include a receptor binding site. Preferably, the IL-17 polypeptide is a mature polypeptide. IL-17 polypeptides may be produced from genetically engineered host cells comprising expression systems by processes well known to those skilled in the art, or may be recovered from natural biological sources. In the present application, the term "polypeptide" includes peptides, polypeptides and proteins. The foregoing may be used interchangeably unless otherwise indicated. The IL-17 polypeptide may in some cases be part of a larger protein, such as, for example, a fusion protein fused to an affinity tag. Antibodies raised against these polypeptides can be obtained by administering the polypeptides to animals, particularly non-human animals, where immunity is essential, using well-known conventional protocols, see, e.g., the Handbook of Experimental Immunology (Handbook of Experimental Immunology, d.m. weir (ed.), Vol4, Blackwell Scientific publications, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cattle or pigs, can be immunized. But generally mice, rabbits, pigs and rats are preferred.

Antibodies useful in the invention include whole antibodies and functionally active fragments or derivatives thereof and may be, but are not limited to, monoclonal, multivalent, multispecific, bispecific, humanized or chimeric antibodies, domain antibodies such as VH, VL, VHH, single chain antibodies, Fv, Fab fragments,Fab 'and F (ab')₂Fragments, and epitope-binding fragments of any of the above. Other antibody fragments include those described in international patent applications WO2005003169, WO2005003170 and WO 2005003171. Other antibody fragments include Fab-Fv and Fab-dsFv fragments as described in WO2009040562 and WO2010035012, respectively. Antibody fragments and Methods for their production are well known to those skilled in the art, see, e.g., Verma et al, 1998, Journal of immunological Methods,216,165-181, Adair and Lawson,2005.therapeutic antibodies, drug Design Reviews-on 2(3):209-217.

Antibodies useful in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that comprise an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD and IgA) or subtype of immunoglobulin molecule.

Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein,1975, Nature,256:495-

The antibodies useful in the present invention may also be produced by the method of single lymphocyte antibody by cloning and expressing immunoglobulin variable region cDNAs produced from single lymphocytes selected for the production of a particular antibody by, for example, the methods described in Babcook, J.et al, 1996, Proc.Natl.Acad.Sci.USA93(15):7843 and 7848l, WO92/02551, WO2004/051268 and International patent application No. WO 2004/106377.

Humanized antibodies are antibody molecules from non-human species having one or more Complementarity Determining Regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g., U.S. Pat. No. 5,585,089; WO 91/09967)

Chimeric antibodies are those antibodies encoded by immunoglobulin genes that have been genetically engineered so that their light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species. These chimeric antibodies may be less antigenic.

Bivalent antibodies can be prepared by methods known in the art (Milstein et al, 1983, Nature305: 537-. Multivalent antibodies may comprise multiple specificities or may be monospecific (see, e.g., WO92/22853 and WO 05/113605).

Antibodies for use in the present invention may also be produced using various phage display methods known in the art and include those disclosed by Brinkman et al (in J.Immunol. methods,1995,182:41-50), Ames et al (J.Immunol. methods,1995,184:177-186), Kettleborough et al (Eur.J.Immunol.1994,24:952-958), Persic et al (Gene,19971879-18), Burton et al (Advances in Immunology,1994,57:191-280) and WO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO 95/82; WO95/20401; and US5,698, 409, 5,223,580,908; 695,695,484; 695,5965,596; 695,484,596; 695,596, 5965,484; 695,484,596, 596, 427; 698,427; and; 698,427). Techniques for the production of single chain antibodies, such as those described in U.S. Pat. No. 4,946,778, may also be suitable for the production of single chain antibodies that bind IL-17A and IL-17F. Likewise, transgenic mice or other organisms, including other mammals, can be used to express humanized antibodies.

Antibody screening can be accomplished using an assay that measures binding to human IL-17A and human IL-17F, such as BIAcore described in the examples herein^TMAnd (5) checking and analyzing. Suitable neutralization assays are known in the art, see, e.g., WO2008/047134 and the examples described herein.

The residues of the antibody variable regions are conventionally encoded according to the system designed by Kabat et al. The system is proposed by Kabat et al, 1987, in Sequences of Proteins of Immunological Interest, US Department of health and Human Services, NIH, USA ("Kabat et al (supra)") henceforth. This coding system is used in this specification unless otherwise specified.

The Kabat residue designations do not always correspond directly to the linear coding of the amino acid amino group. The actual linear amino acid sequence may comprise fewer or additional amino acids than in a strict Kabat encoding corresponding to a shortening or insertion of a structural component, whether a framework or Complementarity Determining Region (CDR) of the variable region structure. The correct Kabat encoding of residues can be determined for a given antibody by alignment of homologous residues in the antibody sequence with "standard" Kabat encoding sequences.

The CDRs of the heavy chain variable region are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat coding system. However, according to Chothia (Chothia, C.and Lesk, A.M.J.mol.biol.,196,901-917 (1987)), the loop corresponding to CDR-H1 extends from residue 26 to residue 32. Thus "CDR-H1" as used herein comprises residues 26-35, which are described by a combination of the Kabat coding system and the topological loop definition of Chothia.

The CDRs of the light chain variable region are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat coding system.

One embodiment of the present invention provides a neutralizing antibody specific for human IL-17A and human IL-17F comprising a light chain wherein the light chain variable region comprises the sequence shown in SEQ ID NO:4 as CDR-L1, the sequence shown in SEQ ID NO:5 as CDR-L2, and the sequence shown in SEQ ID NO:6 as CDR-L3 (see FIG. 1 c).

The antibody molecule of the invention preferably comprises a complementary heavy chain.

Accordingly, one embodiment of the present invention provides a neutralizing antibody specific for human IL-17A and human IL-17F, further comprising a heavy chain, wherein the heavy chain variable region comprises at least one of the CDRs having the sequence shown in SEQ ID NO:1 as CDR-H1, having the sequence shown in SEQ ID NO:2 as CDR-H2, and having the sequence shown in SEQ ID NO:3 as CDR-H3 (see FIG. 1 c).

Another embodiment of the invention provides a neutralizing antibody specific for human IL-17A and human IL-17F comprising a heavy chain, wherein at least two of the CDR-H1, CDR-H12 and CDR-H3 of the variable region of the heavy chain are selected from the group consisting of: the sequence shown in SEQ ID NO.1 was designated as CDR-H1, the sequence shown in SEQ ID NO. 2 was designated as CDR-H2, and the sequence shown in SEQ ID NO. 3 was designated as CDR-H3. For example, an antibody may comprise a heavy chain in which CDR-H1 has the sequence shown in SEQ ID NO.1 and CDR-H2 has the sequence shown in SEQ ID NO. 2. Alternatively, the antibody may comprise a heavy chain in which CDR-H1 has the sequence shown in SEQ ID NO.1 and CDR-H3 has the sequence shown in SEQ ID NO. 3, or the antibody may comprise a heavy chain in which CDR-H2 has the sequence shown in SEQ ID NO. 2 and CDR-H3 has the sequence shown in SEQ ID NO. 3. For the avoidance of doubt, it is to be understood that all permutations are included.

Another embodiment of the present invention provides a neutralizing antibody specific for human IL-17A and human IL-17F comprising a heavy chain wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:1 as CDR-H1, the sequence shown in SEQ ID NO:2 as CDR-H2, and the sequence shown in SEQ ID NO:3 as CDR-H3.

