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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

• the present invention relates to engineered antibody polypeptides. More particularly, the present invention provides antibody polypeptides comprising an antigen binding site, wherein the antigen binding site comprises a human variable domain having at least one New World Primate CDR. In particular the present invention relates to antibody polypeptides directed against TNF- ⁇ .
• TNF- ⁇ Tumor Necrosis Factor- ⁇
• RA rheumatoid arthritis
• Crohn's disease ulcerative colitis and other bowel disorders
• psoriasis toxic shock
• graft versus host disease multiple sclerosis.
• TNF- ⁇ result Ln tissue injury, such as inducing procoagulant activity on vascular endothelial cells (Pobcr, et «/., 1986, i. Immunol. 136:1680-1687), increasing the adherence of neutrophils and lymphocytes (Pobcr, etal., 1987, J. Immunol. 138:3319-3324). and stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Carnussi, et al., 1987, J. Exp. Med. 166: 1390-1404).
• TNF- ⁇ is synthesized as a 26 HD transmembrane precursor protein with an intracellular tail that is cleaved by a TNF- ⁇ -converting mctalloproieinase enzytne and then secreted as a 17 kD soluble protein.
• the active form consists of a homotrimer of the 17 kD monomers which interacts with two different cell surface receptors, p55 TNFRI and p75 TNFR2.
• p55 TNFRI two different cell surface receptors
• p75 TNFR2 cell surface bound precursor form of TNF- ⁇ can mediate some biological effects of the factor.
• Most cells express both p55 and p75 receptors which mediate different biological functions of the ligand.
• the ⁇ 75 receptor is implicated in triggering lymphocyte proliferation, and the p55 receptor is implicated in TNF-mediated cytotoxicity, apoptosis, antiviral activity, fibroblast proliferation and NF- ⁇ B activation (see Locksley et al., 2001, Cell 104: 487- 501 ).
• the TNF receptors are members of a family of membrane proteins including the NGF receptor, Fas antigen, CD27, CD30, CD40, 0x40 and the receptor for the lymphotoxin ⁇ / ⁇ heterodimer. Binding of receptor by the homotrimer induces aggregation of receptors into small clusters of two or three molecules of cither p55 or p75.
• TNF- ⁇ is produced primarily by activated macrophages and T lymphocytes, but also by neutrophils, endothelial cells, keratinocytes and fibroblasts during acute inflammatory reactions.
• TNF- ⁇ is at the apex of the cascade of pro-inflammatory cytokines (Reviewed in Feldmann & Maini, 2001, Ann. Rev. Immunol. 19: 163-196V This cytokine induces the expression or release of additional proinflammatory cytokines, particularly IL-I and IL-6 (see, for example, Rutgeerts el «/., 2004, Gastroenterology 126: 1593-1610).
• TNF- ⁇ inhibits the production of inflammatory cytokines including IL- 1 , IL-6, IL-8 and GM-CSF ( ⁇ rennan et ⁇ L, 1989, Lancet 2: 244-247). Because of Us role in inflammation, TNF- ⁇ has emerged as an important inhibition target in effort's to reduce the symptoms of inflammatory disorders. Various approaches to inhibition of TNF-u for the clinical treatment of disease have been pursued, including particularly the use of soluble TNF-ct receptors and antibodies specific for TNF- ⁇ .
• TNF- ⁇ is highly expressed in inflamed synovium, particularly at ihe cartilage-panmis junction (DiGi ⁇ vine et ⁇ l., 1988, Ann. Rheum. Dis. 47: 768-772; Fircstein et at., 1990, J. Immunol. 144: 3347-3353; and Saxne et al, 1988, Arthritis Rheum.
• TNF- ⁇ increases [he levels of inflammatory cytokines IL-I, IL-6, IL-8 and GM-CSF, TNF- ⁇ can alone trigger joint inflammation and proliferation of fibroblast- 1 ike synoviocytes (Gitter et al, 1989, Immunology (5(5: 196-200), induce collagenase, thereby triggering cartilage destruction (Dayer et al., 1985, J. Exp. Med. 162: 2163-2168; Dayer et al, 1986, J. Clin. Invest.
• TNF- ⁇ induces increased release of CDl 4+ monocytes by the bone marrow.
• Such monocytes can infiltrate joints and amplify the inflammatory response via the RANK (Receptor Activator of NF-KB)-RANiKL signaling pathway, giving rise to osteoclast formation during arthritic inflammacion (reviewed in Anandamjah & Richlin, 2004, Cutr. Opin. Rheumatol. 16: 338-343).
• TNF- ⁇ is an acute phase protein which increases vascular permeability through its induction of IL-8, thereby recruiting macrophage and neutrophils to a siie of infection. Once present. activaLed macrophages continue to produce TNF- ⁇ , thereby maintaining and amplifying the inflammatory response.
• TNF- ⁇ Titration of TNF- ⁇ by the soluble receptor construct etanercept has proved effective fo ⁇ the treatment of RA, but not for treatment of Crohn's disease.
• the antibody TNF-u antagonist infliximab is effective to treat both RA and Crohn's disease.
