JP2009504686A - Chimeric antibodies using the New World primate domain - Google Patents

Abstract

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

Description

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

  Antibodies (immunoglobulins) play an important role in the mammalian immune system. They are produced by mature plasma cells from precursor B cells. An antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked by disulfide bridges. Light chains are called kappa or lambda light chains and heavy chains are called gamma, mu, delta, alpha or epsilon. Each chain consists of a constant and variable region. The variable region confers specificity on the antibody. Within each variable region is a hypervariable region or complementarity determining region (CDR), which is lined with more conserved regions called framework regions. Within each variable region are three CDRs and four framework regions.

  Antibodies are bifunctional molecules, and N-terminal variable fragments from heavy and light chains bind to each other in a specific way to produce a three-dimensional structure that shows affinity for specific epitopes on the antigen surface is doing. The constant region portion is involved in serum half-life and prolongation of antibody effector functions and is implicated in complement binding, phagocytic stimulation, antibody-dependent cytotoxicity, and granulocyte granule release.

  Advances in hybridoma technology have facilitated the production of monoclonal antibodies with specific specificities. Typically, such hybridomas are murine hybridomas.

  A human / mouse chimeric antibody has been generated, in which antibody variable region sequences from the mouse genome are combined with antibody constant region sequences from the human genome. A chimeric antibody displays the binding properties of the parent mouse antibody and effector functions associated with the human constant region. Antibodies are produced by expression in host cells including, for example, Chinese hamster ovary (CHO) cells, NSO myeloma cells, COS cells and SP2 cells.

  Although such chimeric antibodies have been used in human therapy, antibodies against these chimeric antibodies have been produced by human recipients. Such anti-chimeric antibodies adversely affect continuous treatment with chimeric antibodies.

  It has been said that human monoclonal antibodies are expected to be an improvement of mouse monoclonal antibodies for in vivo human therapy. Studies done with antibodies from Old World primates (red-haired monkeys and chimpanzees) have argued that these non-human primate antibodies are structurally similar to human antibodies and are therefore acceptable in humans. (Ehrlich PH et al., Human and primate monoclonal antibodies for in vivo therapy. Clin Chem, 1988, 34: 9 1681 to 1688). Furthermore, since human antibodies are non-immunogenic in redhead monkeys (Ehrich PH et al. Rhesus monkey responses to multiple injections of human monoclonal amtibodies. Hybridoma, 1987, 6: 151-60), the reverse is also applicable. It appears that primate antibodies are non-immunogenic in humans. These monoclonal antibodies are secreted from hybridomas constructed by fusion of lymphocytes with human x mouse heteromyeloma.

  EP 0605 442 discloses chimeric antibodies that bind to human antigens. These antibodies contain all of the variable regions from Old World monkeys and the constant regions of human or chimpanzee antibodies. One of the advantages suggested in this reference for these configurations is the ability to elicit Old World monkey antibodies against human antigens that are less immunogenic in humans compared to antibodies raised in mouse hosts. is there.

  New World primates (infraorder-Platyrrhini) have at least 53 species that are generally divided into two families, Callithricidae and Cebidae. Callithricidae consists of marmosets and tamarins. Cebidae includes squirrel monkeys, titesar monkeys, spider monkeys, woolly monkeys, capuchin monkeys, cucumbers, sakis, night or owl monkeys, and howler monkeys.

  Evolutionary distant primates, such as New World primates, are not only sufficiently different from humans to allow antibodies to human antigens to be generated, but antibodies from such primates are introduced into the human body. In that case, it is sufficiently similar to humans that it has antibodies similar to human antibodies so that the host does not produce an anti-antibody immune response.

  Previous studies were characterized by having an expressed immunoglobulin heavy chain repertoire of Callithrix jacchus marmoset (von Budingen HC et al. Characterization of the expressed immunoglobuilin IGHV repertoire in the New World marmoset Callihrix Jacchus. Immunogenetics, 2001 , 53: 557-563). Six IGHV subgroups with a high degree of sequence homology with their corresponding human IGHV were identified. The framework region is more conserved when compared to the complementarity determining region (CDR). The degree of similarity of IGHV sequences between common marmoset (C. jacchus) and humans is less than between non-human Old World primates and humans.

Domain antibodies Domain antibodies (dAbs) are functional binding units that can be made using an antibody framework and correspond to the variable region of either the heavy chain (V _H ) or light chain (V _L ) of the antibody. To do. Domain antibodies have a molecular weight of about 13 kDa or are smaller than one tenth of the total size of the antibody.

  The immunoglobulin light chain is called the kappa or lambda light chain and the heavy chain is called gamma, mu, delta, alpha or epsilon. The variable region confers specificity on the antibody. Within the variable region of each antibody is a region known as the complementarity-determining region (CDR), which is called the hypervariable region, otherwise called the framework region, and is located alongside more conserved regions. Within each light and heavy chain variable region there are 3 CDRs and 4 framework regions.

In contrast to conventional antibodies, domain antibodies are well expressed in bacterial, yeast, and mammalian systems. Smaller sizes allow for a higher molar amount per gram of product, thus significantly increasing efficacy. In addition, domain antibodies are components for the manufacture of therapeutic products, eg, multi-target domain antibodies in which a construct containing two or more variable domains binds to two or more therapeutic targets, or for pulmonary or oral administration It can be used as a domain antibody.
EP 0605 442 WO 2004/003019 PCT / GB2003 / 002804 U.S. Pat.No. 4,816,567 U.S. Patent No. 5585089 US Patent Application Publication No. 20030039649 International Publication No.93 / 06213 International Publication No.92 / 01047 Ehrlich PH et al., Clin Chem., 1988, 34: 9 1681 ~ 1688 Ehrich PH et al. Hybridoma, 1987, 6: 151-60 von Budingen HC et al. Inmmunogenetics, 2001, 53: 557-563 Schneider H et at, Mol Phylogenet Evol., 1993 Sep; 2 (3): 225-42 Stern, Proc. Natl. Acad. Sci. USA 87: 6808 ~ 6812 (1990) Kong, YY. Et al. Nature 397: 315-323 (1990) Matthews, N. and ML Neale in Lymphokines and Interferons, a Practical Approach, 1987 MJ Clemens, AC Morris and AJH Gearing, eds., IRL Press, p.221 van den Beuken T et al, 2001, J. Mol. Biol, 310, 591 Ward et al, 1989, Nature 341: 544-546 Bird et al., 1988, Science 242: 423-426 Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85: 5879-5883 Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 Poljak, RJ, et al., 1994, Structure, 2: 1121-1123 http://www.imtech.res.in/raghava/propred http://bio.dfci.harvard.edu/Tools/rankpep.html Flower DR, Doytchinova IA. (2004) Immunoinformatics and the prediction of immunogenicity, Drug Discov Today, 9 (2): 82-90 Hammer J et al., 1994, J. Exp. Med., 180: 2353 http://www.ncbi.nlm.nih.gov/ Jespers et al./TECHNOLOGY Vol 12 1994, 899-903 McCafferty et al. 1990, Nature, 348: 552-554 Griffiths et al., 1993, EMBO J, 12: 725-734 Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; a laboratory manual, second edition, Cold Spring Harbor, NY (1989) RA Irving et al., 2001, Journal of Immunological Methods, 248, 31-45 Sambrook, Joseph; and David W. Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press Kipriyanov, SM, et al., 1995 Human Antibodies and Hybridomas, 6: 93-101 Kipriyanov, S. M, et al, 1994 Mol. Immunol., 31: 1047-1058

  The inventors have found that New World primates provide a rich source of binding domains for antibodies to a wide range of antigens, including human antigens. Furthermore, due to sequence homology between humans and New World primates, these New World primate sequences appear to have relatively low immunogenicity in humans.

  In a first aspect, the invention provides a chimeric antibody wherein the antigen-binding portion contains at least two complementarity determining regions (CDRs) and at least three framework regions, and at least one CDR is a New World primate CDR Or an antigen-binding portion thereof.

  Remove one CDR from a human antibody variable region containing at least two CDRs and at least three framework regions and replace it with a New World primate CDR predicted to have low immunogenicity, a chimeric variable region A method for producing a chimeric antibody or antigen-binding portion thereof is provided.

  In a related feature, the method further comprises recovering the chimeric variable region.

