HUMAN ANTIBODIES THAT BIND HUMAN TNFalpha

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DESCRIPTION

Human antibodies that bind to human TNFa.

Background of the invention

Tumor necrosis factor a (TNFa) is a cytokine produced by various cell types, including monocytes and macrophages, which was originally identified based on its ability to induce necrosis of certain mouse tumors (see, for example, Old, L . (1985) Science 230: 630-632). Subsequently, a factor called cachectin, associated with cachexia, was shown to be the same molecule as TNFa. TNFa has been implicated in mediating shock (see, for example, Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57: 505-518; Beutler, B. and Cerami, A. ( 1989) Annu. Rev. Immunol. 7: 625-655). In addition, TNFa has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection, and host versus graft disease (see, for example, Moeller, A., et al. to the. (1990) Cytokine 2: 162-169; US Patent No. 5,231,024 to Moeller et al .; European Patent Publication No. 260 61θ B1 by Moeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10: 411-452; Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45: 491-503).

Due to the deleterious role of human TNFa (hTNFa) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract the activity of hTNFa. In particular, antibodies that bind and neutralize hTNFa have been sought as a means of inhibiting hTNFa activity. Some of the earliest of these antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFa (see, for example, Hahn T; et al., (1985) Proc Natl Acad Sci USA 82: 3814-3818; Liang, CM., Et al. (1996) Biochem. Biophys. Res. Commun. 137: 847-854; Hirai, M., et al. (1987) J. Immunol. Methods 96: 57 -62; Fendly, BM, et al. (1987) Hybridoma 6: 359-370; Moeller, A., et al. (1990) Cytokine 2: 162-169; United States Patent No. 5,231,024 to Moeler et al .; European Patent Publication No. 186 833 B1 by Wallach, D .; European Patent Application Publication No. 218 868 A1 by Old et al .; European Patent Publication No. 260 610 B1 by Moeller, A., et al.). Although these mouse anti-hTNFa antibodies often exhibited high affinity for hTNFa (e.g., Kd <10^-9 M) and were able to neutralize hTNFa activity, their use in vivo may be limited by problems associated with the administration of mouse antibodies to humans, such as a short serum half-life, the inability to induce certain human effector functions, and the induction of an unwanted immune response against the mouse antibody in a human (the "human anti-mouse antibody" (HAMA)).

In an attempt to address the problems associated with the use of fully murine antibodies in humans, anti-hTNFa antibodies have been engineered to be more "human-like". For example, chimeric antibodies have been prepared in which the variable regions of the antibody chains are derived from the mouse and the constant regions of the antibody chains are derived from human (Knight, DM et al. (1993) Mol. Immunol. 30: 1443-1453; PCT Publication No. WO 92/16553 by Daddona, PE, et al.). Furthermore, humanized antibodies have also been prepared, in which the hypervariable domains of the antibody variable regions are derived from the mouse but the rest of the antibody variable regions and constant regions are derived from human (PCT Publication No. WO 92/11383 by Adair, JR, et al.). However, as these chimeric and humanized antibodies retain some murine sequences, they can induce an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods, for example for chronic indications such as the rheumatoid arthritis (see, for example, Elliott, MJ et al. (1994) Lancet 344: 1125-1127; Elliot, MJ et al. (1994) Lancet 344: 1105-1110).

A preferred hTNFa inhibitory agent against murine mAbs or derivatives thereof (eg, chimeric or humanized antibodies) would be a fully human anti-hTNFa antibody, such that an agent does not induce the HAMA reaction, even if used for prolonged periods. . Human monoclonal autoantibodies against hTNFa have been prepared using human hybridoma techniques (Boyle, P., et al. (1993) Cell. Immunol. 152: 556568; Boyle P., et al. (1993) Cell. Immunol. 152: 569-581; European Patent Application Publication No. 614 984 A2 by Boyle, et al.). However, these hybridoma-derived monoclonal autoantibodies were reported to have an affinity for hTNFa that was too low to be calculated by conventional methods, could not bind soluble hTNFa, and could not neutralize hTNFa-induced cytotoxicity (see Boyle, et al .; supra). Furthermore, the success of the human hybridoma technique depends on the natural presence in human peripheral blood of lymphocytes producing autoantibodies specific for hTNFa. Studies have detected serum autoantibodies against hTNFa in humans (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30: 319-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biolj. 10B: 447-452), while others have not (Leusch, HG., Et al. (1991) J. Immunol. Methods 139: 145-147).