• the mere neutralization of soluble TNF- ⁇ is not the only mechanism involved in anti- TNF-based therapeutic efficacy. Rather, the blockade of other pro-inflammatory signals or molecules that arc induced by TNF- ⁇ also plays a role (Rutgeerts etal., supra).
• the administration of infliximab apparently decreases the expression of adhesion molecules, resulting in a decreased infiltration of neutrophils to sites of inflammation.
• infliximab therapy results in the disappearance of inflammatory cells from previously inflamed bowel mucosa in Crohn's disease.
• This disappearance of activated T cells in the lamina intestinal is mediated by apoptosis of cells carrying memhrane-bound TNF- ⁇ following activation of caspascs 8, 9 and ihen 3 in a Fas dependent manner (see Lugering et ciL, 2001, Gastroenterology 121 : 1 145-1157).
• membrane- or receptor- bound TNF- a is an impoit ⁇ nt target for ant ⁇ -TNF- ⁇ therapeutic approaches.
• infliximab binds to activated peripheral blood cells and lamina intestinal cells and induces apoptosis through activation of caspase 3 (sec Van den Brande et a!., 200,3, Gastroenterology 124: 1774- J 785).
• TNF-cz TNF-cz to its receptor triggers a cascade of signaling events, including displacement of inhibitory molecules such as SODD (.silencer of death domains) and binding of the adaptor factors FADD, TRADD, TRAF2, c-fAP, R ⁇ IDD and TRIP plus the kinase RIPl and certain caspases (reviewed by Che” & Goeddcl, 2002, Science 296: 1634-1635, and by Muzio & Saccani in :Mcthods in Molecular Medicine: Tumor Necrosis Factor, Methods and
• the assembled signalling complex can activate either a cell .survival pathway, through NF- ⁇ B activation and subsequent downstream gene activation, or an apoptotic pathway through caspase activation. Similar extracellular downstream cytokine cascades and intracellular .signal transduction pathways can be induced by TNF- ⁇ in other diseases. Thus, for other diseases or disorders in which the TNF-u molecule contributes to the pathology, inhibition of TNF- ⁇ presents an approach to treatment.
• Angiogenesis plays an important role in the active proliferation of inflammatory .synovial tissue.
• VEGF Vascular endothelial growth factor
• VEGF expression in the joints increased upon induction of the disease, and the administration of anti-VEGF airtisera blocked the development of arthritic disease and ameliorated established disease (Sone et al, 2001, Biochcui. Biophys. Res. Comm. 281: 562-568; Lu et aL, 2000, J. Immunol. 164: 5922-5927).
• Antibodies are highly specific for their binding targets and although they are derived from nature's own defence mechanisms, antibodies face several challenges when applied to the treatment of disease in human patients.
• Conventional antibodies are large multi-subunit protein molecules comprising at least four polypeptide chains.
• human IgG has two heavy chains and two light chains that are disulfide bonded to form the functional antibody.
• the size of a conventional IgG is about 150 kD. Because of their relatively large size, complete antibodies (e.g., IgG 7 IgA, IgM, etc.) are limited in their therapeutic usefulness due to problems in, for example, tissue penetration. Considerable efforts have focused on identifying and producing smaller antibody fragments that retain antigen binding function and solubility.
• the heavy and light polypeptide chains of antibodies comprise variable (V) regions that directly participate in antigen interactions, and constant (C) regions that provide structural support and function in non-antigen-specific interactions with immune effectors.
• the antigen binding domain of a conventional antibody is comprised of two separate domains: a heavy chain variable domain (VH) and a light chain variable domain (VL: which can be either VK or V ⁇ ).
• VH heavy chain variable domain
• VL light chain variable domain
• the antigen binding site itself is formed by six polypeptide loops: three from the VH domain (Hl, H2 and H3) and three from the VL domain (Li, L2 and L3).
• a diverse primary repertoire of V genes that encode die VH and VL domains is produced by the combinatorial rearrangement of gene segments.
• C regions include the light chain C regions (referred to as CL regions) and the heavy chain C regions (referred to as CHl , CH2 and CH3 regions).
• CL regions the light chain C regions
• CHl , CH2 and CH3 regions the heavy chain C regions
• a number of smaller antigen binding fragments of naturally occurring antibodies have been identified following protease digestion. These include, for example, the "Fab fragment” (VL-CL-CHl-VH), "Fab' fragment” (a Fab with the heavy chain hinge region) and “F(ab')2 fragment” (a dimer of Fab' fragments joined by the heavy chain hinge region).
• Recombinant methods have been used to generate even smaller antigen-binding fragments, referred to as “single chain Fv” (variable fragment) or "scFv,” consisting of VL and VH joined by a synthetic peptide linker.
• Single Domain Antibodies While the antigen binding unit of a naturally-occurring antibody (e.g., in humans and most other mammals) is generally known to be comprised of a pair of V regions (VL/ V H), camelid species express a large proportion of fully functional, highly specific antibodies that are devoid of light chain .sequences. The camelid heavy chain antibodies are found a ⁇ homodimcrs of a single heavy chain, dimcrizcd via their constant regions.