  In another aspect, the present invention provides a chimeric antibody or antigen-binding portion thereof produced by the method of the present invention.

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

  In yet a further feature, the present invention provides the use of an antibody of the present invention or an antigen-binding portion thereof in a diagnostic application for detecting an antigen associated with a particular disease or condition.

  In another aspect, the method comprises administering to a patient in need of treatment an effective amount of a chimeric antibody or pharmaceutical composition thereof described herein wherein the antibody or antigen-binding portion thereof binds TNF-α. Methods of treating diseases or conditions characterized by human TNF-α activity in human patients are provided.

  In a first aspect, the invention provides a chimeric antibody wherein the antigen-binding portion contains at least two complementarity determining regions (CDRs) and at least three framework regions, and at least one CDR is a New World primate CDR Or an antigen-binding portion thereof.

  In a first aspect, the invention provides a chimeric antibody wherein the antigen-binding portion contains at least two complementarity determining regions (CDRs) and at least three framework regions, and at least one CDR is a New World primate CDR Or an antigen-binding portion thereof.

  The antigen binding portion preferably comprises 3 CDRs and 4 framework regions.

  In certain embodiments of the invention, the chimeric antibody or antigen-binding 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. In another embodiment, CDR2 of the antibody or antigen binding portion thereof is a New World primate CDR.

  In another embodiment of the invention, the at least one New World primate CDR is not derived from a sequence that binds a target antigen.

  In other embodiments of the invention, the framework regions are human sequences. Framework regions that are human sequences include human framework regions or sequences derived from synthetic sequences based on human framework regions.

  It is within the scope of the present invention that the sequence of the antigen binding moiety may be further subjected to affinity maturation in order to improve antigen binding properties such as antigen binding or titer.

Increased binding is indicated by a decrease in K _D (K _off / K _on ) for the antibody or antigen binding portion thereof. Increased efficacy is shown by biological analysis. For example, assays that can be used to measure the effectiveness of an antibody or antigen-binding portion thereof include TNFα-induced L929 cytotoxin neutralization assay, IL-12-induced human PHA-activated peripheral blood mononuclear cells (PBMC) Contains RANKL mediated proliferation analysis and osteoclast differentiation of mouse splenocytes (Stern, Proc. Natl. Acad. Sci. USA 87: 6808-6812 (1990); Kong, YY. Et al. Nature 397: 315 323 (1990); Matthews, N. and ML Neale in Lymphokines and Interferons, a Practical Approach, 1987, MJ Clemens, AC Morris and AJH Gearing, eds., IRL Press, p.221).

  In a further preferred embodiment, at least one CDR is modified to increase binding or titer and to reduce the expected immunogenicity in humans. Preferably, the modified at least one CDR sequence is not a New World primate CDR. Where more than one New World primate CDR is present, it is preferred that at least one New World primate CDR has not been modified.

  In other embodiments of the invention, in addition to at least one CDR sequence, at least one framework region is modified to increase binding and / or to reduce predicted immunogenicity in humans. Is done. Preferably, the modified at least one CDR sequence is not a New World primate CDR sequence.

  In a preferred embodiment, the antigen binding moiety is a domain antibody.

In further embodiments of the invention, the domain antibody is, for example, a hetero- or homodimer (eg, V _H / V _H , V _L / V _L or V _H / V _L ), hetero- or homotrimer. (E.g., _VH / _VH / _VH , _VL / _VL / _VL , _VH / _VH / _VL or _VH / _VL / _VL ), hetero- or homotetramers (e.g., V _H / V _H / V _H / V _H , V _L / V _L / V _L / V _L , V _H / V _H / V _H / V _L or V _H / V _H / V _L / V _L or V _H / V _L / V _L / V _L ), or other higher-order hetero- or homomultimers, may be multimerized. Multimerization can increase the strength of antigen binding, where the binding strength is related to the binding affinity of a multiple binding site or a sum of parts thereof.

  Thus, the present invention provides a domain antibody bound to at least one additional domain antibody. Each domain antibody may bind to the same or different antigen.

Domain antibody multimers may further contain one or more linked domain antibodies, where each domain antibody is a different antigen, a so-called “dual-specific ligand”. Binds to a multispecific ligand comprising For example, a bispecific ligand may contain a pair of _VH domains or a pair of _VL domains. Such bispecific ligands are described in WO 2004/003019 (PCT / GB2003 / 002804) under the name Domantis Ltd, all of which are incorporated herein by reference.

  Preferably, the antibody or antigen binding portion thereof further comprises a human or non-human primate constant region sequence. Examples of non-human primates include but are not limited to chimpanzees, orangutans, and gorillas.

  The present invention also removes one CDR from a human antibody variable region containing at least two CDRs and at least three framework regions and replaces it with a New World primate CDR predicted to have low immunogenicity And a method for producing a chimeric antibody or antigen-binding portion thereof, comprising producing a chimeric variable region.

  In a related feature, the method further comprises recovering the chimeric variable region.

  The selected New World primate CDR is preferably CDR2. The CDR2 sequence is selected from KVSNRAS, RVSNRAS, KVSTRGP, AASNRAS, TSSNLQA, DASSLQP, and YASFLQG. Particularly preferred sequences are KVSNRAS, AASNRAS, TSSNLQA, and KVSTRGP because of their lower expected immunogenicity.

In a further embodiment, the method comprises modifying the sequence of the chimeric variable region to increase binding and / or reduce immunogenicity in humans. Preferably, the New World primate CDR sequence is not altered. Where more than one New World primate CDR sequence is present, it is preferred that at least one New World primate CDR is not modified.
To do.

  In other embodiments of the invention, at least one framework region is modified in addition to at least one CDR sequence to increase binding and / or reduce predicted immunogenicity in humans. Is done. Preferably, the at least one modified CDR sequence is not a New World primate CDR sequence. The present invention also provides a chimeric antibody produced by the method of the present invention or an antigen-binding portion thereof.

As used herein, the term “antibody” is intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. . Each heavy chain includes a heavy chain variable region (HCVR or V _H ) and a heavy chain constant region. The heavy chain constant region includes three domains, C _H 1, C _H 2 and C _H 3. Each light chain includes a light chain variable region (LCVR or _VL ) and a light chain constant region. The light chain constant region comprises one domain, the C _L. The V _H and V _L regions can be further divided into hypervariable regions, which are called complementarity determining regions (CDR) and incorporate more conserved framework regions (FR). Each _VH and _VL region is composed of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term “antigen-binding portion” of an antibody refers to one or more components or derivatives of immunoglobulin that exhibit the ability to bind to an antigen. It has been shown that the antigen binding function of an antibody can be achieved by fragments of a full length antibody. Examples of binding fragments encompassed by the term `` antigen-binding portion '' of an antibody are (i) a Fab fragment, which is a monovalent fragment consisting of V _L , V _H , C _L and C _H 1 domains; (ii) F (ab ′) ₂ fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V _H and C _H 1 domains; (iv) an antibody Fv fragment consisting of V _L and V _H domains of one arm of (v) consisting of one V _H domain or V _L domain (van den Beuken T et al, 2001, J. Mol. Biol, 310, 591) dAb fragment (Ward et al, 1989, Nature 341: 544-546); and (vi) containing an isolated complementarity determining region (CDR). In addition, the two domains of the Fv fragment, _VL and _VH, are encoded by separate genes, which can be ligated by synthetic linkers using recombinant methods, such that a pair of _VL and _VH regions is a single pair. Create a single protein chain that forms a monovalent molecule known as single chain Fv (scFv) (e.g., Bird et al., 1988, Science 242: 423-426 and Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain Fvs are also intended to be encompassed by the term “antigen-binding portion” of an antibody. Other forms of single chain Fvs and related molecules such as dimers or trimers are also encompassed. A dimer is a bivalent antibody in which V _H and V _L domains are expressed on a single polypeptide chain, but uses a linker that is too short to pair between two domains on the same chain Thus, the domains are paired with the complementary domains of other chains to form two antigen-binding portions (e.g., Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA , 90: 6444-6448; see Poljak, RJ, et al., 1994, Structure, 2: 1121-1123).

  The term “chimera” as used herein means that the antibody or antigen-binding portion thereof comprises sequences from two different species.

  In one embodiment, the domain antibody comprises a human framework region and at least one New World primate CDR, more preferably a marmoset CDR.