In one embodiment, the antibody according to the invention comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO.1 as CDR-H1, the sequence shown in SEQ ID NO. 2 as CDR-H2, and the sequence shown in SEQ ID NO. 3 as CDR-H3, wherein the light chain variable region comprises the sequence shown in SEQ ID NO. 4 as CDR-L1, the sequence shown in SEQ ID NO. 5 as CDR-L2, and the sequence shown in SEQ ID NO. 6 as CDR-L3.

In one embodiment, the antibodies provided herein are monoclonal antibodies.

In one embodiment, the antibodies provided herein are CDR grafted antibody molecules comprising each of the CDRs provided in SEQ ID NOS: 1-6. The term "CDR grafted antibody molecule" as used herein refers to an antibody molecule wherein the heavy chain andand/or the light chain comprises one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g., a murine monoclonal antibody) grafted to a variable region framework from the heavy and/or light chain of an acceptor antibody (e.g., a human monoclonal antibody). See Vaughan et al, Nature Biotechnology,16535-539,1998 for review. In one embodiment, only one or more specificity determining residues of any of the CDRs described herein above are transferred into the human antibody framework, rather than the entire CDR (see, e.g., Kashmiri et al, 2005, Methods,36, 25-34). In one embodiment, only the specificity determining residues of one or more of the CDRs described herein above are transferred into the human antibody framework. In another embodiment, only the specificity determining residues of each of the CDRs described herein above are transferred into the human antibody framework.

When the CDRs or specificity determining residues are grafted, any suitable acceptor variable region framework sequence may be used, taking into account the type/type of donor antibody from which the CDRs are derived, including murine, primate and human framework regions. Preferably, the CDR-grafted antibody according to the invention has variable regions comprising human acceptor framework regions and one or more CDRs regions or specificity determining residues as described above. Thus, provided in one embodiment is a neutralizing CDR-grafted antibody, wherein the variable regions comprise human acceptor framework regions and CDRs from a non-human donor.

Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al, supra). For example, KOL and nemw can be used for the heavy chain, REI for the light chain and EU, LAY and POM for both heavy and light chains. Alternatively, human germline sequences may be used; these are inhttp://vbase.mrc-cpe.cam.ac.uk/Can be used as above.

In the CDR-grafted antibodies of the present invention, the acceptor heavy and light chains need not be derived from the same antibody and may include composite chains having framework sequences from different chains if desired.

A preferred framework for the heavy chain of the CDR-grafted antibody of the invention is derived from the human subtype VH3 sequence 1-33-07 previously described in WO2008/047134 along with JH 4. Accordingly, provided is a neutralizing CDR-grafted antibody comprising at least one non-human donor CDR, wherein the heavy chain framework region is derived from human subtype sequences 1-33-07 together with JH 4. The sequence of human JH4 is as follows: (YFDY) WGQGTLVTVSS. The portion of the YFDY motif that is CDR-H3 is not part of framework 4 (ravatch, JV. et al, 1981, Cell,27,583-

Preferred frameworks for the light chain of the CDR-grafted antibodies of the invention are derived from the human germline subtype VK1 sequence 2-1- (1) L4 previously described in WO2008/047134 along with JK 1. Thus, provided is a neutralizing CDR-grafted antibody comprising at least one non-human donor CDR wherein the light chain framework region is derived from the human subtype sequence VK12-1- (1) L4 together with JK 1. The sequence of human JK1 is as follows: (WT) FGQGTKVEIK. The portion of the WT motif that is CDR-L3 is not part of framework 4 (Hieter, PA., et al, 1982, J.biol.chem.,257, 1516-1522)

Likewise, in the CDR-grafted antibodies of the present invention, the framework regions need not have identical sequences to the framework regions of the acceptor antibody. For example, rare residues may become residues that occur more frequently for the classification or typing of receptor chains. Alternatively, selected residues in the acceptor framework regions may be altered so that they correspond to residues found at the same position in the donor antibody (see Reichmann et al, 1998, Nature,332, 323-324). This change should be kept to a minimum necessary to restore the affinity of the donor antibody. Protocols for selecting residues in the acceptor framework regions that may require alteration are set forth in WO 91/09967.

Preferably, in a CDR-grafted antibody molecule according to the present invention, if the acceptor heavy chain has the human VH3 sequences 1-33-07 together with JH4, the acceptor framework region of the heavy chain comprises, in addition to one or more donor CDRs, a donor residue at least position 94 (according to Kabat et al, (supra)). Accordingly, provided is a CDR-grafted antibody, wherein the residue at least at position 94 of the heavy chain variable region is a donor residue.

Preferably, in the CDR-grafted antibody molecule according to the present invention, if the acceptor light chain has the sequence 2-1- (1) L4 together with JK1 of human subtype VK1, no donor residues are transferred, i.e.only the CDRs are transferred. Thus, provided are CDR-grafted antibodies, wherein only the CDRs are transferred to the donor framework.

The donor residues are residues from the donor antibody, i.e. the antibody from which the CDRs were originally derived.

An antibody known as CA028_0496 (previously described in WO 2008/047134) was engineered in the present invention by changing 5 residues on the light chain. 3 residues in the CDRs and 2 in frame. Thus, in one embodiment the light chain variable region comprises an arginine residue at position 30, a serine residue at position 54, an isoleucine residue at position 56, an aspartic acid residue at position 60 and an arginine residue at position 72.

Thus, in one embodiment, an antibody of the invention comprises a light chain, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:7 (gL 7).

In another embodiment, an antibody of the invention comprises a light chain, wherein the light chain variable region comprises a sequence having at least 96% identity to the sequence set forth in SEQ ID NO. 7. In one embodiment, the antibody of the invention comprises a light chain, wherein the light chain variable region comprises a sequence having at least 97, 98, or 99% identity to the sequence set forth in SEQ ID NO. 7.

In one embodiment, the antibody of the invention comprises a heavy chain, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:9 (gH 9).

In another embodiment, the antibody of the invention comprises a heavy chain, wherein the heavy chain variable region comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO. 9. In one embodiment, the antibody of the invention comprises a heavy chain, wherein the heavy chain variable region comprises a sequence having at least 70%, 80%, 90%, 95%, 96, 97, 98 or 99% identity or similarity to the sequence set forth in SEQ ID NO. 9.

In one embodiment, the antibody of the invention comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO 9 and wherein the light chain variable region comprises the sequence shown in SEQ ID NO 7.

In another embodiment, an antibody of the invention comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO. 9 and wherein the light chain variable region comprises a sequence having at least 96% identity to the sequence set forth in SEQ ID NO. 7. Preferably, such antibodies comprise a heavy chain and a light chain, wherein the heavy chain variable region comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98 or 99% identity or similarity to the sequence set forth in SEQ ID NO. 9, and wherein the light chain variable region comprises a sequence having at least 96%, 97%, 98 or 99% identity to the sequence set forth in SEQ ID NO. 7.

As used herein, "identity" refers to the identity of amino acid residues between aligned sequences at any particular position in the sequences. "similarity", as used herein, refers to the similarity of amino acid residues between aligned sequences at any particular position in the sequences. For example, leucine may be replaced by isoleucine or valine. Other amino acids that can often be replaced with another amino acid include, but are not limited to:

phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

lysine, arginine and histidine (amino acids with basic side chains);

aspartic acid and glutamic acid (amino acids with acidic side chains);

asparagine and glutamine (amino acids with amide side chains); and

cysteine and methionine (amino acids with side chains containing sulfur). The degree of identity and similarity can be readily calculated (comparative Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York,1988; Biocomputing. information and Genome Projects, Smith, D.W., ed., Academic Press, New York,1993; Computer Analysis of Sequence Data, Part1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic, 1987; and Sequence Analysis, Prime, Green skin, device, J., New York, N.M., and Stock, 1991).

