EP1945669A1

EP1945669A1 - Chimeric antibodies with new world primate regions

Info

Publication number: EP1945669A1
Application number: EP06774813A
Authority: EP
Inventors: Philip Anthony Jennings; Anthony Gerard Doyle; Adam William Clarke
Original assignee: Arana Therapeutics Ltd
Current assignee: Teva Pharmaceuticals Australia Pty Ltd
Priority date: 2005-08-15
Filing date: 2006-08-15
Publication date: 2008-07-23
Also published as: NZ565904A; AU2006281981A1; NO20080800L; EP1945669A4; ZA200802246B; CN101287762A; BRPI0614867A2; WO2007019621A1; BRPI0614430A2; CN101287763A; RU2008110058A; JP2009504686A; ZA200802247B; MX2008002161A; CA2619245A1; KR20080068005A

Abstract

The present invention provides a chimeric antibody or an antigen-binding portion thereof. The antigen-binding portion comprises at least two complementarity determining regions (CDR) and at least three framework regions, wherein at least one CDR is a New World primate CDR.

Description

CHIMERIC ANTIBODIES WITH NEW WORLD PRIMATE REGIONS

FIELD OF THE INVENTION

The present, invention relates to a chimeric antibody or antigen-binding portion thereof, wherein the antigen binding portion comprises at least two complementarity determining region (CDR) sequences and at least three framework regions, wherein at least one CDR is a New World primate CDR, and to the use of the antibody or antigen -binding portion thereof in treating diseases or disorders.

BACKGROUND OF THE INVENTION

Antibodies (immunoglobulins) play an important role in the immune system of a mammal. They are produced by plasma cells which have developed from precursor B cells. Antibodies consist υf two identical light polypeptide chains and two identical heavy polypeptide chains which are joined by disulfide bridges. The light chains are referred to as either kappa or lambda light chains and the heavy chains as gamma, mu, delta, alpha or cpsilon. Each chain consists of a constant and variable region. The variable region gives the antibody its specificity. Within each variable region are regions of hypervari ability or complementarity determining regions (CDRs) which are flanked by more conserved regions referred to as framework regions. Within each variable region arc three CDRs and four framework regions.

Antibodies are bifunctional molecules, the N-terminai variable segments from the heavy and light chains associate together in a specific manner to generate a three-dimensional structure with affinity for a particular epitope on the surface of an antigen. The constant region segments are responsible for prolonged serum half-life and the effector functions of the antibody and relate to complement binding, stimulation of phagocytosis, antibody- dependent cellular cytotoxicity and triggering of granulocyte granule release.

The development of hybridoma technology has facilitated the production of monoclonal antibodies of a particular specificity. Typically, such hybiϊdomas are murine hybridorπas.

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-chimcric antibodies are detrimental io continued therapy with chimeric antibodies.

It has been suggested that human monoclonal antibodies are expected to be aii 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 (Ehrlich PH el al., Human and primate monoclonal antibodies for in vivo therapy. Clin Chem. 34:9 pg 168J- 1688 (1988)). Furthermore, because human antibodies are non-immunogenic in Rhesus monkeys (Ehrich PH et al.. Rhesus monkey responses to multiple injections of human monoclonal antibodies. Hybridoma 1987; 6:15.1-60), it is likely that the converse is also applicable and primate antibodies will be non-immunogenic in humans. These monoclonal antibodies are secreted by hybrϊdomas constructed by fusing lymphocytes to a human x. mouse heteromyeloma.

EP 0 605442 discloses chimeric antibodies which bind human antigens. These antibodies comprise the whole variable region from an Old World monkey and the constant region of^" a human or chimpanzee antibody. One of the advantages suggested in this reference for these constructs is the ability to raise antibodies in Old World monkeys to human antigens which arc less immunogenic in humans compared with antibodies raised in a mouse host.

New World primates (infraorder- Platyrrhini) comprise at least 53 species commonly divided into two families, the CulUthricidae and Cebidae. The Callilhricidae eonsist of marmosets and tainarins. The Cebidae includes the squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, uakaris, sakis, night or owl monkey and the howler monkey.

Evolulionarily 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 primatc-derived antibodies arc introduced into a human.

Previous studies have characterised the expressed immunoglobulin heavy chain repertoire of the Callithrixjacchun marmoset (von Budingen H-C et al., Characterization of the expressed immunoglobulin IGHV repertoire in the New World marmoset Callilhrix jacchus. Immunogeneties 2001; 53:557-563). Six IGHV subgroups were identified which showed a high degree of sequence similarity to lhcir human IGIlV counterparts. The framework regions were more conserved when compared to the complementarity determining regions (CDRs). The degree of similarity between C, jacchus and human KTH V sequences was less than between non-human Old World primates and humaπa.

Domain antibodies

Domain antibodies (dAb) are functional binding units which can be created using antibody frameworks and correspond to the variable regions of either the heavy (VH) or light (V_L) chains of antibodies. Domain antibodies have a molecular weight of approximately 13 kDa, or less than one tenth the size of a full antibody.

Immunoglobulin light chains are referred to as either kappa or lambda tight chains and the heavy chains as gamma, mu, delta, alpha or epsilon. The variable region gives the antibody its specificity, Within each variable region are regions of hypervari ability, otherwise known as complementarity determining regions (CDRs) which are flanked by more conserved regions referred to as framework regions. Within each light and heavy chain variable region are three CDRs and four framework regions.

In contrast to conventional antibodies, domain antibodies are well 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. In addition, domain antibodies can be used as a building block to create therapeutic products such as multiple targeting domain antibodies in which a construct containing two or more variable domains bind to two or more therapeutic targets, or domain antibodies targeted for pulmonary or oral administration.

SUMMARY OF THE INVENTION The present inventors have found that New World primates provide a rich source of binding domains for antibodies against a range of antigens including human antigens. Further, due to the similarity of the sequences between human and New World primates it is likely that these New World primate sequences will have relatively low immunogenicity in humans. fn a rπai aspect the present invention provides a chimeric antibody or an antigen-binding portion thereof, wherein the antigen-binding portion comprises at least two complementarity determining regions (C-DR) and at least three framework regions, wherein at least one CDR is a New World pϊimate CDR.

In another aspect the present invention provides a method of producing a chimeric antibody υr an antigen-binding portion thereof, the method comprising deleting a CDR from a human antibody variable region comprising id least two CDRs and at least three framework regions and replacing it with a New World primate CDR predicted to he of low immunogenic ity to produce a chimeric variable region,

In a related aspect the method further comprises the step of recovering the chimeric variable region.

In yet another aspect the present invention provides a chimeric antibody or an antigen- binding portion thereof produced according to the method of the present invention.

In a further aspect, the invention provides a pharmaceutical composition comprising an effective amount of tine antibody or antigen-binding portion thereof according to the present invention, together with a pharmaceutically acceptable excipitsnt or diluent.

Jn a still further aspect, the invention provides for die use of an antibody or antigen-binding portion thereof of the present invention in a diagnostic application for detecting an antigen associated with a particular disease or disorder.

In another aspect, the present invention provides a method for treating a disease or disorder characterised by human TNF-α activity in a human subject, comprising administering to the subject in need thereof an effective amount of a chimeric antibody as described herein, or a pharmaceutical composition thereof in which the antibody or antigen-binding portion thereof binds TNF-α.

BRIEF DESCRIPTION OF THE FIGURES figure 1 demonstrates the binding of AB 138 to rat MOG present in rat spinal cord lysatc (lane 2) and not to CHOKISV lysate (lane 3). J.ane 1 contains molecular weight markers. Figure 2 demonstrates the lack of non-specific binding of an OMl-TTJFa monoclonal antibody to the same sample of rat MOG present in rat spinal cord lysate {lunts 2) and CHC)KlSV lysalc (lane 3). Lane I contains molecular weight markers.

Figure 3 shows the acceptor domain antibody amino acid and nucleotide sequence (both strands). The restriction digest sites for Kpn I and San DI, which excises a region including the CDR2, is indicated in the figure. CDR2 residues are indicated in underline.