  Preferably, the New World primate is selected from the group consisting of marmosets, tamarins, squirrel monkeys, titisal monkeys, spider monkeys, woolly monkeys, capuchin monkeys, eucalyptus, genus Saki, cynomolgus monkeys, and howler monkeys. More preferably, the New World primate is a marmoset.

  Methods for producing chimeric antibodies of the present invention are well known to those skilled in the art, for example, see US Pat. No. 4,816,567, US Pat. No. 5585089 and US Patent Application Publication No. 20030039649, which are incorporated herein by reference in their entirety. Become known. Such methods require the use of standard recombinant techniques.

  The antibodies of the invention or antigen-binding portions thereof preferably have the low immunogenicity expected in a human host.

“Low immunogenicity” refers to an antibody response in at least a majority of individuals who receive a sufficient amount of antibody to reduce the effect of continued antibody administration at a time sufficient to achieve a therapeutic effect. It means not triggering.

The level of immunogenicity in humans is predicted using 1% threshold analysis of all alleles using the MHC class II binding prediction program Propred (http://www.imtech.res.in/raghava/propred) be able to. Other available programs are:
Rankpep (http://bio.dfci.harvard.edu/Tools/rankpep.html)
Epibase (Algonomics proprietary software: algonomics.com)
including.

  Low immunogenic molecules will contain 0 or a low number of peptides predicted to bind to MHC class II alleles that are highly expressed in the target population (Flower DR, Doytchinoya 1A. (2004) immunoinformatics and the prediction of immunogenisity, Drug Discov Today, 9 (2): 82-90).

  Molecules with reduced immunogenicity do not contain or have a reduced number of peptides predicted to bind to MHC class II alleles that are highly expressed in the target population compared to the initial donor molecule. Including.

  Functional analysis of MHC class II binding is to test for their ability to generate overlapping peptides and elicit T cell activation corresponding to the protein of interest (T cell proliferation assay) or reporter peptides Can be performed by replacing known MHC class II binding peptides (Hammer J et al., 1994, J. Exp. Med., 180: 2353).

  The present invention further provides methods for amplification of New World primate immunoglobulin genes, eg, from nucleic acids extracted from New World primate lymphocytes using primers specific for the heavy and light chain variable region gene families. Based on the polymerase chain reaction (PCR) method. To produce a New World primate chimeric recombinant antibody, the amplified variable region is cloned into an expression vector containing a human or primate constant region gene. Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, these genes are incorporated into recombinant expression vectors, and the vectors are incorporated into host cells such as Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; Introduce into host cells as described in a laboratory manual, second edition, Cold Spring Harbor, NY (1989).

  Suitable expression vectors are well known to those skilled in the art. New World primate lymphocytes that produce immunoglobulin are typically immortalized by fusion with a myeloma cell line to produce hybridomas.

  Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster 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 induced plants or prokaryotes (bacteria). Such expression systems are well known to those skilled in the art.

The repertoire of _VH , _VL and constant region domains may be a repertoire of naturally occurring immunoglobulin sequences or a synthetic repertoire. A naturally occurring repertoire is, for example, a repertoire prepared from immunoglobulin-expressing cells taken from one or more New World primates. Such a repertoire may be immature, i.e. prepared from neonatal immunoglobulin-expressing cells or rearranged, e.g. prepared from adult primate B cells. If necessary, clones identified from the natural repertoire, or any repertoire that binds to the target antigen, are subjected to mutagenesis and further screening to generate and select mutants with improved binding properties. .

  A synthetic repertoire of immunoglobulin variable domains is prepared by artificially introducing diversity into the cloned variable domain. Such affinity maturation techniques are well known to those skilled in the art (RA Irving et al., 2001, Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapoutics, Journal of Immunological Methods, 248: 31-45).

  The variable regions of New World primate antibody genes or their CDRs supply nucleic acid, e.g., cDNA, supply a primer complementary to the cDNA sequence encoding the 5 'leader sequence of the antibody gene, and contact the cDNA with the primer. Can be cloned by forming a hybrid complex and amplifying the cDNA to produce a nucleic acid encoding the variable region (or CDR region) of the New World primate antibody gene.

  In view of the teachings herein, New World primate variable region sequences can be used as acceptors for transplantation of non-New World primate sequences, in particular, CDR sequences can be used using standard recombinant techniques. Will be recognized by those skilled in the art of the present invention. For example, U.S. Pat.No. 5585089 retains the high affinity of a non-human parent antibody and contains a low immunogenicity containing one or more CDRs from a donor immunoglobulin and a framework region from a human immunoglobulin. A method for making a chimeric antibody is described. U.S. Patent Application Publication No. 20030039649 describes a standard for non-human antibodies as compared to the standard CDR structure type of a human antibody germline as a criterion for selecting an appropriate human framework sequence for a humanized antibody. Based on the use of CDR structure types, humanized methods for making low immunogenic chimeric antibodies containing CDR sequences from non-human and human antibody framework sequences are described. Thus, these principles can be applied to transplant one or more non-New World primate CDRs into a New World primate acceptor variable region.

  CDR sequences can be obtained from genomic DNA isolated from antibodies or from sequences present in databases, such as the National Center for Biotechnology Information protein and nucleic acid databases, The Kabat Database of Sequences of Proteins of Immunological Interest. The CDR sequence may be genomic DNA or cDNA.

  Transplantation methods that replace CDR (s) in acceptor variable sequences are well known to those skilled in the art of the present invention. Typically, the CDRs are grafted into each of the variable light and variable heavy or domain antibodies in the acceptor variable region sequence. A preferred method of the invention involves substitution of CDR1 or more preferably CDR2 through a primer intended for mutagenesis in the variable region sequence. The method consists of annealing a synthetic oligonucleotide encoding the desired mutation to the target region, the synthetic oligonucleotide serving as a primer for initiation of in vitro synthesis of DNA, extending the oligonucleotide with DNA polymerase, and A double helix DNA with a mutation is generated and the sequence is ligated into an appropriate expression vector and cloned (Sambrook, Joseph; and David W. Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press).

In addition, the antibody or antigen-binding portion thereof is part of a larger immunoadhesive molecule formed by the covalent or non-covalent association of an antibody or antibody portion having one or more other proteins or peptides. May be. An example of such an immunoadhesive molecule is the use of the core region of streptavidin to prepare a tetrameric scFv molecule (Kipriyanov, SM, et al., 1995 Human Antibodies and Hybridomas, 6: 93-101) And the use of cysteine residues, marker peptides and C-terminal polyhistidine tags to prepare divalent and biotinylated scFv molecules (Kipriyanov, S. M, et al, 1994 Mol. Immunol., 31: 1047 ~ 1058). Antibody portions such as Fab and F (ab ′) ₂ fragments can be prepared from the whole antibody using a conventional method such as papain digestion or pepsin digestion of the whole antibody. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein and known to those of skill in the art.

  The constant region sequence (Fc portion) is preferably obtained from a human or non-human primate immunoglobulin sequence. The primate sequence may be a New World primate or Old World primate sequence. Suitable Old World primates include chimpanzees, or other humanoid animals, apes, such as gorillas or orangutans, which are phylogenetically very close to humans, and thus have human constant region sequences. Share a high degree of homology. Sequences encoding for human or primate constant regions can be obtained from databases including, for example, The National Center for Biotechnology Information protein and nucleotide databases, The Kabat Database of Sequences of Proteins of Immunological Interest.

  An antibody or antigen-binding portion of the invention can bind to a human or non-human antigen.