The antibody molecules of the invention may comprise whole antibody molecules having full-length heavy and light chains or fragments thereof, such as domain antibodies, e.g., VH, VL, VHH, Fab, modified Fab, Fab ', F (ab')₂Fv or scFv fragment. Other antibody fragments include the Fab-Fv and Fab-dsFv fragments described in WO2009040562 and WO2010035012, respectively. In one embodiment, the antibody fragment of the invention is selected from the group consisting of Fab, Fab ', F (ab')₂scFv or Fv fragments.

It will be appreciated that the antibodies of the invention, particularly the antibody fragments described above, may be incorporated into other antibody formats, particularly multispecific antibodies, such as dual or triple specific antibodies, in which one specificity is provided by the antibodies of the invention, i.e., specificity for IL-17A and IL-17F (including IL-17A/F heterodimers). Accordingly, in one embodiment of the invention there is provided a multispecific antibody comprising one or more antibody fragments as described herein above.

Examples of multispecific antibodies include bivalent, trivalent or tetravalent antibodies, bis-scFv, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, diploid, triploid (see e.g.Holliger and Hudson,2005, NatureBiotech23(9): 1126-. Other multispecific antibodies include Fab-Fv, Fab-dsFv, Fab-Fv, Fab-Fv-Fc and Fab-dsFv-PEG fragments described in WO2009040562, WO2010035012, WO2011/08609, WO2011/030107 and WO2011/061492, respectively.

The constant regions of the antibody molecules of the invention, if present, may be selected for the intended function of the antibody molecule, and in particular for the effector function which may be required. For example, the domain of the constant region may be a domain of human IgA, IgD, IgE, IgG or IgM. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, domains of the constant region of human IgG, particularly IgG1 and IgG3 isotypes, may be used. Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic use and antibody effector functions are not required, for example to simply block IL-17 activity. It will be appreciated that sequence variants of the domains of the constant region may also be used. For example, an IgG4 molecule can be used in which the serine at position 241 has been changed to proline as described in Angal et al, Molecular Immunology,1993,30(1), 105-108. Particularly preferred are IgG1 constant domains. It will also be appreciated by those skilled in the art that antibodies may undergo various post-translational modifications. The type and extent of modification often depends on the host cell line used to express the antibody and the culture conditions. These modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the deletion of a basic residue at the carboxy-terminal end (e.g., lysine or arginine) due to the action of carboxypeptidase (as described by Harris, RJ. journal of Chromatography705:129-134, 1995). Thus, for example, the C-terminal lysine of the heavy chain of the antibody of SEQ ID NO:15 shown in FIG. 2 (a) may be absent.

In one embodiment the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, i.e. κ or λ.

In a preferred embodiment, the antibodies provided herein are neutralizing antibodies specific for human IL-17A and human IL-17F, wherein the heavy chain constant region comprises the human IgG1 constant region. Accordingly, the present invention provides antibodies wherein the heavy chain comprises or consists of the sequence shown in SEQ ID NO. 15 (see FIG. 2 a).

In one embodiment of the invention, the antibody comprises a heavy chain, wherein the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO. 15. Preferably, the antibody comprises a heavy chain, wherein the heavy chain comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO. 15.

In one embodiment, an antibody molecule according to the invention comprises a light chain comprising the sequence shown in SEQ ID NO:11 (see FIG. 1 d).

In one embodiment of the invention, an antibody comprises a light chain, wherein the light chain comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO. 11. Preferably, the antibody comprises a light chain comprising a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO. 11.

One embodiment of the present invention provides an antibody wherein the heavy chain comprises or consists of the sequence shown in SEQ ID NO. 15 and wherein the light chain comprises or consists of the sequence given in SEQ ID NO. 11.

In one embodiment of the invention, an antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO. 15 and the light chain comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO. 11. Preferably, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO. 15, and wherein the light chain comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO. 11.

The antibody molecule of any aspect of the invention preferably has a high affinity, preferably picomolar. It will be appreciated that the binding affinity of an antibody according to the invention to human IL-17A may differ from the binding affinity of the same antibody to human IL-17F and/or IL-17A/F heterodimers. In one example, the antibody molecule of the invention has greater affinity for IL-17A than for IL-17F. In one example, the antibody molecule of the invention has an affinity for IL-17A that is at least 5-fold greater than its affinity for IL-17F. In one example, the antibody molecule of the invention has the same affinity for IL-17A as it has for IL-17F. In one example, the antibody molecules of the invention have picomolar affinity for IL-17A and IL-17F.

Affinity can be measured using any suitable method known in the art, including BIAcore as described in the examples herein^TMIsolated native or recombinant IL-17A and IL-17F present as homodimers are used.

Preferably, the antibody molecules of the invention have a binding affinity for IL-17A of 50pM or less. In one embodiment, the antibody molecule of the invention has a binding affinity for IL-17A of 20pM or less. In one embodiment, the antibody molecule of the invention has a binding affinity for IL-17A of 10pM or less. In one embodiment, the antibody molecule of the invention has a binding affinity for IL-17A of 5pM or less. In one embodiment, the antibody molecule of the invention has a binding affinity of 3.2pM for IL-17A.

Preferably, in one embodiment, the antibody molecule of the invention has a binding affinity for IL-17F of 100pM or less. In one embodiment, the antibody molecule of the invention has a binding affinity for IL-17F of 50pM or less. In one embodiment, the antibody molecule of the invention has a binding affinity for IL-17F of 23 pM.

It will be appreciated that the affinity of the antibodies provided herein may be altered using suitable methods known in the art. The invention therefore also relates to variants of the antibody molecules according to the invention which have an improved affinity for IL-17A and/or IL-17F. These variants can be obtained by a variety of affinity maturation protocols, including mutant CDRs (Yang et al, j.mol.biol.,254392- "403", 1995), strand rearrangement (chain shuffling) (Marks et al, Bio/Technology,10779-783, 1992), using a mutagenic strain of E.coli (Low et al, J.mol.biol.,250359-368, 1996), DNA rearrangement (Pattern et al, Curr. Opin. Biotechnol.,8724-733, 1997), phage display (Thompson et al, J.mol.biol.,25677-88,1996) and sexual PCR (Crameri et al, Nature,391,288-291,1998). Vaughan et al (supra) discussionThese methods of affinity maturation are described.

The antibodies used in the present invention may be conjugated to one or more effector molecules, if desired. It will be appreciated that the effector molecule may comprise a single effector molecule or two or more such molecules may be linked to form a single moiety which may be attached to an antibody of the invention. Where it is desired to obtain an antibody fragment linked to an effector molecule, it may be prepared by standard chemical procedures or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule either directly or via a coupling agent. Techniques for coupling such effector molecules to antibodies are well known to those skilled in the art (see Hellstrom et al, Controlled Drug Delivery,2nd Ed., Robinson et al, eds.,1987, pp.623-53; Thorpe et al, 1982, Immunol. Rev.,62:119-58 and Dubowchik et al, 1999, Pharmacology and therapeutic, 83, 67-123). Specific chemical schemes include, for example, those described in WO93/06231, WO92/22583, WO89/00195, WO89/01476 and WO 03031581. Alternatively, where the effector molecule is a protein or polypeptide, ligation may be achieved using recombinant DNA protocols, such as those described in WO86/01533 and EP 0392745.