• variable domains of these camelid heavy chain antibodies are referred to as VHH domains and retain the ability, when isolated as fragments of the VH chain, to bind antigen with high specificity (Hamers-Casierman et al, 1993, Nature 363: 446-448; Gahroudi et aL, 1997, FEBS Lett, 414: 521 -526).
• Antigen binding single VH domains have also been identified from, for example, a library of murine Vl I genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. c ⁇ i (Ward el al., 19H9, Nature 341: 544- 546). Ward et al.
• dAbs the isolated single VH domains "dAbs," for “domain antibodies”.
• the term “dAb” will refer herein to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen.
• VH, VHH or VL immunoglobulin variable domain
• a “dAb” binds antigen independently of other V domains; however, as the term is iused herein, a “dAb” can be present in a homo- or heteromultimer with other VH or VL domains where the other domains are not required for antigen binding by die dAb, i.e., where the dAb binds antigen independently of the additional VH, VHH or VL domains.
• Single immunoglobulin variable domains for example, VHH, are the smallest antigen-binding antibody unit known.
• human antibodies are preferred, primarily because they are not as likely to provoke an immune response when administered to a patient. Isolated non-camclid VH domains tend to be relatively insoluble and are often poorly expressed. Comparisons of cameiid VHH with the VH. domains of human antibodies reveals several key differences in the framework regions of the camelid VHH domain corresponding to the VR/VL interface of the human VH domains. Mutation of these residues of human VH3 to more closely resemble the VHH sequence (specifically GIy 44 GIu, Leu 45 Arg and Trp 47 GIy) has been performed to produce "camelizcd" human VK domains (Davics & Riechmann, 1994, FEBS Lett.
• Variable domain amino acid numbering used herein is consistent with the Kabat numbering convention (Kabat etal, 1991, Sequences of Immunological Interest, 5th ed. U.S. Dcpt. Health & Human Services, Washington, D.C.).
• WO 03/035694 (Muylderman ⁇ ) reports that a Trp 103 Arg muration improves the solubility of non- camelid VH domains.
• the antigen binding domain of an antibody comprises two separate regions; a heavy chain variable domain (VH) and a light chain variable domain (VL: which can be either VK or V ⁇ ).
• the antigen binding site itself is formed by six polypeptide loops: three from VH domain (Hl, H2 and H3) and three from VL domain (Ll. L2 and L3).
• VH domain Hl, H2 and H3
• VL domain Ll. L2 and L3
• a diverse primary repertoire of V genes that encode the VH and VL domains is produced by the combinatorial rearrangement of gene segments.
• the VI I gene is produced by the recombination of three gene segments, VH, D and Jl 1. In humans, there arc approximately 51 functional VH segments (Cook and Tomlinson, 1995, Immunol. Today, 16: 237), 25 functional D segments (Corbett et al., 1997 J. MoI.
• VH segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the VH domain (H( and H2), whilst the VH, D and JH segments combine to form the third antigen binding loop of the VH domain (H3).
• the VL gene is produced by the recombination of only two gene segments, VL and JL. Iu humans, there are approximately 40 functional VK segments (Schable and Zachau (1993) Biol. Chem.
• the VL segment encodes the region of the polypeptide chain which forms the first and .second antigen binding loops of the VL domain (Ll and L2), wbiJ.st the VL and JL segments combine to font, the third antigen binding loop of the VL domain (L3).
• Antibodies selected from this primary repertoire are believed to be sufficiently diverse to bind almost all anligcns with at least moderate affinity.
• High affinity antibodies are produced by "affinity maturation" of the rearranged genes, in which point mutations are generated and selected by the immune system oil the basis of improved binding.
• H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main- chain conformations for short loop lengths which depend on Qic length and the presence of particular residues, or types of residue, at key positions IQ the loop and the antibody framework (Martin et al., 1996, J. MoI. Biol, 263: 800-815; Shirai et al., 1996, FEBS Letters, 399: 1-8).
• Bispecifio antibodies comprising 'complementary pairs of VH and VL regions are known in Uie art. These bispecific antibodies must comprise two pairs of VH and VLs, each VH/VL pair binding to a single antigen or epitope.
• each antibody species comprises two antigen-binding sites, each fashioned by a complementary pair of VI i and VL domains. Each antibody is thereby able to bind to two different antigens or epitopes at the same time, with the binding to EACH antigen or epitope mediated by a VH and its complementary VL domain.
• Each of diese lecluiiques presents its particular disadvantages; for instance in the cose of hybrid hybridomas, inactive VI f/VL pairs can greatly reduce the fraction of bispecific IgG.
• eross-veactivc antibodies have been so described, usually where the two antigens arc related, in sequence and structure, such as hen egg while lysozyme and turkey lysozyme (McCafferty et ⁇ /., WO 92/01047) or to free hapten and to hapten conjugated to carrier (Griffiths et at., 1994, EMBO J 13:14 3245- 60).
• WO 02/02773 have been so described, usually where the two antigens arc related, in sequence and structure, such as hen egg while lysozyme and turkey lysozyme (McCafferty et ⁇ /., WO 92/01047) or to free hapten and to hapten conjugated to carrier (Griffiths et at., 1994, EMBO J 13:14 3245- 60).
• WO 02/02773 have been so described, usually where the two antigens arc related
• the antibody molecules referred to are antibodies raised or selected against multiple antigens, such that their specificity spans more than a single antigen.