  Preferably, the antigen that binds to the chimeric antibody or antigen-binding portion thereof is an oncofetal antigen, EpCAM, Lewis-Y, Lewis-Y / b, PMSA, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R , Her-2, TRAIL and VEGF receptors, including tumor-associated antigens; CD3, CD4, CD25, CD40, CD49d, MHC class I, MHC class II, GM-CSF, interferon-γ, IL-I, IL-12, IL -13, IL-23, TNF-α, and antigens involved in immune or inflammatory diseases or immune or inflammatory disorders including IgE; glycoprotein IIb / IIIa, P-glycoprotein, purine receptor and CD11a, CD1lb, CD11c, CDl8 Antigens expressed on host cells including adhesion receptors containing CD56, CD58, CD62 or CD144; eotaxin, IL-6, IL-8, TGF-β, C3a, C5a, VEGF, NGF and the like Cytokines, chemokines, growth factors or other soluble physiological regulators or Antigens including their receptors; antigens included in central nervous system diseases or disorders including β-amyloid and prions; non-human-derived antigens containing respiratory syncytial virus protein F, anthrax toxin, rattlesnake venom, and digoxin; For example, it binds to an antigen that is a natural peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid selected from an antigen consisting of a microbial, nanomicrobial or viral antigen or toxin; a chimeric antibody is an agonist or antagonist Acting as a complement, or active against reducing or killing unwanted cells (e.g., anti-CD4) by the action of complement or killer cells (e.g., NK cells), or cytotoxic agents Active or binds to or targets the phagocytic Fc receptor Neutralizes the biological activity of

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

  Alternatively, the antibody or antigen binding portion thereof can bind to a non-human antigen. Preferably the non-human antigen is selected from the group consisting of respiratory syncytial virus F protein, cytomegalovirus, snake venom, and digoxin.

  As used herein, the term `` binds to '' is 1 μM, as measured by, for example, surface plasmon resonance analysis using the BTAcoreTM surface plasmon resonance system and BTAcoreTM velocity evaluation software (e.g., version 2.1). Alternatively, antigen binding by an immunoglobulin variable region of an antibody having a lower dissociation constant (Kd). The affinity or dissociation constant (Kd) for a particular binding interaction is preferably about 500 nM to about 50 pM, more preferably about 500 nM or less, more preferably about 300 nM or less, 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 to about 50 pM, about 10 nM to about 50 pM.

  The antibodies of the present invention are advantageous for human therapy because of the reduced likelihood of inducing a human anti-antibody response.

  Recombinant antibodies produced according to the present invention that bind to the target antigen can be combined with a combination of immunoglobulin libraries (e.g., phage display libraries) to isolate library members that exhibit the desired binding specificity and functional properties. ) Can be identified and isolated by screening. It is understood that all methods using antigen binding moieties or antibody derivatives such as Fabs, scFv and V domain or domain antibodies are within the scope of the present invention. Phage display technology has been extensively described in the art and examples of methods and compounds for generating and screening such libraries and their affinity maturation products are described, for example, in Barbas et al, 1991, Proc Natl. Acad. Sci. USA, 88: 7978-7982; Clarkson et al., 1991, Nature, 352: 624: 628; Dower et al., PCT Publication No. WO 91/17271, US Pat. No. 5,427,908 No. 5, U.S. Pat.No. 5,580,717 and EP 527,839; Fuchs et al, 1991, Bio / Technology, 9: 1370-1372; Garrad et al, 1991 Bio / Technology, 9: 1373: 1377; Garrard el al, PCT Publication Number International Publication 92/09690; Gram et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 3576-3580; Griffiths et al., 1993 EMBO J, 12: 725: 734; Griffiths et al, USA Patent 5,885,793 and EP 589,877; Hawkins et al, 1992, J Mol Biol, 226: 889-896; Hay et al 1992, Hum Antibod Hybridomas, 3: 81-85; Hoogenboom et al, 1991 Nuc Acid Res, 19: 4133-4137; Huse et al., 1 989, Science, 246: 1275-1281; Knappik et al., 2000, J Mol Biol, 296: 57-86; Knappik et al. PCT WO 97/08320; Ladner et al., U.S. Pat.No. 5,223,409 , 5,403,484, 5,571,698, 5,837,500 and EP 436,597; McCafferty et al., 1990, Nature. 348: 552-554; McCafferty et al., PCT Publication No. 5,969,108 and EP 589,877; Salfeld et al, PCT Publication No. 97/29131, US Provisional Application No. 60 / 126,603; and Winter et al. PCT Publication No. 92/20791 and EP 368,684; .

  Recombinant libraries expressing the antibodies of the invention can be expressed on the surface of microorganisms such as yeast or bacteria (see PCT Publication Nos. WO 99/36569 and WO 98/49286).

  As referenced in the state of the art, the selected lymphocyte antibody method or SLAM is another means of rapidly generating high affinity antibodies. Unlike phage display methods, all antibodies are fully divalent. In order to generate New World primate antibodies, New World primates are immunized with a human antigen, eg, a TNFα polypeptide. After immunization, cells are removed and selectively grown in individual microwells. The supernatant is removed from the wells and tested for both binding and function. The gene sequence can be recovered for subsequent manipulation, eg, humanization, Fab fragment, scFv or domain antibody production. Thus, another example is the induction of ligands of the invention by SLAM and its derivatives (Babcook, JS et al 1996, Proc. Natl. Acad. Sci, USA 93; 7843-7848, US Pat. No. 5,627,052 and PCT publication. (International Publication No. 92 / Q2551). Other uses for testing SLAM applications such as panning, such as panning, are also within the scope of the present invention.

  In one expression system, the recombinant peptide / protein library is displayed on ribosomes (eg, Roberts, RW and Szostak, JW1997. Proc. Natl. Acad. Sci. USA. 94: 12297-123202 and (See PCT Publication WO 98/31700). Another such example is preferably, but not specifically limited to, generation and in vitro transcription of DNA libraries (e.g., antibodies and derivatives) prepared from immune cells, proteins and "immunized" mRNAs on ribosomes Translation, affinity selection (e.g. by binding to RSP), mRNA isolation, reverse translation and subsequent amplification (e.g. by polymerase chain reaction or related techniques) . Additional rounds of selection and amplification may optionally be linked to affinity maturation through the introduction of somatic mutations in this system, or other affinity maturation known in the state of the art. The method may be linked to affinity maturation.

  For another example, see the application of emulsion compartmentalisation technology to the antibody production of the present invention. In emulsion fragmentation, in vitro and optical screening methods are combined with co-compartmentalisation of the nucleotides that encode the translated proteins and sequences present in the aqueous phase in the emulsion oil droplets ( PCT Publication International Publication No. 99026711 and International Publication No. 0040712). The main elements for antibody generation and selection are basically similar to in vitro methods of ribosome display.

  The antibodies of the invention or antigen-binding portions thereof can be derivatized or bound to other functional molecules. For example, an antibody or antigen-binding moiety can be linked to one or more other molecular entities, such as other antibodies, detectable drugs, cells, by chemical binding, gene fusion, non-covalent association, or otherwise. It can be operably linked to a protein or peptide that can mediate association of the antibody or antigen-binding portion thereof by a toxic agent, pharmaceutical agent, and / or another molecule (eg, a streptavidin core region or a polyhistidine tag).

  Useful detectable agents in which the antibody or antigen-binding portion thereof may be derivatized include fluorescent compounds. Examples of fluorescent detection agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin and the like. The antibody may be derivatized with a detectable enzyme such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. If the antibody is derivatized with a detectable enzyme, the antibody is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. The antibody can also be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding.

  The present invention extends to non-PEGylated antibody polypeptides to PEGylated antibodies or antibody binding sites that increase half-life and increase resistance to degradation without losing activity (eg, binding affinity).

  As described herein, an antibody or antigen-binding portion thereof is a polymer molecule (preferably used to extend half-life and improve degradation-inhibiting properties using methods known in the art. Can be linked to PEG). The polymer moiety that can be utilized in the present invention may be synthetic or naturally occurring and is not particularly limited, and may be a linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or branched or non-polymerized. Includes branched polysaccharides, such as homo- or heteropolysaccharides. Preferred examples of synthetic polymers that can be used in the present invention include linear or branched poly (ethylene glycol) (PEG), poly (propylene glycol), or poly (vinyl alcohol) and derivatives or substitutes thereof. Particularly preferred substituted polymers for linking to the antibodies described herein include substituted PEGs including methoxy (polyethylene glycol). Naturally occurring polymer moieties that can be used in addition to or in place of PEG include lactose, amylose, dextran or glycogen, or derivatives thereof recognized by those skilled in the art.