The term effector molecule as used herein includes, for example, anti-tumour agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof and the like such as DNA, RNA and fragments thereof, radionuclides, particularly radioiodinates, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds capable of being detected by NMR or ESR spectroscopy.

Examples of effector molecules include cytotoxins or cytotoxic agents, including any agent that is not conducive to (or kills) cells. Examples include combretastatins, dolastatins, epothilone organisms, staurosporins, maytansinoids, spongistatins, rhizoxin, halichondrins, fisetin (roridins), hemiasterins, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax toxin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., dichloromethyldiethylamine, chlorambucil triamcinolone (thiovachloramucil), melphalan (melphalan), carmustine (BSNU) and lomustine (CCNU), cyclophosphamide (cyclothiophanate), busulfan, dibromomannitol (dibromonantol), streptozotocin, mitomycin C, and cisplatin (II) (DDP) cis-platinates), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly actinomycin), bleomycin, mithramycin, Amramycin (AMC), calicheamicin or duocarmycins), and antimitotic agents (e.g., vincristine and vinblastine).

Other effector molecules may include chelating radionuclides such as¹¹¹Indium and⁹⁰yttrium and lutetium¹⁷⁷Bismuth, bismuth²¹³Californium²⁵²Iridium (III)¹⁹²And tungsten¹⁸⁸Rhenium¹⁸⁸(ii) a Or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxanes, and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest, including, but not limited to, immunoglobulins, toxins such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, thrombogenic agents, or anti-angiogenic agents such as angiostatin endostatin, or biological response modifiers such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Nerve Growth Factor (NGF), or other growth factors and immunoglobulins.

Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for positron emission tomography), and nonradioactive paramagnetic metal ions for metal ions coupled to antibodies for diagnosis, see generally U.S. Pat. No. 4,741,900. suitable enzymes include horseradish peroxidase, alkaline phosphatase, β -galactosamine, or acetylcholinesterase, suitable prosthetic groups include streptavidin, avidin, and biotin, suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin, suitable luminescent materials include luminol, suitable bioluminescent materials include luciferase, fluorescein, and aequorin, and suitable radionuclides include luciferase, fluorescein, and aequorin¹²⁵Iodine,¹³¹Iodine,¹¹¹Indium and⁹⁹ are provided.

Another example of an effector molecule may increase the half-life of the antibody in vivo, and/or reduce the immunogenicity of the antibody and/or enhance delivery of the antibody to the immune system through the epithelial barrier. Examples of suitable such effector molecules include polymers, albumin binding proteins or albumin binding compounds as described in WO 05/117984.

When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene, polyoxyalkylene polymer, or branched or unbranched polysaccharide, such as a-or iso-polysaccharide.

Certain optional substituents, which may be present in the above-described synthetic polymers, include one or more hydroxyl, methyl, or methoxy groups.

Specific examples of synthetic polymers include optionally substituted linear or branched poly (ethylene glycol), poly (propylene glycol), poly (vinyl alcohol) or derivatives thereof, in particular optionally substituted poly (ethylene glycol) such as methoxypoly (ethylene glycol) or derivatives thereof.

Specific naturally occurring polymers include lactose, starch, dextran, glycogen or derivatives thereof.

As used herein, "derivative" is intended to include reactive derivatives, such as thiol-selective reactive groups such as maleimides and the like. The reactive group may be attached to the polymer directly or through a linker segment. It will be appreciated that the residue of such a group will in some cases be part of the product as a linking group for the antibody fragment to the polymer.

The size of the polymer may vary as desired, but will generally be in the range of an average molecular weight of 500Da to 50000Da, preferably 5000 to 40000Da and more preferably 20000 to 40000 Da. The polymer size may be selected in particular according to the intended use of the product, e.g. ability to localize to a specific tissue, such as a tumor, or to prolong the circulatory half-life (for review see Chapman,2002, Advanced Drug Delivery Reviews,54, 531-545). Thus, for example, where the product is expected to leave the circulatory system and penetrate tissue, for example for tumour therapy, it may be advantageous to use a polymer of small molecular weight, for example of around 5000 Da. When the product is in the circulation, it may be advantageous to use a high molecular weight polymer, for example having a molecular weight in the range of 20000Da to 40000 Da.

Particular preferred polymers include polyalkylene, such as poly (ethylene glycol) or, in particular, methoxypoly (ethylene glycol) or derivatives thereof, and in particular have a molecular weight in the range of from about 15000Da to about 40000 Da.

In one example, the antibodies used in the present invention are attached to a poly (ethylene glycol) (PEG) moiety. In one particular example, the antibody is an antibody fragment and the PEG molecule may be attached through any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, such as any free amino, imino, sulfhydryl, hydroxyl or carboxyl group. These amino acids may be naturally occurring on antibody fragments, or may be engineered into the fragment using recombinant DNA methods (see, e.g., U.S. Pat. No. 5,219,996; U.S. Pat. No. 5,667,425; WO 98/25971). In one example, the antibody molecule of the invention is a modified Fab fragment, wherein the modification is the addition of one or more amino acids at the C-terminus of its heavy chain to allow for the addition of effector molecules. Preferably, the additional amino acids form a modified hinge region comprising one or more cysteine residues to which effector molecules may be attached. Multiple sites can be used to attach two or more PEG molecules.

Preferably, the PEG molecules are covalently linked through a sulfhydryl group, wherein at least one cysteine residue is located on an antibody fragment. Each multimer molecule attached to an antibody fragment may be covalently linked to the sulfur atom of the cysteine residue located in the fragment. The covalent linkage will typically be a disulfide bond or, in particular, a sulfur-carbon bond. When a thiol group is used as the attachment site, appropriately activated effector molecules may be used, for example thiol-selective derivatives such as maleimide and cysteine derivatives. In preparing the above-described polymer-modified antibody fragments, an activated polymer may be used as a starting material. The activated polymer may be any polymer which includes a thiol reactive group such as an alpha-halocarboxylic acid or an ester such as iodoacetamide, an imine such as maleimide, a vinyl sulfone, or a disulfide. These starting materials can be obtained commercially (e.g., from Nektar, formerly Shearwater Polymers inc., Huntsville, AL, USA) or prepared from commercially available starting materials by conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar as Shearwater; RappPolymer; and SunBio) and M-PEG-SPA (available from Nektar as Shearwater).

In one embodiment, the antibody is a modified Fab fragment which is PEGylated, i.e.has PEG (poly (ethylene glycol)) covalently attached thereto, e.g.according to the method disclosed in EP0948544 [ see also "Poly (ethylene glycol) Chemistry, Biotechnical and biometrical Applications",1992, J.Milton Harris (ed.), Plenum Press, New York, "Poly (ethylene glycol) Chemistry and biological Applications",1997, J.Milton Harris and S.Zallipsky (eds), American chemical Society, WashingDC and "biological Coupling technology for the biological Sciences",1998, M.Assist, publication A.545, New publication, New 2002, Chapter 2002, published, Chapter A.54 ]. In one example, PEG is attached to cysteine of the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently linked to a single thiol group in the modified hinge region. Lysine residues may be covalently linked to maleimide groups and each amino group of a lysine residue may be attached to a methoxy poly (ethylene glycol) polymer of molecular weight about 20000 Da. The total molecular weight of the PEG attached to the Fab fragment may thus be about 40000 Da.