• Each complementary VH/VL pair in the antibodies of WO 02/02773 specifies a single binding specificity for two or more structurally related antigens; the VH and VL domains in such complementary pairs do not " 20 each possess a separate specificity.
• the antibodies thus have a broad single specificity which encompasses two antigens, which are structurally related.
• natural autoantibodies have been described that are polyrcactive (Casali & Notkins, 1989, Ami. Rev. Immunol. 7, 515-531), reacting with at least two (usually more) different antigens or epitopes mat are not structurally related. Jt has also been shown that selections of random
• camel heavy chain single domains are unusual in that they are derived from natural camel antibodies which have no light chains, and indeed the heavy chain single domains are uuable to associate with camel light chains to form complementary VH and VL pairs.
• Single heavy chain variable domains have also been described, derived from natural antibodies which arc normally associated with light chains (from monoclonal antibodies or from repertoires of domains; see EP-A-0368684). These heavy chain variable domains have been shown to interact specifically with one or more related antigens but have not been combined with other heavy or light chain variable domains to create a liga ⁇ d with specificity for two or more different antigens.
• Human/mouse chimeric antibodies have been created in which antibody variable region sequences from the mouse genome are combined with antibody constant region sequences from the human genome.
• the chimeric antibodies exhibit the binding characteristics of the parental mouse antibody, and the effector functions associated with the human constant region.
• the antibodies are produced by expression in a host cell, including for example Chinese Hamster Ovary (CHO), NSO myeloma cells, COS cells and SP2 cells.
• Such chimeric antibodies have been used in human therapy, however antibodies to these chimeric antibodies have been produced by the human recipient. Such anti-chimeric antibodies are detrimental to continued therapy with dumcric antibodies.
• human monoclonal antibodies are expected to be an improvement over mouse monoclonal antibodies for in vivo human therapy. From work done with antibodies from Old World primates (rhesus monkeys and chimpanzees) it has been postulated that these non-human primate antibodies will be tolerated in humans because they arc structurally similar to human antibodies ( ⁇ hrlieh PH era/,, 1988, Human and primate monoclonal antibodies for in viva therapy. Clin Chem.
• EP 0 605442 discloses chimeric antibodies which bind human antigens. These antibodies comprise the whole variable region from an Old World moi ⁇ cey and the constant region of a human or chimpanzee antibody.
• One of the advantages suggested in this reference for lhesc constructs Ls the ability to raise antibodies in Old World monkey.s to human antigens which are less immunogenic in humans compared with antibodies raised in a mouse host.
• Mew World primates comprise at least 53 species commonly divided into two families, the Callithric ⁇ dae and Cebidae.
• the CaUithricxdae consist of marmosets and tamarins.
• the Cebidae includes the squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, uakaris, sakis, night or owl monkey and the howler monkey. n
• Evolutionaitily distant primates such as New World primates, are not only .sufficiently different from humans to allow antibodies against human antigens to be generated, but arc sufficiently similar to humans to have antibodies similar to human antibodies so that the host does not generate an anti-antibody immune response when such primate-derived antibodies are introduced into a human.
• a first aspeet the present invention provides a chimeric antibody polypeptide comprising an antigen binding site, wherein the antigen binding site comprises a human variable domain having at least one New World Primate CDR.
• the present invention provides a method of producing an antibody polypeptide according to the first aspeet of the invention, the method comprising the steps of: (i) providing an acceptor sequence encoding a human variable domain; and
• the present invention provides a chimeric domain antibody (dAb) which hinds human TNF- ⁇ , the dAb comprising an immunoglobulin heavy or light chain variable domain, wherein said variable domain comprises at least one New World Primate CDR.
• dAb chimeric domain antibody
• the present invention provides a pharmaceutical composition comprising an effective amount of an antibody polypeptide according to the first or third aspects of the invention, together with a pharmaceutically acceptable earner or diluent.
• Figure 1 shows the amino acid (SEQ ID No:6) and nucleotide .sequence (SEQ ID No:5) of the acceptor d Ab.
• Figure 2 shows the nucleotide and amino acid sequences of eleven (I I) marmoset and six. (6) Owl monkey VK geuc segments.
• Figure 3 shows the acceptor dAb amino acid and nucleotide sequence (both strands).
• the restriction digest .sites for Kpn 1 and San DI which excises a region including the CDR2 is indicated in the figure.
• CDR2 residues removed are indicated m underline.
• Figure 4 shows sequence alignments showing oligonucleotides used during cloning and final sequence confirmation of the nucleotide (A) and amino acid (B) sequences shown in Figure 2.
• Figure 5 demonstrates the ability of CDR2-grafted d ⁇ bs to inhibit the binding of TNF to recombinant TNF receptor.
• Figure 6 demonstrates the improved ability of Compounds KK) and 123 to neutralise the cytotoxic activity of TNF on mouse L929 fibroblasts relative to Compound 145.
• the present invention provides a chimeric antibody polypeptide comprising an antigen binding site, wherein the antigen binding site comprises a human variable domain having at least one New World Primate CDR.