Figure 4 is a sequence alignment of the domain antibody acceptor sequence with a panel of New World primate derived immunoglobulin sequences performed using AlignX (Vector NTJ, Invitrυgen, Australia). The CDR2 is highlighted in bold text. DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a chimeric antibody or an antigen-binding portion thereof, wherein the antigen-binding portion comprises at least two complementarity determining regions (CDR) and at least three framework regions, wherein at least one CDR is a New World primate CDR. It is preferred that the antigen binding portion comprises three CDRs and four framework regions. It is also preferred that the antigen-binding portion comprises at least one, and preferably two human CDRs.

Iti some embodiments of the present invention, the chimeric antibody or antigen-bmdmg portion thereof comprises one New World primate CDR. In other embodiments, the chimeric antibody or antigen-binding portion thereof comprises two New World primate CDRs. Tn other embodiments CDR2 of the antibody or antigen-binding portion is a New World primate CDR. ϊn other embodiments of the present invention the at least one New World primate CDR is not from a sequence that binds a target antigen. In other embodiments of the present invention the framework regions are human sequences. Framework regions that arc human sequences include sequences derived from human framework regions, or synthetic sequences based on human framework regions.

It is within the scope of the present invention, that the sequence of the antigen hinding portion may be further subject to affinity maturation in order to improve its antigen binding characteristics such as antigen binding or potency. An increase in binding is demonstrated by a decrease in K_D (K_aff/k_nn) for the antibody or antigen binding portion thereof. Λn increase in potency is demonstrated in biological assays. For example, assays thaL can be used to measure the potency of the antibody or antigen-binding portion thereof include the TNFα-induced L929 cytotoxicity neutralisation assay, IL-12-induced human PHΛ-activaled peripheral blood mononuclear cell (PBMC) proliferation assay, and RANKL mediated osteoclast differentiation of mouse splcnocyles (Stem, Proc. Natl. Acad. Sci. USA 87:6808 - 6812 (1990); Kong, Y-Y. et a Nature 397:315 - 323 (1990); Matthews, N. and ML. Neale in Lymphokines and Interferons, a Practical Approach, 1987, MJ. Clemens, A.G. Morris and AJ.H. Gearing, cds., JRL Press, p. 22J)

In a further preferred embodiment at least one framework region is modified to increase binding and/or to reduce predicted immunogcnicity in humans,

In another embodiment at least one CDR sequence is modified to increase binding or potency and or to reduce predicted immunogenicity in humans. It is preferred that where at least one CDR sequence which is modified it is not the New World primate CDR.

Where two or more New World primate CDRs are present then it is preferred that at least one New World primate CDR is not modified.

In other embodiments of the present invention at least one framework region is modi Tied, in addition to at least one CDR sequence, to increase binding and or to reduce predicted immunogenicity in humans. It is preferred that the at the least one CDR sequence which is modified it is not a New World primate CDR sequence.

In a preferred embodiment the antigen-binding portion is a domain antibody.

In a further embodiment of the present invention, the domain antibody may be multirncrised, as for example, hctero- or.homodimers (e.g., V_H/ V_H, V_L/ V_L or V_H/VL), netero- or homolrimcrs (e.g., V_II/ V_H/ V_H, V_L/ V_L/ V _L, V_H/ V_II/ V_L or V_H/ V_L/ V_L ), hetcro- or homotetramers (e.g., V_II/V_H/ V_H/ V_II, V_L/ V_L/ V _L / V _L V_H/V_II/V_H,V_L , V_II/ V_H/ V_L/ V_L or V_H/V_L/V_L/V_L), or higher order hetero- or homomultimers. Multimerisation can increase the strength of antigen binding, wherein the strength of binding is related to the sum of the binding affinities, or part thereof, of the multiple binding sites. Thus, the invention provides a domain antibody wherein the domain antibody is linked, to at least one further domain antibody. Each domain antibody may bind to the same or different antigens. The domain antibody multimers may further comprise oπc or more domain antibodies which are linked and wherein each domain antibody binds to a different antigen, multi- specific Iigands including so-called "dual-specific ljgands". For example, the dual specific Uganda may comprise a pair of V₁₁ domains or a pair of V_L domains. Such dual-speci fie Iigands are described in WO 2004/003019 (PCT/GB2003/002804) in the name of Domanlis Ltd incorporated by reference herein in its entirety.

Preferably, the antibody or antigen-binding portion further comprises a human or non- human primate constant region sequence. Examples of non-human primates include, but are not limited to, chimpanzees, υrangυatangs and baboons. The present invention also provides a method of producing a chimeric antibody or an antigen -binding portion thereof, the method comprising deleting a CDR from a human antibody variable region comprising at least two CDRs and at least three framework regions and replacing it with a New World primate CDR predicted to be of low immunogcnicity to produce a chimeric variable region. Jn a related aspect the method further comprises the step of recovering the chimeric variable region.

It is preferred that the selected New World prunate CDR is CDR2. It is preferred that the CDR2 sequence is selected from KVSNRΛS, RVSNRAS, KVSTRGP, ΛASKRΛS, TSSNLQA, DASSLQP and YASFLQG. Particularly preferred sequences are KVSNRAS, AΛSNRAS, TSSNLQΛ and KVSTRGP due to their predicted lower immunogemcity.

In further embodiments the method further comprises modifying the sequence of the chimeric variable region to increase binding and/or to decrease immuraogenicity in humans. It is preferred that the New World primate CDR sequence is not modified. Where two or more New World primate CDR sequences arc present then it is preferred that at least one New World primate CDR. is not modified.

In other embodiments of the present invention at least one framework region is modified in addition to at least one CDR sequence, to increase binding and or to reduce predicted immunogemcity in humans. It is preferred that the at the least one CDR sequence which is modified it is not a New World primate CDR. scquence.Thc present invention also provides a chimeric antibody or an antigen-binding portion thereof produced by the method of the present invention. The term "antibody" as used herein, is intended to refer to immunoglobulin molecules comprised ot four polypeptide chains, two heavy (H) chains and two light (Iy) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (HCVR or Vn) and a heavy chain constant region. The heavy chain constant region comprises three domains, C_HI, C_H2 and Cn3> Each light chain is comprised of a light chain variable region (LCVR or V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypcrvariabilily, termed complementarity determining regions (CDR), interspersed with regions that arc more conserved, termed framework regions (FR). Each VH and V_L i.s composed of three CDRs and four FRs, arranged from ammo-terminus to carboxy-tcrminutJ in the following order: FR 1 , CDRl, FR2, CDR2, FR3, CDR3, FR4,

The term "antigen-binding portion" of an antibody, as used herein refers to one or more components or derivatives of an immunoglobulin thai 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. Eλamplϋs of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_H1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_H1 domains; (iv) a Fv fragment consisting of the V_t, and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al, 1989, Nature 341 :544-54G) which consists of a single V_H domain, or a V_L domain (van don Beuken T et al, 2(X)I, J. Mo!. Biol, 310, 591); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL 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 V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); (see eg Bird el al , 1988, Science 242:423-426 and Huston el al., 1988 Proc. Natl. Acad. Sci. USA 85:5879^5883). Such single chain Fvs are also intended to be encompassed within the term "antigen-binding portion" of an anti body. Other forms of single chain Fvs and related molecules such as diabodies or triabodies are also encompassed, Diabodies arc bivalent antibodies in which V_H and V_L domains arc expressed on a single polypeptide chain, buL 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., JJolliger, P., et al._r 1993, Proc. NaU. Λcad. Sci. USA, 90:6444-6448; Poljak, RJ., et ^■ aL J 994, Structure, 2:1121-1123). As used herein lhc term "chimeric" means that the antibody υr antigen-binding portion includes sequences from two different species. in one embodiment, the domain antibody comprises a human framework regions and at letust one New World primate CJDRs, more preferably marmoset CDRs. f5 Preferably, the New World primate is selected from the group consisting of marmosets, lamatins, squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, uakaris, saTds. night or owl monkey and the howler monkey. More preferably, the New Wortd primate is a marmoset.

Methods of producing chimeric antibodies according to the invention will be familiar to I (,) persons skilled in the ait, see for example, US Patent No.4,816,567, US Patent No. 5,585,089 and US 20030039649 which are incorporated herein by reference in their entirety. Such methods require the use of standard recombinant techniques.

It is preferred that the antibody or antigen-binding portion thereof according to the present invention has predicted low immunogenic] ty in a human host.