In one embodiment, the invention provides neutralizing antibody molecules specific for human IL-17A and human IL-17F which are modified Fab fragments having a heavy chain comprising the sequence shown in SEQ ID NO. 9 and a light chain comprising the sequence shown in SEQ ID NO. 7 and having a modified hinge region at the C-terminus of its heavy chain comprising at least one cysteine to which an effector molecule may be attached. Preferably, the effector molecule is PEG and it is attached using the methods described in WO98/25971 and WO2004072116, whereby a lysyl-maleimide group is attached to the cysteine residue at the C-terminus of the heavy chain and each amino group of the lysyl residue has covalently attached thereto a methoxypoly (ethylene glycol) having a molecular weight of about 20000 Da. The total molecular weight of the PEG attached to the antibody is therefore about 40000 Da.

In another example, effector molecules may be attached to antibody fragments using the methods described in International patent applications WO2005/003169, WO2005/003170 and WO 2005/003171.

The invention also provides isolated DNA sequences encoding the heavy and/or light chains of the antibody molecules of the invention. Preferably, the DNA sequence encodes the heavy or light chain of an antibody molecule of the invention. The DNA sequences of the present invention may include synthetic DNA such as produced by chemical processes, cDNA, genomic DNA or any combination thereof.

The DNA sequences encoding the antibody molecules of the invention may be obtained by means well known to those skilled in the art. For example, DNA sequences encoding part or all of the antibody heavy and light chains may be synthesized as required by the defined DNA sequences or according to the corresponding amino acid sequences.

DNA encoding acceptor framework sequences is widely available to those skilled in the art and can be readily synthesized based on its known amino acid sequence.

Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibody molecules of the invention. The desired DNA sequence may be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and Polymerase Chain Reaction (PCR) techniques may be used as appropriate.

Examples of suitable sequences are provided by SEQ ID NO 8, 10, 13, 14, 17, 18 and 19.

The invention also relates to cloning or expression vectors comprising one or more of the DNA sequences according to the invention. Accordingly, a cloning or expression vector comprising one or more DNA sequences encoding an antibody of the invention is provided. Preferably, the cloning or expression vector comprises two DNA sequences encoding the light and heavy chains of the antibody molecule of the invention and carrying suitable signal sequences, respectively. Preferably, the vector according to the invention comprises the sequences given in SEQ ID NO. 14 and SEQ ID NO. 18. In one embodiment, the vector according to the invention comprises the sequences given in SEQ ID NO 13 and SEQ ID NO 17.

Common methods for constructing vectors, transfection methods, and culture methods are well known to those skilled in the art. In this respect, reference is made to "Current Protocols in Molecular Biology", 1999, F.M. Ausubel (ed), Wiley Interscience, New York and the Manual produced by Cold spring harbor Publishing.

Host cells comprising one or more cloning or expression vectors containing one or more DNA sequences encoding the antibodies of the invention are also provided. Any suitable host cell/vector system may be used to express the DNA sequences encoding the antibody molecules of the present invention. Bacterial, e.g., E.coli, and other microbial systems may be used or eukaryotic, e.g., mammalian, host cell expression systems may also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

The invention also provides a method of producing an antibody molecule according to the invention comprising culturing a host cell comprising a vector of the invention under conditions suitable to cause expression of the protein from the DNA encoding the antibody molecule of the invention, and isolating the antibody molecule.

The antibody molecule may comprise only heavy or light chain polypeptides, in which case only heavy or light chain polypeptide coding sequences are required to transfect the host cell. For production of a product comprising both heavy and light chains, the cell line may be transfected with two vectors, the first vector encoding the light chain polypeptide and the second vector encoding the heavy chain polypeptide. Alternatively, a single vector may be used, the vector comprising sequences encoding both the light and heavy chains.

Since the antibodies of the invention are useful in the treatment and/or prevention of pathological conditions, the invention also provides pharmaceutical or diagnostic compositions comprising the antibody molecules of the invention in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. Thus, there is provided the use of an antibody according to the invention for the manufacture of a medicament. The compositions will often be supplied as part of a sterile pharmaceutical composition which will typically include a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention may additionally comprise a pharmaceutically acceptable adjuvant.

The invention also provides a method of preparing a pharmaceutical or diagnostic composition comprising adding and mixing the antibody molecules of the invention together with one or more pharmaceutically acceptable excipients, diluents or carriers.

The antibody molecule may be the sole active ingredient in a pharmaceutical or diagnostic composition or may be associated with other active ingredients including other antibody ingredients, such as anti-TNF, anti-IL-1 β, anti-T cells, anti-IFN γ or anti-LPS, or non-antibody ingredients such as xanthine or small molecule inhibitors.

The pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody according to the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent that treats, ameliorates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. For any antibody, a therapeutically effective amount can be determined in cell culture or initially evaluated in animal models, typically rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine the appropriate concentration range and route of administration. Such information can be used to determine useful dosages and routes for administration on humans.

The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, combination of drugs, sensitivity of response, and tolerance/response to treatment. The amount can be determined by routine experimentation and is within the judgment of the clinician. In general, a therapeutically effective amount is from 0.01mg/kg to 50mg/kg, preferably 0.1mg/kg to 20 mg/kg. The pharmaceutical compositions may conveniently be presented in unit doses containing a predetermined amount of the active agent of the invention in each dose.

The compositions may be suitable for use alone in a patient or may be administered in combination (e.g., simultaneously, sequentially or separately) with other agents, drugs or hormones.

The dosage at which the antibody molecule of the invention is administered depends on the nature of the condition to be treated, the degree of inflammation present and the use of the antibody molecule to be prophylactic or to treat an already existing condition.

The frequency of dosage will depend on the half-life of the antibody molecule and the duration of its action. One or more doses per day may be required if the antibody molecule has a short half-life (e.g. 2-10 hours). Alternatively, if the antibody molecule has a long half-life (e.g. 2-15 days) it may only be necessary to administer a dose once a day, once a week or even once every 1 or 2 months.

The pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactivated virus particles.

Pharmaceutically acceptable salts can be used, such as mineral acid salts, for example hydrochloride, hydrobromide, phosphate and sulphate, or organic acids, such as acetate, propionate, malonate and benzoate.

The pharmaceutically acceptable carrier in the therapeutic composition may additionally comprise liquids such as water, physiological saline, glycerol, ethanol. Furthermore, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in these compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include those suitable for parenteral administration, such as by injection or infusion, for example by bolus injection or continuous injection. When the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous medium and it may contain formulatory agents such as suspending, preserving, stabilising and/or dispersing agents. Alternatively, the antibody molecule may be in a dry form for reconstitution with a suitable sterile liquid prior to use.

Once formulated, the compositions of the present invention can be administered directly to a subject. The subject to be treated may be an animal. Preferably, however, the composition is suitable for administration to a human subject.

The pharmaceutical compositions of the present invention may be administered by any of a variety of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal (see, e.g., WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes. Painless hypodermic syringes may also be used to administer the pharmaceutical compositions of the present invention. Typically, therapeutic compositions are prepared as injectable liquid solutions or suspensions. Solid forms may also be prepared which are suitable for use as solutions and suspensions in liquid media prior to injection.