• the present invention provides a chimeric domain antibody (d ⁇ b) which binds human TNF- ⁇ , the dAb comprising an immunoglobulin heavy or light chain variable domain, wherein said variable domain comprises at least one New World Primate CDR.
• the present invention provides a pharmaceutical composition comprising au effective amount of an antibody polypeptide according to the first or third aspects of the invention, together with a pharmaceutical Iy acceptable carrier or dilueni.
• the human variable domain comprises at least one human framework region having an amino acid sequence encoded by a human gcrmline antibody gene segment, or an amino acid sequence comprising up to 5 amino acid differences relative to the amino acid sequence encoded by lhe human gcrmlinc antibody gene segment.
• the human valuable domain preferably comprises f ⁇ iir human framework regions, FRl, FR2, FR3 and FR4 having amino acid sequences encoded by a human gcnnline antibody gene segment, or the amino acid sequences which collectively contain up to 10 amino acid differences relative to the amino acid sequences encoded by said human germline antibody gene seqvnent
• the human gt ⁇ rmlinc antibody gene segment selected from the group consisting of DP47, DP45. DP48 and DPK9.
• the New World Primate CDR may be may CDR, however, it is preferred that the New World Primate CDR is CDR2.
• CDR J CDR J or CDR3.
• New World Primate CDR sequence is a germline New World Primate CDR sequence.
• the antibody polypeptide of the present invention is preferably selected from a d ⁇ b, scFv, Fab, (Fab') 2 , Fv, disulpbide bonded Fv, IgG, and a diabody.
• the antibody polypeptide of the present invention h preferably directed against TNF-o.
• the human variable domain amino acid sequence comprises a Kpnl restriction site spaced from a SanDl restriction site, said CDR of the human variable domain being between the restriction sites.
• the New World Primate CDR sequence is obtainable from New World Primale DN ⁇ by PCR using primer pair VKlBL (SEQ ID No: 11) / VKI BL35a (SEQ ID No:12) or primer pair VKlBL (SEQ ID No:ll / VKlBL35b (SEQ ID No: 13).
• the present invention also provides a chimeric domain antibody (dAb) which binds to human TNF-ci, wherein the dAb is a human dAb thai binds human TNF- ⁇ in which at least one of the CDRs is replaced with the corresponding CDR from a New World Primate.
• dAb chimeric domain antibody
• the present invention also provides a method of producing an antibody polypeptide according to the first aspect of the invention, the method comprising the steps of:
• step (ii) said CDR of said human variable domain is replaced by said donor New World Primate CDR using restriction digestion and annealing of an oligonucleotide encoding the donor CDR into the acceptor sequence. It is preferred that the method further comprises affinity maturing the variable domain produced in step (ii).
• New World Primate CDR refers to a CDR sequence obtained from a New World Primate.
• human framework region refers to a framework region obtained from a human or a human framework region having an amino acid sequence encoded by a human gemiline antibody gene segment, or an amino acid .sequence comprising up to 5 amino acid differences relative to the amino acid sequence encoded by the human gcrniline gene segment.
• the term also encompasses modification of the amino acid sequence of the framework region in order to obtain improved antigen binding characteristics or lower immunogenicity such as disclosed in US 4,816,567, US 5,585,089 and US 20030039649 the disclosures of which are incorporated herein by reference in their entirety. Typically where modifications are made the total number of residues changed will be 10 or less collectively over the frame%vork regions.
• variable domain comprises four framework regions, wherein at least one framework region comprises an amino aeid sequence derived from a corresponding framework region encoded by a human germlinc immunoglobulin gene.
• the four framework regions cojnpri.se amino acid sequences derived from corresponding framework regions encoded by human germline immunoglobulin genes tn yet a further preferred embodiment the human germline immunoglobulin gene is .selected from ihc group consisting of DP47, DP45, DP48 and DPK9.
• domain as used herein is meant a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
• immunoglobulin or antibody "variable domain” as used herein is a term of ait, and includes a folded polypeptide domain comprising sequences characteristic of immunoglobulin or antibody heavy or light chain variable domains and which specifically binds an antigen.
• immunoglobulin refers to a family of polypeptides which retain the immunoglobulin fold characteristic of antibody molecules, which contains two ⁇ sheets and, usually, a conserved disulphide bond.
• the present invention is applicable to all immunoglobulin superfatnily molecules which possess binding domains.
• the present invention relates to antibody polypeptides.
• New World primates comprise at least 53 species commonly divided into two families, the Callithricidae and Cehi ⁇ ae, The Callithrici ⁇ ae consist of marmosets and tamarins.
• the Cebiclae includes the squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, uakaris, sakis. night or owl monkey and the howler monkey.
• Evolutionarily distant primates such as New World primates, are not only sufficiently different from humans to allow antibodies against human antigens to be generated, but are sufficiently similar to humans to have antibodies similar to human antibodies so that the host does not generate an anti-antibody immune response when such primate-derived antibodies are introduced into a human.
• the New World primate CDR is from the family Callithricidae.
• the New World primate CDR is selected from the group consisting of marmosets, tamarins, squirrel monkey, titi monkey, spider monkey, woolly moiikey, capuchin, uakaris, sakis, night or owl monkey and the howle ⁇ monkey. More preferably, the New World primate is a marmoset.