Derivatized forms of polymer molecules include, for example, derivatives having additional groups or reactive groups capable of interacting with amino acid residues of the antibody polypeptides described herein. Such derivatives include N-hydroxysuccinimide (NHS) active esters, succinimidyl propionate polymers, and sulfhydryl selective reactive agents such as maleimides, vinyl sulfones, thiols. Particularly preferred derivatized polymers are PEG polymers having the formula: PEG-O-CH ₂ CH ₂ CH ₂ -CO ₂ -NHS; PEG-O-CH ₂ -NHS; PEG-O-CH ₂ CH ₂ -CO ₂ -NHS; PEG-S-CH ₂ CH ₂ -CO-NHS; PEG-O ₂ CNH-CH (R) -CO ₂ -NHS; PEG-NHCO-CH ₂ CH ₂ -CO-NHS; and PEG-O —CH ₂ —CO ₂ —NHS, where R is ((CH ₂ ) ₄ ) NHCO ₂ (mPEG), but is not limited to these derivatized polymers. PEG polymers can be linear molecules or branched, and multiple PEG moieties are present in a single polymer.

  Reactive groups (eg, MAL, NHS, SPA, VS or thiol) may be attached directly to the PEG polymer or to the PEG via a linker molecule.

  Polymer sizes useful in the present invention can range from 500 Da to 60 kDa, for example, between 1000 Da to 60 kDa, 10 kDa to 60 kDa, 20 kDa to 60 kDa, 30 kDa to 60 kDa, 40 kDa to 60 kDa. And between 50 kDa and 60 kDa. The polymers that can be used in the present invention, in particular PEG, can be linear polymers or have a branched structure.

  Polymer (PEG) molecules useful in the present invention can be attached to the antibody or antigen-binding portion thereof using methods well known in the art. The first step of conjugation of PEG or other polymer moieties to the antibody polypeptide monomers or multimers of the present invention is the substitution of the hydroxy end groups of the PEG polymer by electrophilc-containing functional groups. In particular, PEG polymers bind to cysteine or lysine residues present in antibody polypeptide monomers or multimers. Cysteine or lysine residues can be produced naturally or artificially introduced into antibody polypeptide molecules. For example, a cysteine residue can be introduced into the C-terminus of an antibody polypeptide by recombinant techniques, or a residue at a particular solvent accessible position in an antibody polypeptide can be replaced with cysteine or lysine. Can do.

An antibody can be linked to one or more molecules capable of extending its in vivo half-life. These molecules are linked to the antibody at a site on the antibody other than the antigen binding portion so that they do not interfere with the antigen binding portion or become steric hindrance of the antigen binding portion. Typically, such molecules are polypeptides that are naturally produced in vivo and resist degradation or elimination by endogenous mechanisms. It will be apparent to those skilled in the art to use such naturally occurring fragments or derivatives of molecules and that some are not polypeptides. Molecules that increase half-life can be selected from:
(a) proteins from the extracellular matrix, such as collagen, laminin, integrins and fibronectin;
(b) Proteins found in blood, such as fibrin α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobin, protein, ubiquitin, uteroglobulin, β-2 micro Globulin, plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin and pancreatic kypsin inhibitor;
(c) immune serum proteins such as IgE, IgG, IgM;
(d) transport proteins such as retinol binding protein, alpha-1 microglobulin;
(e) defensins such as β-defensin 1, neutrophil defensins 1, 2 and 3;
(f) proteins found in the blood brain barrier or nerve tissue, such as melanocortin receptors, myelin, ascorbate transporters;
(g) Transferrin receptor specific ligand-neuropharmaceutical fusion protein (see U.S. Patent No. 5977307); brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin-like growth factor 1 (IGF1) receptor, insulin-like growth factor 2 (IGF2) receptor, insulin receptor;
(h) proteins localized in the kidney, such as polycystin, type IV collagen, organic anion transporter K1, Heymann's antigen;
(i) a protein localized in the liver, such as alcohol dehydrogenase, G250;
(j) blood clotting factor X;
(k) α-1 antitrypsin
(l) HNF1α;
(m) proteins localized in the lung, e.g. secretory components (binding to IgA);
(n) a protein localized in the heart, such as HSP27;
(o) a protein localized in the skin, such as keratin;
(p) bone-specific proteins such as bone morphogenetic proteins (BMPs) such as BMP-2, -4, -5, -6, -7 (bone morphogenetic proteins (OP-1) and -8 (OP-2 ));
(q) tumor-specific proteins such as human trophoblast antigen, herceptin receptor, estrogen receptor, cathepsins such as cathepsin B (found in liver and spleen);
(r) Antigen expressed only on activated T cells containing disease-specific proteins such as LAG-3 (lymphocyte activation gene); osteoprotegerin ligand (OPGL) Nature 402 OX304 (a member of the TNF receptor family, expressed on activated T cells and specifically upregulated in human T cell leukemia virus type I (HTLV-I) producing cells) Costimulatory-only T cell molecules known to be-J. Immunol. 2000 Jul 1; 16561); 263-70; CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2, Metalloproteases including murine ftsH (related to arthritis / cancer); acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor / vascular permeability factor ( VEGF / VPF), transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α), angioje , Interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (PlGF), midkine platelet-derived growth factor-BB (PDGF ), Angiogenic factors including fractalkine;
(s) stress protein (heat shock protein);
(t) a protein contained in the Fc transport; and
(u) Vitamins such as B12, biotin.

  In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a chimeric antibody of the invention or antigen-binding portion thereof and one or more pharmaceutically acceptable excipients or diluents. .

  “Pharmaceutically acceptable excipient or diluent” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Including Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. It may contain pharmaceutically acceptable substances such as wetting agents, or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering agents.

  The term `` effective amount '' refers to an antibody or antigen-binding portion thereof (antibody or antibody) sufficient to treat or ameliorate a particular disease or disorder, or one or more symptoms thereof, and / or suppress or reduce the occurrence of the disease or disorder. Amount) including the pharmaceutical composition containing the antigen binding portion.

  The terms “diagnosically effective amount” or “diagnosically effective amount” and synonyms thereof refer to an antibody or antigen binding thereof sufficient to diagnose a particular disease or disorder and / or one or more of its expression. Refers to the amount of a moiety (including a pharmaceutical composition containing an antibody or antigen-binding portion thereof), and diagnosis identifies the presence of a disease or disorder and / or detects the extent or severity of the disease or disorder including. Often, the diagnosis is made in relation to the baseline or background detection level observed for an individual who does not have the disease or disorder. The detection level at the background or baseline level (increased detection level) indicates an indication of presence and in some cases the severity of the condition.

  When used in reference to a method of treatment and use of an antibody or antigen-binding portion thereof (including pharmaceutical compositions containing the antibody or antigen-binding portion thereof), an individual “in need thereof” is diagnosed with a disease or disorder to be treated. Or an individual who has been previously treated for a disease or disorder. With respect to diagnostic methods, an individual “in need thereof” may be an individual suspected of having a disease or disorder, at risk of a disease or disorder, or previously diagnosed with a disease or disorder ( For example, diagnosis can include monitoring the severity of a disease or disorder (eg, progressive / regressive) throughout the period and / or in conjunction with treatment).

  Preferably, the chimeric antibody or antigen binding portion thereof blocks or stimulates receptor function or neutralizes active soluble products such as one or more interleukins, TNF or C5a. More preferably, the active soluble product is human TNF-α.

  Compositions include various liquid, semi-solid or solid dosage forms such as liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes or suppositories. It may be a form. Preferably, the composition is in the form of an injectable solution for vaccination. Administration can be intravenous, subcutaneous, intraperitoneal, intramuscular, transdermal, intrathecal, and intraarterial. Preferably, the dosage form is daily, weekly, every 2 or 3 weeks, or monthly in the range of about 0.001 mg to 10 mg / kg body weight, more preferably about 0.05 to about 5 mg / kg body weight. Weekly dosing.

  The composition may be formulated as a sterile powder for the preparation of a sterile injectable solution.

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

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

  The composition may be formulated for rectal administration.

  The antibody binds to and identifies selected cells in vitro or in vivo, or binds to and destroys selected cells in vivo. Can be administered to invade and destroy. Alternatively, antibodies can be used as immunotoxins and cytotoxic agents such as toxic or chemotherapeutic agents can be delivered to specific cell types such as tumor cells. The preparation of immunotoxins is well known to those skilled in the art.

Cytotoxic preparations commonly used to generate immunotoxicity include radioisotopes such as ¹¹¹ In or ⁹⁰ Y, selenium, ribonuclease, binding domain deletion truncated antibacterial toxins such as Pseudomonas endotoxin, or diphtheria toxin, tubes Phosphorus inhibitors such as calicheamicin (Ozagamycin), maytansinoids (including DM-1), thristatins and taxoids, ribosome inactivating proteins such as ricin, evelin I, saporin and gelonin, and prodrugs such as mel Including faran.