Direct delivery of the composition will generally be accomplished by subcutaneous, intraperitoneal, intravenous or intramuscular injection, or delivered to the interstitial space. The composition may also be administered to the lesion. The dose treatment may be a single dose schedule or a multiple dose schedule.

It will be appreciated that the active ingredient of the composition will be an antibody molecule. In this connection, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition needs to contain an agent that protects the antibody from degradation but which releases the antibody once absorbed through the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J.1991).

In one embodiment, the formulation is provided as an externally applied formulation comprising an inhalant.

Suitable inhalable preparations include inhalable powders, metered aerosols of inhalable solutions containing propellant gas or without propellant gas. The inhalable powders comprising the active substance according to the disclosure may consist of the active substance described above alone or in a mixture with physiologically acceptable excipients.

These inhalable powders may comprise monosaccharides (such as glucose or arabinose), disaccharides (such as lactose, sucrose, maltose), oligo-and polysaccharides (such as dextran), polyols (such as sorbitol, mannitol, xylitol), salts (such as sodium chloride, calcium carbonate) or mixtures of these with each other. The use of mono-or disaccharides, lactose or glucose, is suitable for use, particularly but not exclusively, in the form of their hydrates,

particles deposited in the lung require particles of a size of less than 10 microns, such as 1 to 9 microns, for example 0.1 to 5 μm, especially 1 to 5 μm. The particle size of the active ingredient (e.g., antibody or fragment) is of primary importance.

Propellant gases useful in the preparation of inhalable aerosols are known in the art. Suitable propellant gases are selected from the group consisting of chlorine or fluorine derivatives of hydrocarbons such as n-propane, n-butane or isobutane and halogenated hydrocarbons such as methane, ethane, propane, butane, cyclopropane or cyclobutane. The propellant gases mentioned above may be used alone or in mixtures thereof.

Particularly suitable propellant gas haloalkane derivatives are selected from TG11, TG12, TG134a and TG 227. Among the above halogenated hydrocarbons, TG134a (1,1,1, 2-tetrafluoroethane) and TG227(1,1,1,2,3,3, 3-heptafluoropropane) and mixtures thereof are particularly suitable.

The inhalable aerosols containing propellant gas may also contain other ingredients such as co-solvents, stabilizers, surface active agents (surfactants), antioxidants, lubricants and methods of adjusting pH. All of these ingredients are known in the art.

The inhalable aerosols according to the invention comprising propellant gas may comprise up to 5% by weight of active substance. The aerosols according to the invention comprise, for example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by weight of active ingredient.

Optional external administration to the lung may also be by administration of a liquid solution or suspension formulation, for example using a device such as a nebulizer, e.g., a nebulizer coupled to a compressor (e.g., a Pari LC-Jetplus (R) nebulizer coupled to a Pari Master (R) compressor, manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.)

The antibodies of the invention may be delivered dispersed in a solvent, for example in the form of a solution or suspension. Can be suspended in a suitable physiological solution, such as physiological saline or other pharmaceutically acceptable solvents or buffers. Buffers known in the art may comprise 0.05 to 0.15mg disodium sodium ethylenediaminetetraacetate, 8.0 to 9.0mg NaCl, 0.15 to 0.25mg polysorbate, 0.25 to 0.30mg anhydrous citric acid, and 0.45 to 0.55mg sodium citrate per 1ml of water to achieve a pH of about 4.0 to 5.0. Suspensions may be employed, for example, lyophilized antibodies.

The formulation of the therapeutic suspension or solution may also comprise one or more excipients. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (such as serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The solution or suspension may be encapsulated in liposomes or biodegradable microspheres. The formulations are typically provided in a substantially sterile form using aseptic manufacturing processes.

May include manufacture and sterilization by filtration of the buffered solvent/solution used for the formulation, sterile suspension of the antibody in a sterile buffered solvent solution, and dispersion of the formulation into sterile containers by methods familiar to those skilled in the art.

The spray formulations according to the present disclosure are provided as single metered units (e.g., sealed plastic containers or vials), for example, packaged in foiled envelopes. Each vial contains one unit dose by volume of solvent/solution buffer (e.g., 2 ml).

The antibodies disclosed herein may be suitable for delivery by spraying.

It is also contemplated that the antibodies of the invention may be administered by use of gene therapy. To achieve this, DNA sequences encoding the heavy and light chains of the antibody molecule are introduced into the patient under the control of suitable DNA components so that the chains of the antibody can be expressed from the DNA sequences and assembled in situ.

The invention also provides antibody molecules for use in the control of inflammatory diseases. Preferably, the antibody molecule may be used to reduce an inflammatory process or to prevent an inflammatory process.

The invention also provides antibody molecules according to the invention for use in the treatment or prophylaxis of pathological disorders mediated by IL-17A and/or IL-17F or associated with increased levels of IL-17A and/or IL-17F. Preferably, the pathological condition is selected from the group consisting of infections (viral, bacterial, fungal and parasitic), infection-related endotoxic shock, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic Juvenile Idiopathic Arthritis (JIA), Systemic Lupus Erythematosus (SLE), asthma, Chronic Obstructive Airway Disease (COAD), Chronic Obstructive Pulmonary Disease (COPD), acute lung injury, chronic pelvic inflammatory disease, alzheimer's disease, crohn's disease, inflammatory bowel disease, irritable bowel syndrome, ulcerative colitis, Castleman's disease, ankylosing spondylitis and other spondyloarthropathies, dermatomyositis, myocarditis, uveitis, exophthalmos, autoimmune thyroiditis, induration, celiac disease, gallbladder disease, Tibetan hair disease, peritonitis, psoriasis, atopic dermatitis, vasculitis, surgical adhesions, stroke, autoimmune diabetes, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis and Guillain-Barre syndrome, other autoimmune disorders, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, fibrotic disorders (including pulmonary fibrosis, liver fibrosis, kidney fibrosis, scleroderma or systemic sclerosis), cancer (solid tumors such as malignant melanoma, hepatoblastoma, sarcoma, squamous cell carcinoma, transitional cell carcinoma, ovarian cancer and hematologic malignancies and in particular acute myeloid leukemia, chronic lymphocytic leukemia, gastric cancer and colon cancer), heart disease including ischemic diseases such as myocardial infarction and atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, periodontitis and hypochlororhydia.

In one embodiment, the antibodies of the invention are useful for the treatment and prevention of a pathological disorder selected from the group consisting of arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic Juvenile Idiopathic Arthritis (JIA), Systemic Lupus Erythematosus (SLE), asthma, chronic obstructive airway disease, chronic obstructive pulmonary disease, atopic dermatitis, scleroderma, systemic sclerosis, pulmonary fibrosis, inflammatory bowel disease, ankylosing spondylitis and other spondyloarthropathies and cancers.

In one embodiment, the antibodies of the invention are used for the treatment and prevention of a pathological disorder selected from the group consisting of: arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic Juvenile Idiopathic Arthritis (JIA), Systemic Lupus Erythematosus (SLE), asthma, chronic obstructive airways disease, chronic obstructive pulmonary disease, atopic dermatitis, scleroderma, systemic sclerosis, pulmonary fibrosis, crohn's disease, ulcerative colitis, ankylosing spondylitis and other spondyloarthropathies and cancers.