• the at least one New World primate CDR is substantially identical to a CDR encoded by a New World primate germline immunoglobulin gene.
• antibody as used herein, is intended to refer to immunoglobulin molecules comprised of two heavy chains or immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two lighr (L) chains inter- connected by disulfide bonds.
• Each heavy chain is comprised of a heavy chain variable region (HCVR or V H ) and a heavy chain constant region.
• the heavy chain constant region comprises 11
• Each light chain is comprised of a light chain variable region (LCVR or VL) and a light chain constant region.
• the light chain constant region is comprised of one domain, C ⁇ .
• the V H and VL regions can be further subdivided into regions of hypcrvariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• FR framework regions
• Each V H and V L is composed of three CDRs and four FRs, arranged from aroino-tcrminus to earbuxy-le ⁇ ninus in the following order: FRl, CDR 1 , FR2, CDR2, FR3, CDR3, FR4,
• antibody polypeptide refers to a polypeptide comprising one or more components or derivatives of an immunoglobulin that exhibit the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody.
• binding fragments encompassed within the term "antibody polypeptide” include (i) a Fab fragment, a monovalent fragment consisting of the VL, V H , C and C H ⁇ domains; (ii) a F(ab'h fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (i ⁇ ) a Fd fragment consisting of the Vn and CuI domains; (iv) a Fv fragment consisting of the V ⁇ and V H domains of a single arm of an antibody; (v) a d ⁇ b fragment (Ward et al, 1989, Nature 341 :544-546) which consists of a single V H domain, or a V L domain (van den Beucken et al, 2001 , J.
• iwo domains of the Fv fragment, Vi. and V H arc coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Vi. and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); (see eg Bird et al, 1988, Science 242:423-426 and Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883).
• scFv single chain Fv
• Sucb single chain Fvs are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
• Other forms of single chain Fvs and related molecules such as diabodies or triabodies are also encompassed.
• Diabodies are bivalent antibodies in which V H and Vr. domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, et al., 1993, Proc. Natl. Acad. Sci.
• the antibody polypeptide is selected from, the group consisting of a dAb, scFv, Fab, F(ab')2. Pv, di.sulphide bonded Fv, a diabody and IgG.
• the antibody polypeptide further comprises a human or non-human primate constant region sequence.
• non-human primates include, but arc uot limited to, chimpanzees, o ⁇ mguaiangs and baboons.
• the constant region sequence is preferably obtained from a human or non- human primate immunoglobulin sequence.
• the primate sequence may be a New World primate or an Old World primate sequence. Suitable Old World primates include chimpanzee, or other hoininid ape eg. gorilla or orang utan, which because of their close phyl ⁇ genetic proximity to humans, share a high degree of homology with the human constant region sequence. Sequences which encode for human or primate constant regions are available from databases including e.g. The National Centre for Biotechnology Information protein and nucleotide databases, The Kabat Database of Sequences of Proteins of Immunological Interest.
• the antibody polypeptide is a domain antibody (dAb).
• Domain antibodies are small functioning binding units of antibodies and correspond to the variable regions of either the heavy (Vn) or light (Vi.) chains of antibodies. Domain antibodies have a molecular weight, of approximately 13 kDa, or less than one tenth the size of a full antibody.
• Antibody light chains are referred to as either kappa or lambda light chains and the heavy chains as gamma, mu, delta, alpha or epsilon.
• the variable region gives the antibody its specificity.
• regions of hypervariabilily otherwise known as complementarity determining regions (CDRs) which are flanked by more conserved regions referred to as framework regions.
• CDRs complementarity determining regions
• framework regions Within each variable region are three CDRs and four framework regions.
• domain antibodies are wel) expressed in bacterial, yeast and mammalian systems. Their small size allows for higher molar quantities per gram of product, thus providing a significant increase in potency per dose.
• domain antibodies can be used as a buiiding block to create lhcrapctitic products such as multiple targeting dAbs in which a construct containing two or more variable domains bind to two or more therapeutic targets, ⁇ r dAbs targeted for pulmonary or oral administration.
• an increase' in binding is demonstrated by a decrease in Kp (k ⁇ /k ⁇ ) for the antibody or antigen binding portion thereof.
• An increase in potency is demonstrated in biological assays.
• assays that can be used Io measure the potency ot lhe antibody or antigen-binding portion thereof include the TNF ⁇ -mduced L929 cytotoxicity neutralisation assay, TL-12-induced human PHA-aciivated peripheral blood mononuclear cell (PBMC) proliferation assay, and RANKL mediated osteoclast differentiation of mouse splcnocytcs (Stem, 1990, Proe. Natl. Acad. Sci. USA 87:6808 - 6812; Kong, et uL, 1990, Nature 397:315 - 323; Matthews and Nealc in Lymphokines and Interferons, a Practical
• Tlic CDR sequences may be obtained from several sources, for example, databases e.g. The National Centre for Biotechnology Information protein and nucleotide databases www.ncbi.nlm.nih.gov. The Kabat Database of Sequences of Proteins of Immunological Merest www.kabatdatabase.com, or the IMCJT database www.imgt.cines.fr.