An alternative to natural human anti-hTNFa antibodies would be a recombinant hTNFa antibody. Recombinant human antibodies have been described that bind to hTNFa with relatively low affinity (i.e., K_d ~10^-7 M) and a fast dissociation rate (i.e., K_off ~10^-2sec^-1) (Griffiths, AD et al (1993) EMBO

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J. 12: 725-734). However, due to their relatively rapid dissociation kinetics, these antibodies may not be suitable for therapeutic use. Furthermore, a recombinant human anti-hTNFa has been described that does not neutralize the activity of hTNFa, but instead increases the binding of hTNFa to the surface of cells and enhances the internalization of hTNFa (Lidbury, A., et al. (1994) Biotechnol. Ther. 5: 27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.).

Consequently, there is still a need for human antibodies, such as recombinant human antibodies, which bind soluble hTNFa with high affinity and slow dissociation kinetics and which have the ability to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and hTNFa-induced cell activation.

Summary of the invention

This invention provides human antibodies, preferably recombinant human antibodies that specifically bind to human TNFa. The antibodies of the invention are characterized by binding to hTNFa with high affinity and slow dissociation kinetics and by neutralization of hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and cell activation induced by hTNFa. The antibodies of the invention are further characterized by binding to hTNFa, but not hTNL / 1 (lymphotoxin) and by having the ability to bind other primate TNFa and non-primate TNFa in addition to human TNFa.

Antibodies of the invention may be full length (eg, an IgG1 or IgG4 antibody) or may comprise only one antigen-binding protein (eg, a Fab, F (ab ') 2 or scFv fragment). The most preferred recombinant antibody of the invention, designated D2E7, has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO. N °: 4. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO. °: 2.

In one embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, that dissociates from human TNFa with a Kd of 1 x 10^-8 M or less and a rate constant Koff of 1 x 10^-3 s^-1 or lower, both determined by surface plasmon resonance and neutralizes the cytotoxicity of human TNFa in a conventional in vitro L929 assay with an IC50 of 1 x 10^-7 M or less. More preferably, the isolated human antibody, or the antigen-binding portion thereof, dissociates from human TNFa with a Koff of 5 x 10^-4 s^-1 or less, or even more preferably with a Koff of 1 x 10^-4 s^-1 or less. More preferably, the isolated human antibody, or the antigen-binding portion thereof, neutralizes the cytotoxicity of human TNFa in a standard in vitro L929 assay with an IC50 of 1 x 10^-8 M or less, or even more preferably with an IC50 of 1 x 10^-9 M or less and even more preferably with an IC50 of 5 x 10^-10 M or less.

In another embodiment, the invention provides a human antibody, or an antigen-binding portion thereof, with the following characteristics:

a) dissociates from human TNFa with a Koff of 1 x 10^-3 s^-1 or less, determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1,4,5 , 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9.

c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4 , 5, 6, 8, 9, 10, or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11, and / or 12.

More preferably, the antibody, or the antigen-binding portion thereof, dissociates from human TNFa with a Koff of 5 x 10^-4 s^-1 or less. Even more preferably, the antibody, or the antigen-binding portion thereof, dissociates from human TNFa with a Koff of 1 x 10^-4 s^-1 or less.