  The invention also provides the use of a chimeric antibody or antigen-binding portion thereof in diagnostic applications to detect an antigen associated with a particular disease or disorder.

  More preferably, according to the third aspect, as described herein, the present invention provides a method for diagnosing a subject having an antigen associated with a particular disease or disorder, wherein There is provided the use of a chimeric antibody or antigen-binding portion thereof comprising administering a diagnostically effective amount of the portion or pharmaceutical composition. Preferably the subject is a human.

  For example, a labeled antibody or antigen-binding portion thereof can be used to determine the presence of an antigen in a biological sample, such as serum or plasma, or an elevated level of antigen (e.g., TNF-α), using conventional immunoassays such as It can be used for detection using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry.

  Preferably, the antigen that binds to the chimeric antibody or antigen-binding portion thereof is an oncofetal antigen, EpCAM, Lewis-Y, Lewis-Y / b, PMSA, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R , Her-2, TRAIL and VEGF receptors, including tumor-associated antigens; CD3, CD4, CD25, CD40, CD49d, MHC class I, MHC class II, GM-CSF, interferon-γ, IL-I, IL-12, IL -13, IL-23, TNF-α and antigens involved in immune or inflammatory diseases or immune or inflammatory disorders including IgE; glycoprotein IIb / IIIa, P-glycoprotein, purine receptor and CD11a, CD11b, CD11c, CDl8 Antigens expressed on host cells including adhesion receptors containing CD56, CD58, CD62 or CD144; eotaxin, IL-6, IL-8, TGF-β, C3a, C5a, VEGF, NGF and the like Cytokines, chemokines, growth factors or other soluble physiological regulators or Antigens including their receptors; antigens included in central nervous system diseases or disorders including β-amyloid and prions; non-human-derived antigens containing respiratory syncytial virus protein F, anthrax toxin, rattlesnake venom, and digoxin; For example, it binds to an antigen that is a natural peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid selected from an antigen consisting of a microbial, nanomicrobial or viral antigen or toxin; a chimeric antibody is an agonist or antagonist Acting as a complement, or active against reducing or killing unwanted cells (e.g., anti-CD4) by the action of complement or killer cells (e.g., NK cells), or cytotoxic agents Active or binds to or targets the phagocytic Fc receptor Neutralizes the biological activity of

  The anti-human TNF-α chimeric antibody or antigen-binding portion thereof according to the present invention can also be used for cell culture applications that require inhibition of TNF-α activity.

  The invention also relates to human TNF-α activity in a human subject comprising administering to a subject in need thereof a pharmaceutical composition according to the invention wherein the chimeric antibody or antigen-binding portion thereof binds to TNF-α. Methods of treating the disease or disorder characterized are provided.

  As used herein, the term “disease or disorder characterized by human TNF-α activity” refers to a disease or disorder that indicates the presence of TNF-α in a subject suffering from the disease or disorder, or a disease or disorder. It is meant to include a disease or disorder that is suspected of being involved in the disorder, is included in the pathophysiology of the disease or disorder, or is a factor that exacerbates the disease or disorder. Thus, a disease or disorder in which TNF-α activity is detrimental is a disease or disorder in which inhibition of TNF-α activity is expected to alleviate the symptoms and / or progression of the disease or disorder. Such a disease or disorder can be evidenced, for example, by increasing the concentration of TNF-α in a biological fluid of a subject suffering from the disease or disorder (e.g., in the subject's serum, plasma, joint fluid, etc. For example, the antibody of the present invention specific to TNF-α can be detected.

  A disease or disorder characterized by human TNF-α activity is the presence of TNF-α in a subject afflicted with the disease or disorder is involved in the pathophysiology of the disease or disorder or exacerbates the disease or disorder It is meant to include a disease or disorder indicated or suspected of being a factor. Preferably, the disease or disorder characterized by human TNF-α activity is sepsis, including septic shock, endotoxic shock, Gram-negative sepsis and toxic shock syndrome; rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis , Psoriasis and gouty arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome; fever and myalgias from infection and cachex associated with infection Infectious diseases including quality; Graft-versus-host rejection; Tumor growth or metastasis; Adult respiratory distress syndrome, Shock lung, Chronic inflammatory lung disease, Lung diseases including pulmonary sarcoidosis, Pulmonary fibrosis and silicosis; Crohn's disease And inflammatory bowel disease including ulcerative colitis; heart disease; inflammatory bone disease, hepatitis, coagulopathy, burns, reperfusion injury, keloid formation and scarring It is selected from the group consisting of tissue formation.

  Supplementary active compounds can also be incorporated into the compositions. The antibody or antibody-binding fragment is one or more additional therapeutic agents, such as one or more chemical agents that inhibit the production or activity of cytokines or cell surface molecules or alternatively human TNF-α. May be co-formulated with antibodies that bind to the target and / or may be administered separately and sequentially.

  In another aspect, the invention provides a pharmaceutical composition containing a therapeutically effective amount of an antibody of the invention or antigen-binding portion thereof, or a therapeutically effective amount of a chimeric antibody or antigen-binding portion thereof, and packaging and use. A kit including instructions is provided. In certain embodiments, the instructions for use include instructions on how to effectively administer a therapeutically effective amount of a chimeric antibody of the invention or antigen-binding portion thereof.

  Throughout this specification, the word “comprise” or variations thereof, such as “comprises” or “comprising” refers to an element, integer or step, or group of elements, plural Is meant to imply excluding other elements, integers or stages, or groups of elements, multiple integers or stages.

  All publications mentioned in this specification are herein incorporated by reference. Any discussion, action, material, device, product, etc. of the literature contained herein provides the context for the present invention exclusively. Any or all of these matters form part of the prior art that exists in Australia or elsewhere before the priority date of each claim of this application, or are common general knowledge in the field relevant to the present invention. It does not admit that there is.

  In order that the nature of the present invention may be more clearly understood, preferred forms thereof are illustrated by the following non-limiting examples.

Fusion Materials and Methods Gene Synthesis and Cloning of Marmoset Variable Region into Human Constant Region
The V _H chain of the MOG-specific marmoset derivatized antibody (accession number: AAM54057, SEQ ID NO: 1) is added to the human constant region (human IgGl heavy chain C _H 1, hinge, C _H 2 & C _H 3 domain (NCBI accession No. P01857 etc.) (SEQ ID NO: 2)). This is done by optimizing the expression of mammalian cells using Gene Optimizer technology, and the amino acid sequence in a newly synthesized (de novo) DNA sequence by a combination of synthetic oligonucleotides (GeneArt, Germany). Was achieved by reverse translation. In optimizing the DNA sequence, specific restriction enzyme sites Asc f and Tth 111 were included to allow further manipulation of the V _H region. After gene synthesis, all sequences including Kozak sequences were cloned into the multiple cloning site of the pEE6.4 GS accessory vector (Lonza Biologies). The _VL chain (accession number: AAM54058, SEQ ID NO: 3) of the MOG-specific marmoset derivatized antibody was expressed by the human kappa light chain constant region (NCBI accession number AAA58989 etc.) (SEQ ID NO: 4). DNA encoding the light chain ( _VL kappa) amino acid sequence was prepared in the same manner as the heavy chain described above. During DNA sequence optimization and synthesis, a specific restriction enzyme site Bsi WI / Rsr II was included to allow further manipulation of the _VL region. After gene synthesis, all sequences including Kozak sequences were cloned into the multiple cloning site of the pEE12.4 GS expression vector (Lonza Biologies). For stable expression, two single gene vectors (pEE6.4-V _H -IgG ₁ and pEE12.4-V _L -Kappa) were combined into a double gene vector. This was done by digesting the heavy chain expression cassette (hCMV-MIE promoter, Kozak sequence, marmoset V _H , human constant region and SV40 polyA site) from the pEE6.4 backbone using Not I and BamH I. The resulting fragment is used with a light chain expression cassette (hCMV-MIE promoter, Kozak, using Not I and BamH I sites to create a vector that expresses both the heavy and light chains of AB 138 (SEQ ID NOs: 5 and 6). The sequence, marmoset V _L , human kappa constant region and SV40 polyA site) was subcloned into the pEE12.4-V _L -kappa vector downstream.