15 By "low immunogenioily" il is meant that the antibody docs not raise an antibody response in at. least the majority of individuals receiving the antibody of sufficient magnitude to reduce the effectiveness of continued administration of the antibody for a sufficient time to achieve therapeutic efficacy.

The level of immunogenicity in humans may predicted using the MHC class II binding 0 prediction program Propred (http://www.imtech.res.in/raghava/propred) using a 1% threshold value analysis of all alleles. Other programs which may bo used include:

Rankpep (http://bio.dfci. harvard.edu/Tools/rankperxhtml) Epibase (Algonomies proprietary software: algonomics.com)

Low immunogenicity molecules will contain no or low numbers of peptides predicted to 5 bind to MlJC class Il alleles that are highly expressed in the target population (Flower DR, Doytchinova IA. (2004) Immunoinformatics and the prediction of immunogenicity, Drug Discov Today, 9(2): 82-90).

Reduced immunogenicity molecules will contain no or a reduced numbers of peptides predicted to bind to MHC class Il alleles that are highly expressed in the target population, 0 relative to the starting donor molecule. Functional analysis of MHC class ΪI binding can be performed by generating overlapping peptides corresponding to lhc protein of interest and testing these for their ability to evoke T cell activation (T ce)! proliferation assay) or displace a reporter peptide, a known Ml IC class l!-binding peptide (Hammer J el at., 1994, J. Kxp. Med., 180:2353). The present invention is further based on a method for amplification of New World primate immunoglobulin genes, for example by polymerase chain reaction (PCR) from nucleic acid extracted from New World primate lymphocytes using primers specific for heavy and light chain variable region gene families. The amplified variable region is then cloned into an expression vector containing a human or primate constant region gene for the production of New World primate chimeric recombinant antibody. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Krilsch and Maniotis (eds), Molecular Cloning: a laboratory manual, second edition, Cold Spring Harbor, N.Y (1989). Suitable expression vectors will be familiar to those skilled in the art. The New World pi imalc lymphocytes producing the immunoglobulins are typically immortalised by fusion with a myeloma cell line to generate a hybridoma.

Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese I lamster Ovary (CHO), NSO myeloma cells, COS cells and SP2 cells. In addition to mammalian expression systems, the present invention also contemplates the use of non-mammalian expression systems such as those which arc plant or prokaryotic (bacterial) derived. Such expression systems would be familiar to persons skilled in the art.

The repertoire of Vn, V_L and constant region domains can be a naturally occurring repertoire of immunoglobulin sequences or a synthetic repertoire. A nanirally occurring repertoire is one prepared, for example, from immunoglobulin expressing cells harvested from one or more primates. Such repertoires can be naive ie. prepared from newborn immunoglobulin expressing cells, or rearranged ie. prepared from, for example, adult primate B cells. If desired, clones identified from a natural repertoire, or any repertoire that bind the target antigen are then subject to mutagenesis and further screening in order to produce and select variants with improved binding characteristics.

Synthetic repertoires of immunoglobulin variable domains are prepared by artificially introducing diversity into a cloned variable domain. Such affinity maturation techniques will be familiar to persons skilled in lhe art (Irving R. A. et ai (2001) Ribosorne display and affinity maturation: from antibodies Io single V-domains and steps towards cancer therapeutics, Journal of Immunological Methods, 248: 31-45).

The variable region, or a CDR thereof, of a New World primate antibody gene may be cloned by providing nucleic acid eg. cDNΛ, providing a primer complementary to the cDN A sequence encoding a 5' leader sequence of an antibody gene, contacting that cDNA and the primer to form a hybrid complex and amplifying the cDNA to produce nucleic acid encoding the variable region (or CDR region) of the New World primate antibody gene.

It will be appreciated by persons skilled in the art of the present invention, the non-New World primate variable region sequence may be used as an acceptor for grafting New World primate sequences, in particular, CDR sequences using standard recombinant techniques. For example, US Patent No. 5,585,089 describes methods for creating low immuiiogenicity chimeric antibodies that retain the high affinity of the non-human parent antibody and contain one or more CDRs from a donor immunoglobulin and a framework region from a human immunoglobulin. United States publication no. 20030039649 describes a humanisation method for creating low immiinogenicity chimeric antibodies containing CDR sequences from a non-human antibody and framework sequences of human antibodies based on using canonical CDR structure types of the non-human antibody in comparison to germline canonical CDR structure types of human antibodies as the basis for selecting the appropriate human framework sequences for a humanised antibody. Accordingly, these principles can be applied to the grafting of one Or more New World primate CDRs into a -non-New World primate acceptor variable region.

The CDR sequences may be obtained from the genomic UNA isolated from an antibody, or from sequences present in a database e.g. The National Centre for Biotechnology Information protein and nucleotide databases, The Kabat Database of Sequences of Proteins of Immunological Interest The CDR sequence may be a genomic DNA or a cDNA.

Methods for grafting a replacement CDR(s) into an acceptor variable sequence will be familiar to persons skilled in the an of the present invention. Typically, the CDRs will be grafted into acceptor variable region sequences for each of a variable light chain and a variable heavy chain or a sitiglc chain in the case of a domain antibody. The preferred method of the present invention involves replacement of either CDRl or, more preferably, CDR2 in a variable region sequence via primer directed mutagenesis. The method consists of annealing a synthetic oligonucleotide encoding a desired mutation to a target region where it servea as a primer for initiation of DNA synthesis in vitrυ, extending the oligonucleotide by a DNΛ polymerase io generate a double-stranded I⁾NA that carries the desired mutation, and ligating and cloning the sequence inlo an appropriate expression vector (Sambroolc, Joseph; and David W. Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd cd., Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory Press)..

Still Further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesioπ molecule, formed by covalent or noncovalcnt association of the antibody ^■ or antibody portion with one or more other proteins or peptides. Examples of such irnmunoadhesion molecules include use of the streplavidin core region to make a tctrameric seFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-tcrminal polyhislidinc tag to make bivalent and biotinylatcd scFv molecules (Kipriyanov, S. M., el al. (ϊ994) Mσl. Immunol. 31 :1047-1058). Anlibody portions, such as Fab and JF(ab')₂ fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and iromunυadhcsion molecules can be obtained using standard recombinant DNΛ techniques, as described herein as is known to the skilled artisan,

The constant region sequence (Fe portion) 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 hominid ape eg. gorilla or orang utan, which because of their close phylogenetic 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. 'Che National Centre for Biotechnology Information protein and nucleotide databases, The Kabat Database of^" Sequences of Proteins of Immunological Interest.

The antibody or antigen-binding portion according to the invention is capable of binding to a human or non-human antigen. Preferably, the antigen to which the chimeric antibody or antigen-binding portion thereof binds, is peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid in nature, selected from a tumour-associated antigen including carcinoembryonic antigen, EpCAM, Lewis-Y, I-ewis-Y/b, PMSA, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R, Her-2, TRAIL and VEGF receptors, an antigen involved in an immune or inflammatory disease or disorder including CD3, CD4, CD25, CD40, CD49d, MHC class I₇ MHC.: class II, GM- CSF, intcrfcron-γ. IL-I, IL-12, IL-13, IL-23, TNF-α, and IgE, an antigen expressed on a host cell including glycoprotein Ub/AIIa, P-glycoprotcin, purinergjc receptors and adhesion receptors including CDlIa, CDl Ib, CDl Ic, CD18, CD56, CD58, CD62 or CD144, an antigen comprising a cytokine, chemokinc, gi'owth factor or other soluble physiological modulator or a receptor thereof including eotaxiπ, 11_^6, IL-8, TGF-p¹, C3ii, C5n, VEGF, NGF and their receptors, an antigen involved in central nervous system diseases or disorders including β-amyloid and prions, an antigen of non-human origin such as microbial, nanobial or viral antigens or toxins including respiratory sync^ytial vims protein F, anthrax toxin, rattle snake venom and digoxin; wherein the chimeric antibody acts as an agonist or antagonist or is active to either deplete (kill or eliminate) undesired cells (eg. anti-CD4) by acting with complement, or killer cells (eg. NK cells) or is active as a cytotoxic agent υr to cause Fu-reeeptor binding by a phagocyte or neutralizes biological activity of its target.

More preferably, the antigen is TNFα, preferably human TNFo.