In one embodiment, the antibodies of the invention are used for the treatment and prevention of a pathological disorder selected from the group consisting of: arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic Juvenile Idiopathic Arthritis (JIA), Systemic Lupus Erythematosus (SLE), asthma, chronic obstructive airways disease, chronic obstructive pulmonary disease, atopic dermatitis, scleroderma, systemic sclerosis, pulmonary fibrosis, crohn's disease, ulcerative colitis, ankylosing spondylitis and other spondyloarthropathies.

In one embodiment the pathological disorder is rheumatoid arthritis.

In one embodiment the pathological disorder is crohn's disease.

In one embodiment the pathological disorder is ulcerative colitis.

In one example, the antibodies of the invention are used to treat inflammation or immune-related diseases. In one example, the inflammatory or immune-related disease is selected from rheumatoid arthritis, crohn's disease, ulcerative colitis.

The invention also provides said antibody molecule according to the invention for use in the treatment or prevention of pain, in particular pain associated with inflammation.

The invention further provides the use of an antibody molecule according to the invention for the preparation of a medicament for the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F or associated with increased levels of IL-17A and/or IL-17F. Preferably, the pathological disorder is one of the medical indications described herein above. The invention further provides the use of an antibody molecule according to the invention for the preparation of a medicament for the treatment or prophylaxis of pain, in particular pain associated with inflammation.

The antibody molecules of the invention may be used in any therapy requiring a reduction in the effects of IL-17A and/or IL-17F in the human or animal body. IL-17A and/or IL-17F may circulate in the body or may be present at an undesirably high level of localization at a particular site in the body, such as a site of inflammation.

The antibody molecule according to the invention is preferably used for the control of inflammatory diseases, autoimmune diseases or cancer.

The invention also provides a method of treating a human or animal subject undergoing or at risk of an IL-17A and/or IL-17F mediated disorder, said method comprising administering to the subject an effective amount of an antibody molecule according to the invention.

The antibody molecules according to the invention may also be used for diagnosis, for example in vivo diagnosis and imaging of disease states involving IL-17A and/or IL-17F.

The invention is further described by way of example only in the following examples, which refer to the accompanying drawings, in which:

FIG. 1 shows a schematic view of a

a) Light chain V region of antibody CA 028-0496 g 3(SEQ ID NO:7)

b) Heavy chain V region of antibody CA 028-0496 g 3(SEQ ID NO:9)

c) CDRH1(SEQ ID NO:1), CDRH2(SEQ ID NO:2), CDRH3(SEQ ID NO:3), CDRL1(SEQ ID NO:4), CDRL2(SEQ ID NO:5), CDRL3(SEQ ID NO:6) of antibody CA028_496g 3.

d) The light chain of antibody CA028_496g 3(SEQ ID NO: 11).

e) The light chain of antibody CA028_496g 3(SEQ ID NO:12) including a signal sequence.

FIG. 2

a) The heavy chain of antibody CA028_496g 3(SEQ ID NO: 15).

b) The heavy chain of antibody CA028_496g 3(SEQ ID NO:16) including a signal sequence.

c) DNA (NO signal sequence) encoding the light chain of antibody CA028_496g 3(SEQ ID NO: 13).

FIG. 3

a) DNA encoding the light chain of antibody CA028_496g 3(SEQ ID NO:14) including a signal sequence

b) DNA encoding the light chain variable region of antibody CA028_496g 3(SEQ ID NO:8)

c) DNA encoding the heavy chain variable region of antibody CA028_496g 3(SEQ ID NO:10) including a signal sequence

FIG. 4 DNA (including exons) encoding the heavy chain of antibody CA 028-496 g3 without signal sequence (SEQ ID NO:17)

FIG. 5 DNA (including exon and signal sequence) encoding the heavy chain of antibody CA028_496g 3(SEQ ID NO:18)

FIG. 6 cDNA encoding the heavy chain of antibody CA028_496g 3(SEQ ID NO:19) including a signal sequence.

FIG. 7 effects of antibodies CA028_0496 (designated 496g1 in the legend) and CA028_00496.g3 (designated 496.g3 in the legend) on the induction of IL-6 production from HeLa cells by human IL-17F.

Example 1: production of improved neutralizing antibodies that bind IL-17A and IL-17F

The isolation and humanization of antibody CA028_0496 has been described previously in WO 2008/047134. CA028_0496 is a neutralizing antibody that binds to humanization of IL-17A and IL-17F and comprises the grafted variable regions gL7 and gH9, the sequences of which are provided in WO 2008/047134. The heavy chain acceptor framework is human germline sequence VH31-33-07 with framework 4 from this portion of the human JH region germline JH 4. The light chain acceptor framework is the human germline sequence VK12-1- (1) L4 with framework 4 from this portion of the human JK region germline JK 1.

Antibody CA028_00496 was affinity matured to improve the affinity of the antibody for IL-17F while retaining affinity for IL-17A. In contrast to antibody CA028_00496, the antibody after affinity maturation was called CA028_00496.g3 and was expressed as IgG1 instead of IgG 4. The genes were modified by oligonucleotide directed mutagenesis to generate affinity matured versions. The affinity matured light chain variable region (gL 57) gene sequence was subcloned into the human light chain expression vector pkh10.1 of UCBCelltech, which contained DNA encoding the human C-Kappa constant region (Km 3 allo-anti-immunoglobulin). The unaltered heavy chain variable region gH9 (gH 9) sequence was subcloned into UCB Celltech expression vector pVhg1FL, which contains DNA encoding the human heavy chain gamma-1 constant region. Lipofectamine was used according to the manufacturer's instructions (InVitrogen, catalog No. 11668)^TM2000 the plasmid was co-transfected into CHO cells.

The final sequence of the variable region after affinity maturation of antibody CA028_00496.g3 is given in fig. 1a and 1 b. In antibody CA028_00496.g3, the heavy chain variable region sequence is identical to that in the parent antibody CA028_00496. In contrast, the light chain variable region differs by 5 amino acids. The 5 residues that differ between the light chain of antibody CA028_00496 and antibody CA028_00496.g3 are underlined in fig. 1 a.

Example 2 BIACORE

The assay format was to capture antibody CA 028-00496. g3 by an immobilized anti-human IgG Fc specific antibody, followed by titration of human IL-17A and IL-17F on the captured surface, as described below.

Biomolecular interaction Analysis (Biamolecular interaction Analysis) was performed by Biacore3000 (Biacore AB). The measurement was carried out at 25 ℃. Goat anti-human IgG Fc specific affinity purification F (ab')₂(Jackson Immuno Research) was immobilized onto a CM5 sensor chip (Biacore AB) by amino coupling chemistry to a level of approximately 6000 Reaction Units (RU). HBS-EP buffer (10mM HEPES pH7.4,0.15M NaCl,3mM EDTA,0.005% surfactant P20, Biacore AB) was used as the running buffer at a flow rate of 10. mu.L/min (min). 10 μ L of 0.5 μ g/mL CA028_00496.g3 was injected for capture by immobilized anti-human IgG Fc. Human IL-17A (UCB produced internally) was titrated at a flow rate of 30 μ L/min for 3min on the captured CA 028-00496. g3, starting at 5nM, followed by 20min desorption. Human IL-17F (R)&D systems) titrated on captured CA028_00496.g3 for 3min at a flow rate of 30 μ L/min starting at 10nM, followed by 5min desorption. The surface was regenerated at a flow rate of 10. mu.L/min by injection of 10. mu.L of 40mM HCl followed by injection of 5. mu.L of 5mM NaOH.