• the CDR regions can be predicted from the V H and Vi, domain repertoire (see for example Kabat and Wu, 1971, Ann. NY Acad. Sci. 190:382-393).
• the CDR sequence may be a genomic DNA or a cDNA.
• the preferred method of the present invention involves replacement of the CDR2 in the variable region domain via primer directed mutagenesis. This method consists of annealing a synthetic oligonucleotide encoding a desired mutations Lo a target region where it serves as a primer for initiation of DNA synthesis in vitro ⁇ extending the oligonucleotide by a DNA polymerase to generate a double-stranded DNA that carries the desired mutations, and ligating and cloning the sequence into an appropriate expression vector.
• variable domain sequence into which the CDR is grafted is the "dAb acceptor sequence" (designated Compound J 28; SEQ ID No:6) provided in Figure 1.
• dAb acceptor sequence designated Compound J 28; SEQ ID No:6
• chimeric is meant that the antibody polypeptide or domain antibody includes sequences from more than one species.
• the anii-hunian TNF- ⁇ dAb according to the invention can be used to delect human TNF- ⁇ for example in a biological sample, such as scrum or plasma using a conventional immunoassay, such as an enzyme linked immunosorbent assay (KLISA), a radioimmunoassay (RlA) or tissue irnmunohistochcmistry.
• KLISA enzyme linked immunosorbent assay
• RlA radioimmunoassay
• tissue irnmunohistochcmistry tissue irnmunohistochcmistry.
• the anti-human TNF- ⁇ d ⁇ b according to the invention can be assayed in biological fluids by a competition immunoassay using recombinant human TNF- ⁇ standards label led with a detectable substance and an vin label led anti-human TNF- ⁇ antibody.
• the anti-human TNF- ⁇ dAb according to the invention may also be used to delect TNF- ⁇ from species other than humans eg. chimpanzee, marmoset, rhesus, mouse, pig,
• the anti-human TNF- ⁇ dAb according to the invention may also be used in cell culture applications where it is desired to inhibit TNF- ⁇ activity.
• the invention also provides a method for treating a disorder characterised by human TNF- ⁇ activity in a human subject, comprising administering lo the subject a pharmaceutical composition according to the second aspect of the invention.
• a disorder characterised by human TNP- ⁇ activity is intended to include diseases and other disorders in which the presence of TNF- ⁇ in a subject suffering froin the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor which contributes to a worsening of the disorder.
• the disorder characterised by human TNF- ⁇ activity is selected from the group consisting of inflammation, inflammatory diseases, sepsis, including septic shock, e ⁇ dotoxic shock, gram negative sepsis and toxic shock syndrome; autoimmune disease, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome; infectious disease, including fever and myalgias due to infection and cachexia secondary to infection; graft versus host disease; tumour growth or metastasis; pulmonary disorders including adult respiratory distress syndrome, shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis; hiflammaiory bowel disorders including Crohn's disease and ulcerative colitis; cardiac disorders; inflammatory bone disorders, hepatitis, coagulation disturbances, burns, repeifasion injury
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of the antibody polypeptide according to the first aspect of the invention or a chimeric domain antibody according to the third aspect of the invention, together with a pharmaceutically acceptable carrier or diluent.
• a pharmaceutically acceptable carrier includes any aod all solvents, dispersion media, coatings, antibacterial and antifungal -agents, isotonic and absorption delaying agents, and the like which are physiologically compatible.
• pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like as well as combinations thereof.
• isotonic agents for example, sugars, polyalcohols such as inannitol, sorbitol, or sodium chloride in the composition.
• Pharmaceutically acceptable substances such as welling or minor amounts of auxiliary substances such, as wetting or emulsifying agents, preservatives or buffers.
• the composition may be in a variety of forms, including liquid, semi-solid and solid dosage forms, such as liquid solutions (eg injectable and infusible solutions), dispersions or suspensions, tablets, pills, powder, liposomes and suppositories.
• the composition is in the form of an injectable solution for immunization.
• the administration may be intravenous, subcutaneous, intraperitoneal, intramuscular, transdermal, intrathecal, and mtra-artevial.
• Therapeutic compositions iypkaily must be sterile and stable under the conditions of manufacture and storage.
• the compositions can be formulated as a solution, microemul.siort, dispersion, liposome, or other ordered structure suitable to high drug concentration.
• Sterile injectable solutions can be prepared by incorporating the active compound (i.e. antibody polypeptide) into the required amount in an appropriate solvent with one or a combination of ingredients listed above, followed by filtered sterilisation.
• composition may also be formulated as a sterile powder for the preparation of sterile injectable solutions.
• the proper fluidity of a solution can be maintained by for example, use of a coating such as lecithin and/or surfactants.
• the active compound mjiy be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, a ⁇ d microencapsulated delivery systems.
• a carrier that will protect the compound against rapid release
• Compatible polymers may be used such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoeslers and polylactic acid.
• composition may also be formulated for oral administration.
• the antibody polypeptide may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
• composition may also be formulated for rectal administration.
• Supplementary active compounds can also be incorporated into the composition.