Transfection For each transfection, 175 μl of Lipofectamine 2000 was added to 5 mL of Optimem I medium in wells of a 6-well plate (Invitrogen catalog numbers, 11668-027 and 31985-062). 70 μl of expression vector (70 μg) in the second well was added to 5 mL of Optimem I vehicle. After 5 minutes incubation at room temperature, the contents in the two wells were mixed with each other and incubated for an additional 20 minutes. After this second incubation, the entire transfection mixture was added to a T175 tissue culture flask containing CHOK1SV cells. Cells were incubated for 72 to 96 hours and supernatants were collected. The supernatant was centrifuged at 4000 × g for 5 minutes to give pelleted cell debris and sterile filtered through a 0.22 μm cartridge filter.

Antibody purification The supernatant was passed through a HiTrap protein A column (Amersham Biosciences, catalog number: 17-0402-01) three times at a flow rate of 1 mL / min. The column was then washed with 20 mM sodium phosphate for 40 minutes at 1 mL / min. The collected fractions were used to elute the antibody with 0.1 M citric acid pH 3.5 and immediately neutralized with 1 M Tris-HCl pH 9.0. The antibody sample was then desalted with a PD-10 column (Amersham Biosciences, catalog number: 17-0851-01). Analysis of the antibody by SDS-PAGE and size exclusion HPLC confirmed the correct molecular weight, the presence of the assembled antibody and the concentration of the antibody.

Western blot analysis
The ability of AB138 to retain binding to the M26 antigen, rat MOG (myelin-oligodendrocyte glycoprotein) was tested by Western blot. 130 mg rat spinal cord (IMVS, Australia) was homogenized in 1.8 ml CelLytic M cell lysis reagent (SIGMA, C2978) and incubated at 4 ° C. for 30 minutes. Furthermore, the lysate was homogenized by drawing several times with a 27 g 1/2 needle and then centrifuging at 13000 g for 30 minutes at 4 ° C. The pellet and supernatant were diluted in SDS-PAGE sample buffer (125 mM Tris-HCl pH 6.8, 5% SDS, 0.25% bromophenol blue, 25% glycerol). At the same time, 200 μl of CHOKISV cells were spun down at 13000 × g for 1 minute at 4 ° C. in 1 × 10 ⁶ live cells per ml and resuspended in 200 μl CelLytic M lysis reagent (SIGMA) . After centrifugation at 4 ° C. and 13000 × g for 30 minutes, the supernatant was mixed with an appropriate amount of SDS-PAGE sample buffer. All samples were run on a 4-20% Novex pre-cast gel (Invitrogen, Australia) for 2 hours at 120 V along with samples of sample molecular weight markers. The protein was then transferred to PVDF (BioRad, Australia) using Western blot equipment in 1x Tris-Glycine buffer (BioRad, Cat 161 + -0771) containing 20% methanol for 2 hours at 4 ° C and 250 mA. Transcribed. Membranes were blocked by incubating for 1 hour at room temperature with PBS containing 5% skim milk powder. Next, the membrane was washed 3 times with 1 × PBS and incubated overnight at 4 ° C. with PBS containing 10 μg / mL AB138. Membranes were incubated for 1 hour at room temperature with goat anti-human IgG (H + L) HRP conjugate (Sigma, Australia) diluted 1: 5000 in 1 × PBS after washing. After washing, bound antibody was detected using an ECL Western blot analysis system (Amersham Biosciences Cat: RPN2109). To identify non-specific binding events, parallel experiments were performed replacing AB138 with an isotype matched, non-specific negative control antibody (anti-TNFα monoclonal antibody).

Results After protein expression and purification, Western blot analysis was performed on AB138 to determine whether binding affinity for rat MOG was retained. AB138 bound to a protein of approximately 25 kDa size present in the clear lysate of rat spinal cord and bound to a protein not present in the clear lysate of CHOKISV (FIG. 1). The negative control antibody does not bind to any lysate present protein, and the interaction between AB138 and a protein with a size of 25 kDa is associated with an artificial or non-specific binder associated with the human constant region. It was shown that it was not relevant (Figure 2). This protein is consistent with the predicted size of rat MOG minus a single sequence (24.9 kDa). This result indicates that AB138 retaining affinity for rat MOG is present in rat spinal cord lysate, and that the marmoset human fusion antibody can retain the antigen-binding ability.

  It can be appreciated by those skilled in the art that rat MOG can be produced using the ability of AB138 to bind to rat MOG as measured in recombinant DNA technology and binding assays such as ELISA, Biacore analysis.

CDR2 substitution of domain antibodies Using standard recombinant DNA methods, the CDR2 of the acceptor anti-TNF-α domain antibody (Basran et al., WO 2004/081026; SEQ ID NO: 7; FIG. 3) is replaced with the donor New World primate immunology. By substituting with globulin derived CDR2, a partially engineered domain antibody can be produced.

  Applying the rules of Kabat (Sequences of Proteins of Immunological Intcrest, H. Kabat et al., U.S. Department of Health and Human Services, 1983), CDR2 is identified on the acceptor anti-TNF-α domain antibody (SASELQS). Domain antibody acceptor sequences are then aligned against a panel of New World primate immunoglobulin sequences. These sequences are derived from Ma Aotus Nancymaac (SEQ ID NO: 8-18) and common marmoset (SEQ ID NO: 19-24) (FIG. 4). The CDR2 sequences of New World primate immunoglobulins that differ from those of the acceptor CDR2 sequence can be identified as SASTLQT, DASSLQP, GASTRAT, KVSNRAS, RVSNRAS, KVSTRGP, AASNRAS, TSSNLQA, KASTLQS, AASTLQS, YASSLQS, YASFLQG (Table 1). Perform a BLAST analysis (http://www.neci.nlm.nih.gov/BLAST) on each of these donor New World primate CDR2 sequences to remove sequences that exactly match the immunoglobulin sequences. . Sequences unique to New World primates were KVSNRAS, RVSNRAS, KVSTRGP, AASNRAS, TSSNLQA, DASSLQP, YASFLQG (Table 1).

  Acceptor CDR2 and potential donor CDR2 can be obtained by MHC class II binding prediction program Propred (http://www.imtech.rcs.in/raghavu/propred) using 1% threshold analysis of all alleles. We investigated its predicted immunogenicity in humans. From this analysis, the acceptor CDR2 SASELQS is predicted to be bound by 11 alleles (DRB1_0306, DRB1_0307, DRB1_0308, DRB1_0311, DRB1_0401, DRB1_0426, DRB1_0806, DRB1_0813, DRB1_1501, DRB1_1502, DRB1_1HC). Forms part of the peptide LIYSASELQ. Donor CRD2 sequence KVSNRAS is part of the sequence LIYKVSNRAS, which is predicted to bind MHC class II encoded by 9 alleles (DRB1_0309, DRB1_0402, DRB1_0802, DRB1_0804, DRB1_0806, DRB1_0813, DRB1_1301, DRB1_1327, DRB1_1328) . The donor CDR2 sequence AASNRAS forms part of the sequence LIYAASNRA predicted to bind MHC class II encoded by 6 alleles (DRB1_0402, DRB1_0404, DRB1_0408, DRB1_0423, DRB1_0813, DRB1_1506). Donor CDR2 sequence TSSNLQA is predicted to bind MHC class II LI that is partly associated with MHC class II encoded by 10 alleles (DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0410, DRB1_0423, DRB1_0426, DRB1_0813, DRB1_1501, DRB1_1502, DRB1_1506). Form. The donor CDR2 sequence KVSTRGP forms part of the sequence LLIYKVSTR predicted to bind MHC class II encoded by 8 alleles (DRB1_0309, DRB1_0802, DRB1_0804, DRB1_0806, DRB1_0813, DRB1_1301, DRB1_1327, DRB1_1328). Therefore, acceptor CDR2 can be replaced with low expected immunogenic donor CDR2, including KVSNRAS, AASNRAS, TSSNLQA, and KVSTRGP.

  Using recombinant DNA methods, the acceptor CDR2 is replaced with a donor CDR2 sequence to generate a partially engineered domain antibody (SEQ ID NOs: 25-31). Examples of recombinant DNA methods include those described by Winter et al. (US Pat. No. 5,225,539), including but not limited to methods such as site-directed mutagenesis and oligoaniling. Protein expression of domain antibodies can be performed in E. coli BL21 (DE3) pLys (Novagen, Germany) using an appropriate vector for expression such as pET21d (+) (Novagen, Germany), or Basran et al. ( It can be carried out by other methods known to those skilled in the art, such as those described in WO 2004/081026). Following bacterial cell lysis, domain antibodies are purified using protein L (Pierce, USA).