TABLE 1 affinity of CA028_496.g3 for human IL-17F and IL-17A

Binding curves minus the double reference background were analyzed using BIAevaluation software (version 4.1) according to standard procedures. Kinetic parameters were determined by a fitting algorithm. The data are detailed in table 1, with the average values highlighted in grey.

The affinity value determined for the original antibody CA 028-0496 binding to IL-17A was 16pM and for IL-17F was 1750 pM. In contrast, the improved antibody CA 028-0496 g3 had an affinity for IL-17A of 3.2pM and for IL-17F of 23 pM. Antibody CA028_0496 improved the affinity for IL-17F by more than 70-fold without decreasing the affinity of the antibody for IL-17A. In fact, the affinity for IL-17A increased by a factor of 5.

The affinity of CA028_0496g3 for the IL-17A/F heterodimer (prepared as described in WO 2008/047134) was also improved, where the affinity was found to be 26pM (data not shown).

Example 3

The efficacy of CA028_00496.g1 (previously described in WO 2008/047134) and CA028_00496.g3 for neutralizing human IL-17F was determined using HeLa cell bioassay. HeLa cells were obtained from the ATCC cell bank (ATCC CCL-2). Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, gentamicin and glutamate. 1x10⁴Cells were plated onto 96-well flat-bottom tissue culture plates, cells were incubated overnight and washed once in assay buffer HeLa cells were stimulated with a combination of recombinant human IL-17F (125 ng/ml) and tumor necrosis factor- α (TNF- α) (1 ng/ml) in the presence of different concentrations of the antibody for 48 hours in the HeLa cell line IL-17F synergistically with TNF- α to induce IL-6 production, IL-6 was quantitated using a specific MSD assay kit, using the MesoScale Discovery (MSD) assay technique and calculated IC₅₀The values measure the final amount of secreted IL-6. CA028_00496.g1 and CA028_00496.g3 showed concentration-dependent inhibition of the biological activity of IL-17F as measured in the bioassay of HeLa cells (fig. 7). The activity of CA028_00496.g1 and CA028_00496.g3 in HeLa cell assays was expressed as the dose required to inhibit 50% of the activity of IL-17F (IC)₅₀). IC of CA028_00496.g1₅₀IC of 92mg/mL and CA0496.g3₅₀It was 4 ng/mL.

The ability of CA028_00496.g3 to neutralize IL-17A was confirmed using the same assay as previously described for CA028_00496.g1 in WO2008/047134, in which IL-17F was replaced by IL-17A (data not shown).

Claims (31)

1. A neutralizing antibody that binds human IL-17A and human IL-17F having a light chain and a heavy chain, wherein the light chain variable region has the sequence shown in SEQ ID NO. 7 and the heavy chain variable region has the sequence shown in SEQ ID NO. 9.

2. The antibody of claim 1, which also binds to an IL-17A/IL-17F heterodimer.

3. The antibody of claim 1 or 2, wherein the antibody is a whole antibody or a functionally active fragment thereof.

4. The antibody of claim 3, wherein the antibody fragment is selected from the group consisting of Fab, Fab ', F (ab')₂scFv and Fv fragments.

5.The antibody of claim 1, wherein the antibody is multispecific.

6. A neutralizing antibody that binds human IL-17A and human IL-17F having a heavy chain comprising the sequence set forth in SEQ ID NO. 15 and a light chain comprising the sequence set forth in SEQ ID NO. 11.

7. An isolated DNA sequence encoding the heavy and light chains of the antibody of any one of claims 1-6.

8. A cloning or expression vector comprising a DNA sequence according to claim 7.

9. The vector of claim 8, wherein the vector comprises the sequences shown in SEQ ID NO 13 and SEQ ID NO 17.

10. A host cell comprising one or more cloning or expression vectors according to claim 8 or claim 9.

11. A method for producing an antibody according to any one of claims 1-6, comprising culturing the host cell of claim 10 and isolating the antibody.

12. A pharmaceutical composition comprising an antibody according to any one of claims 1-6 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

13. The pharmaceutical composition of claim 12, further comprising an additional active ingredient.

14. An antibody according to claim 1 or a pharmaceutical composition according to claim 12 or 13 for use in therapy.

15. The antibody according to claim 1 or the pharmaceutical composition according to claim 12 or 13 for use in the treatment or prevention of a pathological condition, selected from the group consisting of infection, infection-related endotoxic shock, arthritis, chronic obstructive airways disease, acute lung injury, chronic pelvic inflammatory disease, alzheimer's disease, inflammatory bowel disease, irritable bowel syndrome, castleman's disease, spondyloarthropathy, dermatomyositis, myocarditis, uveitis, celiac disease, gallbladder disease, hirsutism, peritonitis, psoriasis, atopic dermatitis, vasculitis, surgical adhesions, stroke, type I diabetes, lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, trauma, transplant rejection, fibrotic disorders, cancer, heart disease, intravascular coagulation, bone resorption, osteoporosis and periodontitis.

16. The antibody or the pharmaceutical composition of claim 15, wherein the infection is selected from the group consisting of viral, bacterial, fungal, and parasitic infections.

17. The antibody or the pharmaceutical composition of claim 15, wherein the arthritis is selected from the group consisting of: rheumatoid arthritis, psoriatic arthritis and systemic juvenile idiopathic arthritis.

18. The antibody or the pharmaceutical composition of claim 15, wherein the chronic obstructive airways disease is selected from the group consisting of: asthma and chronic obstructive pulmonary disease.

19. The antibody or the pharmaceutical composition of claim 15, wherein the inflammatory bowel disease is selected from the group consisting of: crohn's disease and ulcerative colitis.

20. The antibody or the pharmaceutical composition of claim 15, wherein the autoimmune disorder is selected from the group consisting of: systemic lupus erythematosus, autoimmune thyroiditis, autoimmune diabetes and ankylosing spondylitis.

21. The antibody or the pharmaceutical composition of claim 15, wherein the immune-mediated inflammatory disorder of the central and peripheral nervous system is selected from the group consisting of: multiple sclerosis and Guillain-Barre syndrome.

22. The antibody or the pharmaceutical composition of claim 15, wherein the wound is a surgical wound.

23. The antibody or the pharmaceutical composition of claim 15, wherein the cancer is a solid tumor.

24. The antibody or the pharmaceutical composition of claim 23, wherein the solid tumor is selected from the group consisting of: malignant melanoma, hepatoblastoma, sarcoma, squamous cell carcinoma, transitional cell carcinoma, ovarian cancer, gastric cancer and colon cancer.

25. The antibody or the pharmaceutical composition of claim 15, wherein the cancer is hematological malignancy.

26. The antibody or the pharmaceutical composition of claim 25, wherein the cancer hematologic malignancy is selected from: acute myelogenous leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia.

27. The antibody or the pharmaceutical composition of claim 15, wherein the cardiac disease is an ischemic disease.

28. The antibody or the pharmaceutical composition of claim 27, wherein the ischemic disease is selected from the group consisting of: myocardial infarction and atherosclerosis.

29. The antibody or the pharmaceutical composition of claim 15, wherein the transplant rejection is graft versus host disease.

30. The antibody or the pharmaceutical composition of claim 15, wherein the fibrotic disorder comprises pulmonary fibrosis, liver fibrosis, kidney fibrosis, scleroderma, or systemic sclerosis.

31. Use of an antibody according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 12 or 13 for the preparation of a medicament for the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F or associated with increased levels of IL-17A and/or IL-17F.