Alternatively the chimeric antibody or antigen-binding portion thereof may bind a non-human antigen, Preferrably the non-human antigen is selected from the group consisting of respiratory syncytial virus F protein, cytomegalovirus, snake venoms and digoxin. The term "binds to" as used herein, is intended to refer to the binding of an antigen by an immunoglobulin variable region of an antibody with a dissociation constant (Kø) of lμlvϊ or lower as measured by surface plasmon resonance analysis using, for example a EIΛeorc™ surface plasmon resonance system and BIΛcorc™ kinetic evaluation software (eg. version 2.1). The affinity or dissociation constant (Ku) for a specific binding interaction is preferably about 500 nM to about 50 pM, more preferably about 500 nM or lower, more preferably about 300 nM or lower and preferably at least about 300 nM to about 50 pM, about 200 nM to about 50 pM, and more preferably at least about 100 nM to about 50 pM, about 75 nM lo about 50 pM, about 10 nM to about 50 pM.

The antibodies of the present invention are advantageous in human therapy because the likelihood of induction of a human anti-antibody response will be reduced. Recombinant antibodies produced according to the invention that hind a target antigen can be identified and isolated by screening a combinatorial immunoglobulin library (eg a phage display library) to isolate library members that exhibit the desired binding specificity and lϊmctional behaviour. It will be understood that all approaches where antigen-binding portions or derivatives of antibodies are used, eg Fabs, scFv and V domains or domain antibodies, lie within the scope of the present invention. The phage display technique has bcen described extensively in lhe art and examples of methods and compounds for generating and screening such libraries and affinity maturing the products of them can be found in, for example, Barbus et al. (1991) PNAS 88:7978-7982: Clarkson et al. (1991) Nature 352:624:628; Dower ct al. PCT. 91/17271 , U.S. Patent No. 5,427,908, U.S. Patent Nυ. 5,580,717 and EP 527,839; Fuchs et a!. (1991) Bio/Technology 9:1370-1372; Garrad et al. (1991) Bio/Technology 9: 1373:1377; Garrard ct al. PCT WO 92/09690; Gram et al. (1992) PNAS 89:3576-3580; Griffiths et al. (1993) EMBO J 12:725:734; Grittiths et al. U.S. Patent No. 5,885,793 and EP 589,877; Hawkins et al. (1992) J MoI Biol 226:889-896; ! lay et al, (1992) Hum Anlibod Hybridomas 3:81 -H5; Hoogenboom ct al. ( 1991 ) Nue Λeid Res 19:4133-4137; I fuse et al. (1989) Science 246:1275-1281; Knappik et al. (2000) J MoI Biol 296:57-86; Knappik el al. PCT WO 97/08320; Ladner ct al. U.S. Patent Ko. .'5,223,409, No. 5,403,484, No. 5,571,698, No. 5,837,500 and EP 436,597; McCafferty ct al (1990) Nature 348:552-554; McCafferty et al. PCT. WO 92/01047, U.S. Patent No. 5,969,108 and EP 589,877; Salfcld et al PCr WO 97/29131, U.S. Provisional Application No. 60/126,603; and Winter ct al. PCT WO 92/20791 and EP 368,684;

Recombinant libraries expressing the antibodies of the invention can be expressed on the surface of microorganisms eg. yeast or bacteria (sec PCT publications WO99/36569 and 98/49286), The Selected Lymphocyte Antibody Method or SLAM as it is referred to in the state of the art, is another means of generating high affinity antibodies rapidly. Unlike phage display approaches all antibodies are fully divalent. In order to generate New World primate antibodies, New World primates arc immunised with a human antigen eg. a TNFo: polypeptide. Following immunisation cells are removed and selectively proliferated in individual micro wells. Supematants arc removed from wells and tested for both binding and function. Gene sequences can be recovered for subsequent manipulations eg, humanisaliofl, Fab fragment, scFv or domain antibody generation. Thus another example is the derivation of the ligand of the invention by SLAM and its derivatives (Babcook, J.S. et al 1996, Proc. Natl. Acad. Sci, USΛ 93; 7843-7848, US Patent 5,627,052 and PCT publication WO92/0255t). Adaptations of SLAM, such as the use of alternatives to testing supernatants sυcli as panning, also lie within the scope of this invention.

JLti one expression system the recombinant pepfide/protein library is displayed on ribosomes (for examples see Roberts, RW and Szostak, J.W.I 997. Proc.NatJ.Acad.Sci.USA. 94:12297 - 123202 and PCT Publication No. WO98/31700). Thus another example involves the generation and in vitro transcription of a DNA library (eg of antibodies and derivatives) preferably prepared from immunised cells, but not so limited), translation of the library such that the protein and "immunised" mRNΛs stay on the ribosome, affinity selection (eg by binding to RSP), mRNΛ isolation, reverse translation and subsequent amplification (eg by polymerase chain reaction or related technology). Additional rounds of selection and amplification can be coupled as necessary ^■ to affinity maturation through introduction of somatic mutation in this system or by other methods of affinity maturation as known in the state of the art.

Another example sees the application of emulsion compartmenlah'sation technology to the generation of the antibodies of the invention. In emulsion cornparlmcnlalisation, in vitro and optical sorting methods are combined with co-eompartmenUtlisation of translated protein and its nucleotide coding sequence in aqueous phase within an oil droplet in an emulsion (sec PCT publications no's WO99026711 and WO0040712). The main elements for the generation and selection of antibodies are essentially similar to the in vitro method of ήbosomc display.

The antibody or antigen-binding portion thereof according to the invention can be derivatised or linked to another functional molecule. For example, the antibody or antigen- binding portion can be functionally linked by chemical coupling, genetic fusion, noncovalenl association or otherwise, to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or anli gen-binding portion thereof with another molecule (such as a streptavidin core region or a polyhistidine tag).

Useful delectable agents with which an antibody or antigen-binding portion thereof may be derivatised include fluorescent compounds. Exemplary fluorescent delectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-ϊ- napthalenesulfonyl chloride, phycoeiythrin and the like. An antibody may also be derivatised wilh delectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. An antibody may also be dcrivatised with biolin, and detected through indirect measurement of avidin or streptavidin binding.

The present invention also extends to PEGylatcd antibodies or antibody-binding portion which provide increased half-life and resistance to degradation without a loss in activity (eg. binding affinity) relative to non-PEGylatcd antibody polypeptides.

The antibody or antibody-binding portion as described herein can be coupled, using methods known in the art, to polymer molecules (preferably PEG) useful for achieving the increased half-life and degradation resistance properties. Polymer moieties which can be utilised in the invention can be synthetic or naturally occurring and include, but are not limited to, straight or branched chai n polyaikylcnc, polyalkeny lene or polyoxyalkylcne polymeis, or a branched or unbranched polysaccharide such as a horno-or heterapolysaccharidc. Preferred examples of synthetic polymers which can be used in the invention include straight or branched chain polyethylene glycol) (PEG), polypropylene glycol), or poly(vϊnyl alcohol) and derivatives or substituted forms thereof. Particularly preferred substituted polymers for linkage to antibodies as described herein include substituted PEG, including methoxy(polyethylene glycol). Naturally occurring polymer moieties which can be used in addition to or in place υf PEG include lactose, amylose, dextran, or glycogen, as well as derivatives thereof which would be recognised by persons skilled in the art. Dcrivatised forms of polymer molecules include, for example, derivatives which have additional moieties or reactive groups present therein to permit interaction with amino acid residues of the antibody polypeptides described herein. Such derivatives include N- hydraxylsuccinimidc (NIIS) active esters, succinimidyl propionate polymers, and sulfhydryl-sclcctivc reactive agents such as maleimide, vinyl sulfonc, and thiol. Particularly preferred derivatized polymers include, but are not limited to PEG polymeis having the formulae: PEG-O-CH₂CH₂CH₂-CO₂-N HS; PEG-O-CH₂-NHS; PEO-O- CH₂CH_Z-CO₂-NHS; PKG-S-CH₂CH₂-CO-NI IS; PKG-O₂CNH-CH(R)-CO₂-NHS; PKG- NHCO-CH₂CH₂-CO-NHS; and PEG-O-CH₂-CO₂-NHS; where R is (CH₂)₄)NHC0₂(mPEG), PBG polymers can be linear molecules, or can be branched wherein multiple PEG moieties are present in a single polymer.