In another embodiment, the invention provides a human antibody, or an antigen-binding portion thereof, with an LCVR having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with an HCVR having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position, 2, 3, 4, 5, 6, 8, 9, 10 or 11. More preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO. : 5 and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6. Even more preferably, the LCVR further has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8.

In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, with an LCVR comprising the amino acid sequence of SEQ ID NO: 1 and an HCVR comprising

ES 2 198 552 T3 is the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the antibody has an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In other embodiments, the antibody is a Fab fragment, an F (ab ') 2 fragment, or a single chain Fv fragment.

In other embodiments, the invention provides antibodies, or antigen-binding portions thereof, with an LCVR having a CDR3 domain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO. NO: 11, SEQ ID NO: 12, SEQ ID NO: 13; SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with an HCVR having a CDR3 domain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: : 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34 and SEQ ID No: 35.

In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that neutralizes the activity of human TNFa but not human TNIye (lymphotoxin). In a preferred embodiment, the human antibody, or the antigen-binding portion thereof, neutralizes the activity of human TNFa, chimpanzee TNFa, and at least one additional primate TNFa selected from the group consisting of baboon TNFa, marmoset, cynomolgus monkey TNFa and rhesus TNFa. Preferably, the antibody also neutralizes the activity of at least one non-primate TNFa. For example, in one sub-embodiment, the isolated human antibody, or the antigen-binding portion thereof, also neutralizes the activity of canine TNFa. In another sub-embodiment, the isolated human antibody, or the antigen-binding portion thereof, also neutralizes the activity of pig TNFα. In another sub-embodiment, the isolated human antibody, or the antigen-binding portion thereof, also neutralizes the activity of mouse TNFα.

Another aspect of the invention relates to nucleic acid molecules that modify the antibodies, or antigen-binding moieties, of the invention. A preferred nucleic acid of the invention, encoding a D2E7 LCVR, has the nucleotide sequence shown in Figure 7 and SEQ ID NO: 36. Another preferred nucleic acid of the invention, encoding a D2E7 HCVR, has the sequence of nucleotides shown in Figure 8 and SEQ ID No. 37. The invention also includes recombinant expression vectors carrying the nucleic acids encoding antibodies of the invention, and host cells into which such vectors have been introduced, as well as methods for obtaining the antibodies of the invention by culturing the host cells. of the invention.

Another aspect of the invention relates to methods for inhibiting human TNFa activity using an antibody, or an antigen-binding portion thereof, of the invention. In one embodiment, the method comprises contacting human TNFa with the antibody of the invention, or the antigen-binding portion thereof, such that the activity of human TNFa is inhibited. In another embodiment, the method comprises administering an antibody of the invention, or an antigen-binding portion thereof, to a human suffering from a disorder in which the activity of TNFa is deleterious, such that the activity is inhibited. of human TNFa in the human subject. The disorder can be, for example, sepsis, an autoimmune disease (for example, rheumatoid arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic syndrome), an infectious disease, a malignancy, transplant rejection or disease host versus graft, lung disorder, bone disorder, intestinal disorder, or heart disorder.

Brief description of the drawings

Figures 1A and 1B show the amino acid sequences of the D2E7 light chain variable region (D2E7 VL; also shown in SEQ ID NO: 1), D2E7 VL alanine scanning mutants (LD2E7 * .A1, LD2E7 * .A3, LD2E7 * .A4, LD2E7 * .A5, LD2E7 * .A7 and LD2E7 * .A8), the light chain variable region of the D2E7-related antibody 2SD4 (2SD4 VL; also shown in SEQ ID NO: 9) and other D2E7-related light chain variable regions (EP B12, VL10E4, VL100A9, VL100D2, VL10F4, LOE5, VLLOF9, VLL0F10, VLLOG7, VLLOG9, VLLOH1, VLLOH7, VL1B17, VL1B1 , VL1C7, VL0.1F4, VL0.1H8, LOE7, LOE7.A and LOE7. T). Figure 1A shows the FR1, CDR1, FR2 and CDR2 domains. Figure 1B shows the FR3, CDR3 and FR4 domains. The CDR1 ("CDR L1"), CDR2 ("CDR L2") and CDR3 ("CDR L3") domains of the light chain are boxed.