• the antibody polypeptide may be co-formulated with aud/or co-administered with one or more additional therapeutic agents eg. anti-inllammatory compounds, soluble TNF- ⁇ receptor or a chemical agent, that inhibits human TNF- ⁇ production, or antibodies that bind other targets such as cytokines or cell surface molecules.
• additional therapeutic agents eg. anti-inllammatory compounds, soluble TNF- ⁇ receptor or a chemical agent, that inhibits human TNF- ⁇ production, or antibodies that bind other targets such as cytokines or cell surface molecules.
• a soluble iimiiiuiochcniical reagent such as protein A, C, G or L.
• An effective amount may include a therapeutically effective amount or prophylactically effective amount of the antibody polypeptide of the invention.
• ⁇ therapeutically effective amount refers to an amount, effective at dosages and for periods of time accessary, to achieve the desired therapeutic result.
• a prophylactically effective amount refers to an amount effective, at dosages and for periods of lime necessary, fo achieve the desired prophylactic result.
• composition is administered to mammals, preferably humans or primates.
• Marmoset (genus Callithrix, species unknown) and Owl monkey (Aolus trivirgatus) ⁇ cnomic DNA were obtained from the European Collection of Cell Cultures (ECACC), catalogue numbers 85011419 and 90110510 respectively. Marmoset DNA was derived from eel! line B95-8 while Owl monkey DNA came from cell line OMK 637-69.
• Genomic PCR (30 cycles) was performed using Taq polymerase with either primer pair VKIBLxVKI B L35a or VKlBLxVKlBL35b. There was overlap between the sequences cloned and the two primer .sets used.
• PCR products were cloned into Invitrogen's TOPO TA cloning kit (Cat No K4500-01) and sequenced with M13 forward and pUC reverse primers, Sequence was confirmed in forward and reverse directions. In order to further confirm key .sequences were not subject to PCR errors, the PCR and cloning process was repeated twice for marmoset sequences. Nucleotide (SEQ ID Nos:14-24 and .SRQ ID Nos:36-41) and amino acid (SEQ DD Nos:25- 35 and SEQ ID Nos:42-47) sequences are given in Figure 2. Marmoset sequences 1, 2 and 3 were confirmed. Sequences 4, 5, 6, 7 and 8 were seen only in the initial PCR. Sequences 9, IO and 1 1 were seen only in the repeat (i.e. second) PCR and cloning.
• CDR sequences namely YAATKLQS (SEQ JD No: J ) from Owl monkey sequence 1 (SEQ JD No:42), YEASSLQS (SEQ ID No:2) from Owl monkey sequence 2 (SEQ ID No:43), YEASKLQS (SEQ FD NO:3) from Marmoset sequence 1 (SEQ ID No:25), and YSASNLET (SEQ ID No:4) from Marmoset sequence 2 (SEQ ID No:26), were chosen from the amino acid sequences shown in Figure 2 as indicated.
• acceptor variable region (anli-TNF domain antibody) sequence in the expression vector was digested (25 ⁇ g) sequentially with Kpnl and SanDI which excises the majority of FR2 as well as CDR2 as indicated on the restriction digest map. The vector was then gel purified to remove the excised wild-type FR2 and CDR2 sequence.
• Olig ⁇ annealing was performed by incubating oligo pairs (500 pmol of each as shown in Figure 4A and 4B) at 95°C for 5 minutes followed by 65°C for 5 minutes and then allowed to reach room temperature slowly on a hot block. Overlaps ware then filled in during a Klenow reaction in the presence of dNTPs.
• the marmoset CDR-grafted dAb Compound 145 (SEQ ID No:7> was affinity matured by constructing 14 separate libraries, each a diversification of the sequence of SEQ ID No:7 at a single amino acid residue. The selected residues are shown shaded below,
• the wells were washed with phosphate buffered saline (PBS) containing 0.05% Twccn-20 and then blocked with 1% BSA in PBS before being incubated with 100 ng/rnl TNF receptor ⁇ Fc fuhion protein (R&D Systems, Minneapolis, USA), Each d ⁇ b was mixed with TNF which was added to the washed wells at a final concentration of 10 ng/ml.
• PBS phosphate buffered saline
• TNF binding was detected with 0,2 vng/ml biotinylaled anli- TNF antibody (HyCult biotechnology, Ubcn, Netherlands) followed by 1 in 500 dilution of horse radish peroxidase labelled slreplavidin (Amcrshain Rioscie ⁇ ces, UK) and (lien incubation with TMB substrate (KPL, Gaithcrsburg, USA).
• the reaction was slopped by the -addition of HCl and the absorbancc was read at 450nin.
• Anti-TNF dAb activity lead to a decrease in TNF binding and therefore a decrease in absorbance compared with the TNF only control (Figure 5).
• Anti-TNF dAbs identified by the minilibrary diversification approach including Compounds 100 (SEQ ID No:9) and 123 (SEQ ID No: 8), were also tested for the ability to neutralise the cytotoxic activity of TNF on mouse L929 fibroblasts (Evans, T., 2000, Molecular Biotechnology 15, 243-248)- Briefly, L929 cells plated in microtitre plates were incubated overnight with anti-TNF dAb, 100 pg/ml TNF and I ing/rnl actinomycin D (Sigma, Poole, UK).

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