The reactive group (e.g., MAL, NHS, SPΛ, VS, or Thiol) may be attached directly to the PRG polymer or may be attached to PEG via a linker molecule. The size of polymers useful in the invention can be in the range of between 500 Da to 60 kDa, for example, between 1000 Da and 60 kDa, 10 kDa and 60 kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40 kDa and 60 kDa, and up to between 50 kDa and 60 kDa. The polymers used in the invention, particularly PEG, can be straight chain polymers or may possess a branched conformation.

The polymer (PEG) molecules useful in the invention can be attached to an antibody or antigen-binding portion thereof using methods which are well known in the art. The first step in the attachment of PEG or other polymer moieties to an antibody polypeptide monomer or multimer of the invention is the substitution of the hydroxyl end-groups of the PEG polymer by clectrophiie-containing functional groups. Particularly, PEG polymers are attached to cither cysteine or lysine residues present in the antibody polypeptide monomers or multimers. The cysteine and lysine residues can be naturally occurring, or can be engineered into the antibody polypeptide molecule. For example, cysteine residues can be recombinantly engineered at the C-terminus of an antibody polypeptide, or residues at specific solvent accessible locations in an antibody polypeptide can be substituted with cysteine or lysine.

The antibody may be linked to one or more molecules which can increase its half -life in vivo. These molecules arc linked to the antibody at a site on the antibody other than the antigen binding site, so that they do not interfcrc/sterically hinder the antigen-binding site. Typically, such molecules are polypeptides which occur naturally in vivυ and which resist degradation or removal by endogenous mechanisms. It will be obvious to one skilled in the art that fragments or derivatives of such naturally occurring molecules may be used, and that some may not be polypeptides. Molecules which increase half life may be selected from the following: (a) proteins from the extracellular matrix, eg. collagen, Jaminin, integrin and fibronectin;

(b) proteins found in blood, eg, fibrin α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, scrum amyloid protein A, hcptaglobin, protein, ubiquitin, utcroglobulin, β-2 microglobulin, plasminogen, lysozymc, cystatin C, alpha-1-antitrypsin and pancreatic kypsin inhibitor;

(c) immune serum proteins, eg. IgE, IgG, IgM;

(d) transport proteins, eg. rctinol binding protein, α-1 microglobulin;

(e) defensins, eg. beta-dcfcnsin 1, Neutrophil defcnsins 1, 2 and 3;

(f) proteins found at the blood brain barrier or in neural tissues, eg. melanocortin receptor, myelin, ascorbatc transporter; (g) transferrin receptor specific ligand-neuropharmaccutJcai agent fusion proteins (see US5977307); brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin- like growth factor 1 (IGF 1) receptor, iπsulin-ϋke growth factor 2 (IGF 2) receptor, insulin receptor; (h) proteins localised to the kidney, eg. polyeyslin, type IV collagen, organic anion transporter Kl, Heymann's antigen;

(i) proteins localised to the liver, eg, alcohol dehydrogenase, G250;

(j) blood coagulation factor X;

(k) α-1 antitrypsin; (l) HNF lα;

(m) proteins localised to the lung, eg. secretory component (binds IgA);

(n) proteins localised to the Hearing. USP 27;

(o) proteins localised to the skin, eg, keratin;

(p) bone specific proteins, such as bone morphogeny proteins (BM Ps) eg. BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-I) and -8 (OP-2);

(q) tumour specific proteins, eg. human trophoblast antigen, herceptin receptor, oestrogen receptor, cathepsiπs eg cathepsin B (found in liver and spleen);

(r) disease-specific proteins, eg. antigens expressed only on activated T- cells: including LAG-3 (lymphocyte activation gene), ostcoprotegerin ligand (OPGL) sce Natuie 402, 304-309, 199S); OX40 (a member of the TNFα receptor family, expressed on activated T cells and the only costimulatory T cell molecule known to be specifically up-regulated in human '1' cell leukaemia virus type^l (HTLV-I)-producing cells - see J. Immunol. 2000 JuI 1; J6561):263-70; metalloproteases (associated with arthritis/cancers), including CCΪ6512 DroKophila, human paraplegin, human RsH, human AFG3L2, murine flsH; angiogenic giowth factors, including acidic fibroblast growth factor (FGF-I), basic fibroblast growth factor (FGF-2), Vascular endothelial growth factor/vascular permeability factor (VEGF/VPK), transforming growth factor-α (TGF-α), tumor necrosis factor-alpha (TNFα), angiogenin, inierlcukin-3 (IL-3), intcrleukin-8 (IL-8), platelet derived endothelial growth factor (PD- BCGF), placental growth factor (PlGF), midkine platelet-derived growth factor-BJβ (FDGF), fractalkine;

(s) stress proteins (heat shock proteins);

(t) proteins involved in Fc transport; and

(u) vitamins eg B12, Biotin. fn another aspect, the invention provides a pharmaceutical composition comprising an effective amount of the chimeric antibody or antigen-binding portion thereof according to the present invention, together with a pharmaceutically acceptable excipient or diluent. Λ "pharmaceutically acceptable excipient or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and Lhe like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, Baline, phosphate buffered saline, dextrose, glycerol, ethanυl, and the like as well as combinations thereof. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as rπanπitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives! or buffers. The term "effective amount" refers to an amount of an antibody or antigen binding portion thereof (including pharmaceutical compositions comprising the antibody or antigen binding portion thereof) sufficient to treat a specified disease of disorder or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder.

The term "diagnostically effective amount" or "amounts effective for diagnosis" and cognates thereof, refers to an amount of a antibody or antigen binding portion thereof (including pharmaceutical compositions comprising the antibody or antigen binding portion thereof) sufficient to diagnose a specified disease or disorder and/or one or more of its rnamfestutiona, where diagnosis includes identification of the existence of the disease or disorder and/or detection of the extent or severity of the disease or disorder. Often, diagnosis will be carried out with reference to a baseline or background detection level observed for individuals without the disease or disorder. Levels of detection above background or baseline levels (elevated levels of detection) are indicative of the presence and, in some cases, The severity of the condition.

When used with respect to methods of treatment and the use of the antibody or antigen binding portion thereof (including pharmaceutical compositions comprising the antibody or antigen binding portion thereof), an individual "in need thereof" may be an individual who has been diagnosed with or previously treated for the disease or disorder to be treated. With respect to methods of diagnosis, an individual "in need thereof may be an individual who is suspected to have a disease or disorder, is at risk for a disease or disorder, or has previously been diagnosed with the disease or disorder (e.g., diagnosis can include monitoring of the severity (e.g., progression/regression) of the disease or disorder over time and/or in conjunction with therapy).

It is preferred that the chimeric antibody or antigen-binding portion thereof blocks or stimulates receptors functions or neutralizes active soluble products, such as one or more of the inlerleukins, TN Fa or C5a. More preferably, the active soluble product is human TN Fa.

The composition may be in a variety of forms, including liquid, semi-solid or solid dosage forms, such as liquid solutions (eg injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes or suppositories. Preferably, lhe composition is in the form of an injectable solution for immunization. The administration may be intravenous, subcutaneous, intraperitoneal, intramuscular, transdermal, intrathecal, and intraarterial. Preferably the dosage form is in the range of from about 0.001 nig to about 10 mg/lfg body weight administered daily, weekly, bi- or tri-wcekly or monthly, mote preferably about 0.05 to about 5 mg/kg body weight weekly.

The composition may also be formulated as a sterile powderlbr the preparation of sterile injectable solutions.

In certain embodiments, the active compound may be prepared with a carder that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Compatible polymers may be used such as ethylene vinyl acetate, polyanbydrides, polyglycolic acid, collagen, potyorthocsters or polylactic acid.

The composition may also be formulated for oral administration. In this embodiment, the antibody may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.

The composition may also be formulated for rectal administration.

The antibody rαay be administered in order to bind to and identify selected cells in vitro and in vivo, to bind to and destroy selected cells i?ι vlvό, or in order to penetrate into and destroy selected cells in vivo. Alternatively, the antibody may be used as an immunotoxin to deliver a cytotoxic agent eg. a toxin or cliemolherapcutie agent IO a particular cell type such as a tumour cell. Production of irnrnunotoxins would be familiar to persons sltilled in the art.