  Following purification, engineered domain antibodies are analyzed for maintenance of TNFα binding ability by methods known to those skilled in the art, such as the L929 neutralization assay, or the TNFα receptor I binding assay.

  To improve the binding affinity of engineered domain antibodies, affinity maturation can be performed by amino acid substitutions of framework residues that surround and stabilize CDR2, or by other methods known to those skilled in the art (Winter , US 5,225,539; Griffiths et al., US 5,885,793; Rajpal, A et al., 2005 A general method for greatly improving the affinity of antibodies by using comninatorial libraries, Proc Natl Acad Sci USA 102 (24) 8466-71; Irving RA et al., ( 2001) Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics, Journal of Immunological Methods, 248: 31-45).

  It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the invention shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Let's go. The embodiments of the present invention are therefore to be considered in all respects only as illustrative and not restrictive.

FIG. 4 shows the binding of AB138 to rat MOG present in rat spinal cord lysate (lane 2) and non-binding to rat MOG present in CHOKlSV lysate (lane 3). Lane 1 contains molecular weight markers. FIG. 5 shows the lack of non-specific binding of anti-TNFα monoclonal antibody to rat MOG present in rat spinal cord lysate (lane 2) and CHOKlSV lysate (lane 3) of the same sample. Lane 1 contains molecular weight markers. It is a figure which shows an acceptor domain antibody amino acid and a nucleotide sequence (both chains). The restriction cleavage sites of KpnI and SanDI that excise the region containing CDR2 are shown in the figure. The CDR2 residue is underlined. FIG. 5 shows alignment of domain antibody acceptor sequences using a panel of New World primate immunoglobulin sequences performed using AlignX (Vector NTJ, Invitrogen, Australia). CDR2 is highlighted in bold.

Claims (30)

  A chimeric antibody or antigen-binding portion thereof, wherein the antigen-binding portion contains 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.   2. The chimeric antibody or antigen-binding portion thereof according to claim 1, wherein the antigen-binding portion contains 3 CDRs and 4 framework regions.   The chimeric antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the antigen-binding portion contains at least one CDR which is a human CDR.   4. The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antigen-binding portion contains two CDRs that are human CDRs.   The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein CDR2 is New World primate CDR2.   6. The chimeric antibody or antigen-binding portion thereof according to claim 5, wherein the CDR2 sequence is selected from the group consisting of KVSNRAS, RVSNRAS, KVSTRGP, AASNRAS, TSSNLQA, DASSLQP, and YASFLQG.   The CDR2 sequence is selected from the group consisting of KVSNRAS, AASNRAS, TSSNLQA, and KVSTRGP. The chimeric antibody or antigen-binding portion thereof according to claim 6.   The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 7, wherein the framework region is a human sequence.   9. The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 8, wherein at least one framework region is modified to increase binding.   10. The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 9, wherein at least one framework region is modified to reduce the predicted immunogenicity in humans.   11. The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 10, wherein at least one CDR sequence is modified to increase binding without modifying at least one New World primate CDR sequence.   12. The chimera according to any one of claims 1 to 11, wherein at least one CDR sequence is modified to reduce predicted immunogenicity in humans without modifying at least one New World primate CDR sequence. An antibody or antigen-binding portion thereof.   13. The chimeric antibody or antigen-binding portion thereof of claim 11 or 12, wherein at least one CDR sequence to be modified is not a New World primate CDR.   The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 13, wherein the antigen-binding portion is a domain antibody.   The chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 14, further comprising a human or non-human primate constant region sequence.   16. The New World primate according to any one of claims 1 to 15, wherein the New World primate is selected from the group consisting of marmosets, tamarins, squirrel monkeys, sea cucumbers, genus Titi monkeys, spider monkeys, woolly monkeys, capuchin monkeys, monkeys, and howler monkeys. Or the antigen-binding portion thereof.   17. The chimeric antibody or antigen-binding portion thereof of claim 16, wherein the New World primate is a marmoset.   Carcinoembryonic antigen, EpCAM, Lewis-Y, Lewis-Y / b, PMSA, CD20, CD30, CD33, CD38, CD52, CD154, tumor-associated antigens including EGF-R, Her-2, TRAIL and VEGF receptors; CD3 Immunity, including CD4, CD25, CD40, CD49d, MHC class I, MHC class II, GM-CSF, interferon-γ, IL-I, IL-12, IL-13, IL-23, TNF-α, and IgE Or an antigen involved in an inflammatory disease or immune or inflammatory disorder; a host cell comprising glycoprotein IIb / IIIa, P-glycoprotein, purine receptor and adhesion receptor comprising CD11a, CD1lb, CD11c, CDl8, CD56, CD58, CD62 or CD144 Antigens expressed on; cytokines, chemokines, growth factors or other soluble physiological, including eotaxin, IL-6, IL-8, TGF-β, C3a, C5a, VEGF, NGF and their receptors Antigens containing modulators or their receptors; central nerves containing β-amyloid and prions Antigens involved in systemic diseases or disorders; selected from non-human-derived antigens, including respiratory syncytial virus protein F, anthrax toxin, rattlesnake venom, and digoxin, for example, antigens consisting of microbial, nanomicrobial or viral antigens or toxins 18. The chimeric antibody or antigen-binding portion according to any one of claims 1 to 17, which binds to an antigen that is a natural peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid.   19. The chimeric antibody or antigen-binding portion thereof according to claim 18, which binds to TNFα.   Remove one CDR from a human antibody variable region containing at least two CDRs and at least three framework regions and replace it with a New World primate CDR predicted to have low immunogenicity, a chimeric variable region Producing a chimeric antibody or antigen-binding portion thereof.   21. The method of claim 20, further comprising the step of recovering the chimeric variable region.   The method according to claim 20 or 21, wherein the New World primate CDR is CDR2.   23. The method of any one of claims 20-22, further comprising modifying the chimeric variable region sequence to increase binding without modifying the New World primate CDR sequence.   24. The method of any one of claims 20 to 23, further comprising modifying the chimeric variable region sequence to reduce immunogenicity in humans without altering the New World primate CDR sequence.   25. Any one of claims 20 to 24, wherein the New World primate is selected from the group consisting of marmosets, tamarins, squirrel monkeys, titis monkeys, spider monkeys, woolly monkeys, capuchin monkeys, sea cucumbers, genus Sakis, monkeys, and howler monkeys. The method according to item.   26. The method of claim 25, wherein the New World primate is a marmoset.   Tumors wherein the antibody comprises carcinoembryonic antigen, EpCAM, Lewis-Y, Lewis-Y / b, PMSA, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R, Her-2, TRAIL and VEGF receptors Related antigens; CD3, CD4, CD25, CD40, CD49d, MHC class I, MHC class II, GM-CSF, interferon-γ, IL-I, IL-12, IL-13, IL-23, TNF-α, and Antigens involved in immune or inflammatory diseases or immune or inflammatory disorders including IgE; glycoprotein IIb / IIIa, P-glycoprotein, purine receptors and adhesion receptors including CD11a, CD1lb, CD11c, CDl8, CD56, CD58, CD62 or CD144 Antigens expressed on host cells, including: eotaxin, IL-6, IL-8, TGF-β, C3a, C5a, VEGF, NGF and their receptors, cytokines, chemokines, growth factors or others Soluble physiological regulators or antigens containing their receptors; β-amyloid and prions Antigens involved in central nervous system diseases or disorders, including non-human antigens, including respiratory syncytial virus protein F, anthrax toxin, rattlesnake venom, and digoxin, such as microbial, nanomicrobial or viral antigens or toxins 27. A method according to any one of claims 20 to 26, wherein the method binds to an antigen selected from antigens, which is a natural peptide, protein, carbohydrate, glycoprotein, lipid or glycolipid.   28. The method of claim 27, wherein the antibody binds TNFα.   A chimeric antibody or an antigen-binding portion thereof produced by the method according to any one of claims 20 to 28.   A kit comprising the chimeric antibody or antigen-binding portion thereof according to any one of claims 1 to 19, packaging and instructions for use.

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