Cytotoxic agents commonly used to generate immunotoxins include radioactive isotopes such as ¹¹¹In or Y, selenium, libonucleascs, binding domain - deleted truncated microbial toxins such as Pseudomonas exotoxin or Diphtheria toxin, tubulin inhibitors such as calioheamicin (ozagamicin), maytansinoids (including DM-I), auristatins, and taxoids, ribosome inactivating proteins such as ricin, ebulin 1, saporin and gclonin, and prodrugs such as melphalan,

In the preferred embodiment, the composition is administered- to a human.

The present invention also provides Tor Lhe use of the chimeric antibody or antigen-binding portion thereof in a diagnostic application for detecting an antigen associated with a particular disease or disorder,

More particularly, the invention provides for the use of the chimeric antibody or antigen-binding portion thereof in a method for diagnosing a subject having an antigen associated with a particular disease or disorder, comprising administering to said subject a diagnostically effective amount of a pharmaceutical composition according to the third aspect. Preferably the subject is a human.

For example, the chimeric antibody υr antigen-binding fragment thereof, preferably labelled, can be used to detect the presence υf an antigen, or elevated levels of an antigen (e.g. TNFu) in a biological sample, such as serum or plasma using a convention immunoassay, such as an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) or tissue immunohistoehemistry.

Preferably, the antigen to which the chimeric antibody or antigen-binding portion thereof binds, is peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid in nature, sclccTcd from a tumour-associated antigen including carcinoembryonic antigen, EpCΛM, Lβwis-Y, Lewis- Y/b, PMSΛ, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R, Her-2, TRAIL and VEGF receptors, an antigen involved in an immune or tnflanimatory disease or disorder including CD3, CD4, CD25, CD40, CD49d, MHC class I, MHC class II, GM- CSF, inlerferon-γ, IL-I, IL- 12, IL-13, IL-23, TNh'-α, and IgR, an antigen expressed on a host cell including glycoprotein llb/llla, P-glycoprotcin, purinergic receptors and adhesion receptors including CDl Ia, CDl Ib, CD 1 Ic, CD 1 S, CD56, CD58, CD62 or CD144, an antigen comprising a cytokine, chemokinc, growth factor or other soluble physiological modulator or a receptor thereof including eotaxin, JLL-6, IL-8, TGF-β, C3a, C5a, VRdF, NGF and their receptors, an antigen involved in central nervous system diseases or disorders including β-aτnyloid and prions, an antigen of non-human origin such as microbial, nanobial or viral antigens or toxins including respiratory syneitial virus protein F.. anthrax toxin, rattle snake venom and digoxin; wherein the chimeric antibody acts as an agonist or antagonist or is active Io either deplete (kill or eliminate) undesired cells (eg. aπti-CD4) by acting with complement, or killer cells (eg. NK cells) oris active as a cytotoxic agent or to cause Fe-receptor binding by a phagocyte or neutralizes biological activity of its target.

The anti-human TNFα chimeric antibody or antigen binding portion thereof according to the invention may also be used in cell culture applications where it is desired to inhibit TNFn activity.

The present invention also provides a method for treating a disease or disorder characterised by human TNFα activity in a human subject, comprising administering to the subject in need thereof a pharmaceutical composition according to the present invention in which the chimeric antibody or antigen-binding portion thereof binds TNFre.

The term "disease or disorder characterised by human TNFα activity" as used herein is intended to include diseases or disorders in which the presence of TNFα in a subject suffering from the disease or disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disease or disorder or a factor that contributes to the worsening of the disease or disorder. Accordingly, a disease or disorder in which TNFα activity is detrimental is a disease or disorder in which inhibition of TNFre activity is expected to al leviate symptoms and/or progression of the disease or disorder. Such diseases or disorders may be evidenced, for example, by an increase in the concentration of TNFα in a biological fluid of a subject suffering from the disease or disorder (c.g., an increase in the concentration of TNFα in serum, plasma, synoviid fluid etc of the subject), which can be detected, for example, using a chimeric antibody of the invention specific for TNFα

A disease or disorder characterised by human TNFα activity is intended to include diseases or disorders and other disease or disorder in which the presence of TNFα in a subject suffering from the disease or disorder has been shown to be, or is suspected of being, cither responsible for the pathophysiology of the disease or disorder or a factor which contributes to a worsening of the disease or disorder. Preferably, the disease or disorder characterised by human TNFα activity is selected from the group consisting of sepsis, including septic shock, endotoxic shock, gram negative sepsis and toxic shock syndrome; autoimmune disease, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, psoriasis 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 disease including adult respiratory distress syndrome, shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis; inflammatory bowel disease including Crohn's disease and ulcerative colitis; cardiac disease; inflammatory bone disease, hepatitis, coagulation disturbances, burns, reperfusion injury, keloid formation and scar tissue formation.

Supplementary active compounds can also be incorporated into lhc composition. The ^' antibody or antibody-binding fragment may be co-formulalcd with and/or administered simultaneously, separately or sequentially with one or more additional therapeutic agents eg. antibodies that bind to other targets sυch as cytokines or cell surface molecules or alternatively one or more chemical agents that inhibit human TNfFa production or activity.

Jn another aspect, lhe invention provides a kit comprising a therapeutically effective amount of a chimeric antibody or antigen-binding portion of lhc invention, or a pharmaceutical composition comprising a therapeutically effective amount of a chimeric antibody or antigen-binding portion thereof, together with packaging and instructions for use. In certain embodiments, the instructions for use include instructions for how to effectively administer a therapeutic amount of the chimeric antibody or antigen-binding portion of flic invention,

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. In order ihai the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples. EXAMPLE 1

Fusion of a marmoset variable region to a human constant region

Materials andi methods

Gene Synthesis and Cloning The V_K chain (Accession Number: AAM54057. SEQ LD NO: 1 ) of lhe MOG specific marmoset derived antibody was expressed with a human constant region (human IgGI heavy chain C_H1., hinge, Cn2 & C_H3 domains (such as NCBI accession number POl 857) (SEQ ID NO; 2». This was achieved by back translation of flic amino acid sequence into a DNA sequence which was optimized for mammalian cell expression using GeneOptimizer technology and synthesized de novo by assembly of synthetic oligonucleotides (GencArl, Germany). During DNΛ sequence optimisation lhe specific restriction enzyme sites Aac I and Tth I UI were included to allow for future manipulation of the V_H region. Following gene synthesis the whole sequence including a Kozak sequence was cloned into the multiple cloning site of the pEE6,4 GS accessory vector (Lonza Biologies). The Vr. chain (Accession Number: A AM54058, SEQ ID NO: 3) of the MOG specific marmoset derived antibody was expressed with a human kappa light chain constant region (such as NCBI accession number AAA58989) (SEQ ID NO: 4). DNA encoding the light chain (V_L- Kappa) amino acid sequence was prepared as described above for tlic heavy chain. During DNA sequence optimization and synthesis lhe specific restriction enzyme sites Bsi WI / Rsr IJ were included Io allow future manipuation of the V^ region. Following gene synthesis the whole sequence including a Kozak sequence was cloned into lhe multiple cloning site of the pEE12.4 GS expression vector (Lonza Biologies). For stable expression the two single gene vectors (pHE6.4-V_H-IgGi and pEE12.4-V_f Kappa) were combined into a double gene vector. This was done by digesting out of the pt£E6.4 backbone lhe heavy chain expression cassette (hCM V-MIE promoter, Kozak sequence, marmoset V_H, human constant region and S V40 polyA site) using Not I and BcnriΑ I. The resultant fragment was subcloned using Not I and BamH 1 sites into the pEHI2.4-Vu-Kappa vector downstream of the light chain expression cassette (hCMV-MTE promoter, Kozak sequence, marmoset V_L,, human Kappa constant region and SV40 polyA site) creating a vector expressing both lhe heavy and light chains of AB \ 38 (SEQ ID NOs: 5 and 6).

Transfection For each transtection 175μl of Lipofectaminc 2000 was added to 5rnL nf OpLirnetn I media (Invitrogen Cat Noa. 11668-027 and 31985-062) in a well or a 6 weJt plate. In a second well 70μl of the expression vector (7θμg) was added to 5 πiL, of Optimerπ I media. Following a 5 minute room temperature incubation, the contents of the two wells were mixed together and left for a further 20 minute incubation. Following this second incubation the whole transfection mixture was added to a T175 tissue culture flask containing the CHOKlSV cells. Cells were incubated for 72 to 96 hours and supcrnatants harvested. Supematants were certlrifugcd at 4,000 x g for 5 minutes to pellet cell debris, and were filter sterilised through 0.22 μm cartridge filter. Antibody Purification

The supernatant was passed over a HiTrap Protein A column (Amersham Bioseienecs, Cat No; 17-0402-01) three times at a flow rate of 1 mL/min. The column was then washed with 20 mlVt sodium phosphate for 40 niins at I mL/min. The antibody was eluted with 0.1 M citric acid pH 3.5 with fractions collected and immediately neutralised with IM Tris- I ICl ptf 9.0. Antibody samples were then desalted on a PD-10 column (Λmcrsham Biosciences, Cat No: 17-0851-01). Analysis of the antibody by SDS-PΛGE and size- exclusion HPLC confirmed the correct molecular weight, presence of assembled antibody and the concentration of antibody.

Western Biol analysis The ability of AB 138 to retain binding to the antigen of M26, rat MOG (myelin- oligodendrocyte glycoprotein), was investigated by Western Blot. 130 mg of rat spinal cord (IMVS, Australia) was homogenized in 1.8 ml CeILytic M Cell Lysis Reagent (SlGM A, C2978) and incubated for 30 minutes at 4°C. Farther homogenization was performed by drawing the lysate through a 27gI/2 needle several times followed by centrifugalion at 4"C and 13000g for 30 minutes. The pellet and supernatant was diluted into SDS-PΛGE sample buffer (125 πuM Tris-HCl pH 6.8, 5% SDS, 0.25% bromophenol blue, 25% glycerol). Along with this 200 μ! CHOKlSV cells at 1 X 10⁶ viable cells per ml were spun down at 13000 x g at 4°C for 1 minute and rcsuspended in 200 μl CeILytic M Cell J.ysis Reagent (SIGMA). Following centrifugatioπ at 4"C and 13000 x g for 30 minutes the supernatant was mixed with the appropriate amount of SDS-PAGE sample buffer. All samples, along with a sample of molecular weight markers, were run on a 4- 20% Novex prc-cast gel (Invilrogen, Australia) for 2 hours at 120V. Proteins were then transferred to PVDF (BioRad, Australia) using a western blot apparatus in 1 X Tris- Glycine Buffer with 20% methanol (BioRad, Cat 161+-0771) at 4⁰C at 250 mΛ for 2 hours. The membrane was then blocked by incubation with 5 % skim milk powder in PBS for 1 h at room temperature, The membrane was then washed with { X PBS three times followed by an overnight incubation at 4^υC with AB 138 in PBS at I0 ug/mL. After washing, the membrane was incubated with Goat Anti-human IgG (1.1+L) HRP conjugate (Sigma, Australia) diluted 1:5000 in I XPBS for I hour at room temperature. Following washing, bound antibody was detected using the ECL Western Blotting Analysis System, (Amcrsham Biosciences Cat: RPN2109). Λ parallel experiment was performed in which AB 138 was replaced with an iso type-matched irrelevant specificity negative control antibody (anti-TNFα monoclonal antibody) in order to identify any non-specific binding events.

Results

After successful protein expression and purification, western blot analysis was performed on AB138 to determine if it retained binding affinity to rat MOG. AB 138 bound a protein with approximate size of 25 kDa present in the rat spinal cord cleared lysatc, a protein not present in cleared CHOKlSV lysale (Figurc 1). The negative control antibody did not bind to protein present in either Iysate indicating the interaction between AB 138 and the protein of size 25 kDa was not due to artifact or non-specific binding events associated with the human constant region (Figure 2). This protein matches the expected size of rat MOG minus the signal sequence (24,9 kDa). This result indicates that AB138 retained affinity for rat MOG present in rat spinal cord Iysate and demonstrates that a marmoset human fusion antibody can retain antigen binding ability.

It can be appreciated by someone skilled in the art that rat MOG could be produced using recombinant DNA technology and the ability of AR 138 to bind rat MOG determined in binding assays such as JELISΛ or Biacorc analysis. EXAMPLE Z

CDR2 Substitution of a domain antibody

Standard recombinant DNA technology can be used to produce a locally engineered domain antibody by substitution of the CDR2 of an acceptor anti-TNFα domain antibody (Basran el al. WO 2004/081026; SRQ ID NO: 7; Figure 3) with a CDR2 from a donor New World primate immunoglobulin.

Applying the rules of Kabat (Sequences of Proteins of Immunological Interest" K. Kabal eL al., U.S. Department of Health and Human Services, 1983) the CDR2 is identified on the acceptor anti-TNT-α domain antibody (SASELQS). The domain antibody acceptor sequence is then aligned against a panel of New World primate immunoglobulin sequences. These sequences arc derived from the Ma's night monkey (Λotus nancymaac) (SEQ !D NOs:.8 - I 8) and from the common marmoset (Callithrix jacchus) (SEQ IT) NOs: 19 - 24 ) (Figure 4). The CDR2 sequences of the New World primate immunoglobulins that differ from that of Lhe acceptor CDR2 sequence can be identified as SASTLQT, DASSLQP, GASTRAT, KVSNRAS, RVSNRAS, KVSTRGF, AΛSNRAS, TSSNLQA, KASTLQS, AASTLQS, YASSLQS, YASFLQG) (Table I). BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) oil each of these donor New World primate CDR2 sequences is performed to remove sequences that are exact matches for human immunoglobulin sequences. Sequences unique to New World primates were KVSNRAS, RVSNRAS, KVSTRCGP, AΛSNRAS, TSSNLQA, DASSLQP, YASFLQG (Table 1 ).

Table 1 : New World primate CDR2 sequences and their suitability as donor sequences. The acceptor CDR2 and the potential donor CDR2s are examined for their predicted immunogenicity in humans by the MHC class U binding prediction program Proprcd (http://www.imleeh.res.in/raghava/prot>rcd) using a 1% threshold value analysis of all alleles. From this analysis the acceptor CDR2, SASELQS, forms part of the peptide, LIYSASFϊLQ, which is predicted to bind MiIC class II encoded by 1 1 alleles (DRBl .0306, DRBl_0307. DRB1_O3O8, DRB1_O311, DRBl-0401 , DRB1_O426, DRB1_0806, DRBl_0813, DKBlJ 50 L, DRB I .1502, DRB1_1506). The donor CDR2 sequence, KVSNRAS, forms part υf a sequence, LIYK VSNRAS, which is predicted to bind MHC class !l encoded by 9 alleles (DRB 1 _0309, DRB1_O4O2, DRB I _0802, DRB 1_0804, DRB 1. 0806, DRB l_0813, DRB 1 _1301 , DRB IJ 327, DRB I J328). The donor CDR2 sequence, ΛASNRAS, forms part of a sequence, LlY AASNRA, which is predicted to bind MHC class Il encoded by 6 alleles (DRBl_0402, DRBl_0404, DRBl_040S, DRB1_O423, DIIB1_O813, DRB IJ 506). The donor CDR2 sequence, TSSNLQA, forms part of a sequence, Ll YTSSNLQA, which is predicted to bind MHC class l[ encoded by 10 alleles (DRBlJMO 1 , DRB l_0402, DRB1_0404, DRB I JWlO₇ DRBl JH23, DRBl J)42(>, DRBl_0813, DRB1J50I , DRBl J502, DRBl J 506). The donor CDR2 sequence, KVSTRGP, forms part of a sequence LUYKVSTR, which is predicted to bind MUC class II encoded by 8 alleles (DRB1_0309, DRBI_0802, DR.B1J0804, DRB1JJ806, DRB1JJ813, DRB 1 J301, DRB1_1327, DRB1_1328). Hence, the acceptor CDR2 can be replaced with a donor CDR2 of lower predicted immunogenicity, including KVSNRAS, A ASNRAS, TSSNLQΛ and KVSTRGP.

Using recombinant DNA technology, the acceptor CDR2 is replaced with the donor CDR2 sequences, generating the locally engineered domain antibodies (SEQ ID No: 25 - 31), Examples of recombinant DNΛ technology include those described by Winter et al. (US 5,225,539), and include, but is not limited to, techniques such as sile-dirceled mutagenesis and oligo annealing. Protein expression of the domain antibodies is then performed in E .colt BL21 (DE3) pLys (Novagcn, Germany) using a suitable vector for expression such as pBT21d(+) (Novagen, Germany), or by other such methods known in the art such as those describe by Basran et al. (WO 2004/0S1026). Following bacterial cell lysis the domain antibodies are purified using Protein L (Pierce, USA) chromatography. Following purification the engineered domain antibodies are analysed for retention of TNFα binding ability by methods known in the art, such as the L929 neutralisation assay or the TNFα receptor I binding assay.

To improve the binding affinity of the engineered domain antibodies, affinity maturation could be performed by amino acid substitution of the framework residues surrounding and stabilising CDR2 or by other methods known in the art.(Winter et al. (US 5,225,539); Griffiths et al (US 5,885,793); Rajpal, A. et at (2005) A general method for greatly improving the affinity ot antibodies by using combinatorial libraries, PrM Natl Acad Sci U S A , 102(24) 8466-71 ; Irving R.A. el al. (2001) Rtbosome display and affinity maturation: from antibodies to single V-dυmains and steps towards cancer therapeutics, Journal of Immunological Methods, 248: 31 -45). It will be appreciated by persons skilled in ihe art that numerous variations and/or modifications may be made to the invention as shown^' in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments arc, therefore, to be considered in all respects as illustrative and not. restrictive.

Claims

CLATMS;-

1. A chimeric antibody or an antigen-binding portion thereof, wherein the antigen- binding portion comprises at least two complementarity determining regions (CDR) and at ieaKt three framework regions, wherein at least, one CDR is a New World primate CDR,

1. Λ chimeric antibody or an antigen-binding portion thereof according to claim 1 wherein the antigen binding portion comprises three CDRs and four framework regions.

3. A chimeric antibody or an antigen-binding portion thereof according to claim 1 or claim 2 wherein the antigen-binding portion comprises at least one CDR which is human CDR.

4. A chimeric antibody or an antigen -binding portion thereof according to any one of claims 1 to 3 wherein the antigen-binding portion comprises two CDRs which are a human CDRs. 5. A chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to 4 wherein CDR2 is a New World primate CDR2.

6. A chimeric antibody or an antigen-binding portion thereof according to claim 5 wherein the CDR2 sequence is selected from the group consisting of KVSNRAS, RVSNRAS, KVSTRGP, AASNRΛS, "! SSNLQA, DASSLQP and YASFLQG. 7. A chimeric antibody or an antigen-binding portion thereof according to claim 6 wherein the CDR2 sequence is selected from the group consisting of KVSNRAS, AΛSNRAS, TSSNLQA and KVSTRGP.

8. A chimeric antibody or an antigen-binding portion thereof according to atvy one of claims t to 7 wherein the framework regions are human sequences. 9. A chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to S wherein at least one framework region is modified Io increase binding.

10. Λ chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to 9 wherein at least one framework region is modified Io reduce predicted immunogenicity in humans.

1 1. A chimeric anLi body or an antigen-binding portion thereof according to any one of claims 1 lo 10 wherein at least one CDR sequence is modified to increase binding, provided thai Lhe at least one New World primate CDR sequence is not modified. ■

12. A chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to 11 wherein at least one CDR sequence is modified to reduce predicted immunogcπicity in humans, provided that the at least one New World primate CDR sequence is noL modified.

13. Λ chimeric antibody or an antigen-binding portion thereof according to claim 11 or claim 12 wherein the al least oαc CDR sequence which is modified is not the New World primate CDR.

14. A chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to 13 wherein the antigen-binding portion is a domain antibody,

15. Λ chimeric antibody or an antigen-binding portion thereof according to any one of claims I to 14 wherein the antibody or antigen-binding portion further comprises a human or non-human primate constant region sequence.

16. A chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to 15 wherein the New World primate is selected from the group consisting of marmosets, lamarins, squirrel monkey, uakaris, sakis, titi monkey, spider monkey, woolly monkey, capuchin, night or owl monkey and the howler monkey.

17. A chimeric antibody or an antigen-binding portion thereof according to claim 16 wherein the New World primate is a marmoset.

18. Λ chimeric antibody or an antigen-binding portion thereof according to any one of claims 1 to 17 wherein the antibody binds an antigen that is peptide, protein, carbohydrate, glycoprotein, lipid or glycoUpid in -nature, selected from a tumour- associated antigen including carcinυembryorrie antigen, EpCΛM, Lewis-Y, Lewis- Y/b, PMSΛ, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R, Her-2_, TRAIL and VEGF receptors, an antigen involved in an immune or inflammatory disease or disorder including CD3, CD4, CD25, CD40, CD49d, MUC class T, MHC class II, GM-CSF, interfcron-γ, IL-I, IL- 12, IL-13, IL-23, TNF-_Ot, and IgB_, an antigen expressed on a host cell including glycoprotein Ilb/Ula, P-glycoprotem, purinergic receptors and adhesion receptors including CDlIa, CDHb, CDl Ic_, i CD 18, CD56, CD58, CD62 or CD 144, an antigen comprising a cytokine, chemokine, growth factor or other soluble physiological modulator or a receptor thereof including eυlaxin, IL-6, IL-8, TGF-β, C3a, C5a, VEGF, NGF and their receptors, an antigen involved in central nervous system diseases or disorders including β-amyloid and prions, an antigen of non-human origin such as microbial, nanobial or viral antigens or toxins including respiratory syncilial virus protein F, anthrax toxin, rattle snake venom and digoxin,

19. A chimeric antibody or an antigen-binding portion thereof according to claim 18, wherein the antibody binds to TNFα. 20. A method of producing a chimeric antibody or an antigen-binding portion thereof, the method comprising deleting a CDR from a human antibody variable region comprising at least two CDRs and at least three framework regions and replacing it with a New World primate CDR predicted to be of low immunogenici Iy to produce a chimeric variable region. 21. The method according to claim 20 wherein the method further comprises the step of recovering the chimeric variable region.

22. The method according to claim 20 or claim 21 wherein the New World primate CDR is CDR2.

23. The method according to any one of claims 20 to 22 further comprising the step of modifying the sequence of the chimeric variable region to increase binding, provided that the New World primate CDR sequence is not modified.

24. The method according to any one of claims 20 to 23 further comprising the step of modifying the sequence of the chimeric variable region to decrease immunogenicity in humans, provided that the at least one New World primate CDR. sequence is not modified.

25. The method according to any one of claims 20 to 24 wherein the New World primate is selected from the group consisting of marmosets, lamarins, squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, uakari.s, sakis, night or owl monkey and the howler monkey. 26. The method according to claim 25 wherein the New World primate is a marmoset.

27. The method according to any one of claims 20 to 16 wherein the antibody binds to an antigen that is peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid in nature, selected from a tumour-associated antigen including carciπocmbryonic antigen, EpCAM, Lcwis-Y, Lewis- Y/b, PMSA, CD20, CD3Q, CD33, CD38,

S CD52, CD 154, EGF-R, tfer-2, TRA \L and VFOF receptors, an antigen invol ved in an immune or intlamimatory disease or disorder including CD3, CD4, CD25, CD40, CD49d, MfIC class I, MIlC class II, CM-CSF, IL-I, IH2, EL-13, IL-23, TNF-α, and IgE, an antigen expressed on a host cell including glycoprotein Ilb/IIla, P-glyco protein, purinergic receptors and adhesion receptors 0 including CDl Ia, CDlIb, CD I Ie, CDiS, CD56, CD58, CD62 or CD144, an antigen comprising a cytokine, chcinokine, growth factor or other soluble physiological modulator or a receptor thereof including cotaxin, IL-6, IL-8, TOF- P, C3a, C5a, VEGF, NGF and their receptors, an antigen involved in central nervous system diseases or disorders including β-amyloid and prions, an antigen 5 of non-human origin such as microbial, nanobial or viral antigens or toxins including respiratory syncytial virus protein F₇ anthrax, toxin, rattle snake venom and digoxin.

28. The method according to claim 27, wherein the antibody binds to TJN Fa.

29. A chimeric antibody or an antigen-binding portion thereof produced by the 0 method according to any one of claims 20 to 28.

30. A kit comprising a chimeric antibody or antigen-binding portion according to any one of claims 1 to 19, or a pharmaceutical composition thereof, packaging and instructions for use.