Figures 2A and 2B show the amino acid sequences of the D2E7 VH heavy chain variable region; also shown in SEQ ID NO: 2), D2E7 VH Alanine Scan Mutants (HD2E7 * .A1, HD2E7 * .A2, HD2E7 * .A3, HD2E7 * .A4, HD2E7 * .A5, HD2E7 * .A6 , HD2E7 * .A7, HD2E7 * .A8 and HD2E7 * .A9), the heavy chain variable region of the D2E7-related antibody 2SD4 (2SD4 VH; also shown in SEQ ID NO: 10) and other D2E7-related heavy chain variable regions (VH1B11, VH1D8, VH1A11, VH1B12, VH1-D2, VH1E4, VH1F6, VH1G1, 3C-H2, VH1-D2.N and VH1-D2.Y). Figure 2A shows the FR1, CDR1, FR2 and CDR2 domains. Figure 2B shows the FR3, CDR3 and FR4 domains. The CDR1 ("CDR H1"), CDR2 ("CDR H2") and CDR3 ("CDR H3") domains of the heavy chain are boxed.

Figure 3 is a graph depicting the inhibition of TNFa-induced L929 cytotoxicity by anti-human hTNFa antibody D2E7, compared to murine anti-hTNFa antibody MAK 195.

Figure 4 is a graph depicting the inhibition of rhTNFa binding to hTNFa receptors in U937 cells or anti-hTNFa antibody D2E7, compared to anti-hTNFa antibody MAK 195.

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Figure 5 is a graph depicting the inhibition of TNFa-induced ELAM-1 expression in HUVEC by anti-hTNFa antibody D2E7, compared to anti-hTNFa antibody MAK 191.

Figure 6 is a bar graph depicting protection from TNFa-induced lethality in mice sensitized to D-galactosamine by administration of anti-hTNFa antibody D2E7 (black bars), compared to anti-hTNFa antibody MAK 195. murine hTNFa (hatched bars).

Figure 7 shows the nucleotide sequence of the D2E7 light chain variable region, with the predicted amino acid sequence below the nucleotide sequence. The CDR L1, CDR L2 and CDR L3 regions are underlined.

Figure 8 shows the nucleotide sequence of the D2E7 heavy chain variable region, with the predicted amino acid sequence below the nucleotide sequence. The CDR H1, CDR H2 and CDR H3 regions are underlined.

Figure 9 is a graph depicting the effect of D2E7 antibody treatment on mean joint size of Tg197 transgenic mice as a polyarthritis model.

Detailed description of the invention

This invention relates to isolated human antibodies, or antigen-binding portions thereof, that bind to human TNFa with high affinity, low dissociation rate, and high neutralization capacity. Various aspects of the invention relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for obtaining such antibodies and fragments. The invention also includes methods of using the antibodies of the invention to detect human TNFa or to inhibit the activity of human TNFa in vitro or in vivo.

In order that the present invention may be more easily understood, certain terms are first defined.

The term "human TNFa" (abbreviated herein as hTNFa or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a secreted form of 17 kD and a membrane-associated form of 26 kD, whose biologically active form is composed of a trimer of 17 kD molecules linked non-covalently. The structure of hTNFa is further described, for example, in Pennica, D., et al. (1894) Nature 312: 724-729; Davis, J. M., et al. 81987) Biochemistry 26: 1322-1326; and Jones, EY, et al. (1989) Nature 338: 225-228. The term "human TNFa" is intended to include recombinant human TNFa (rhTNFa) which can be prepared by conventional recombinant expression methods or commercially available (RyD Systems, Catalog No. 210-TA, Minneapolis, MN).

The term "antibody", as used herein, is intended to refer to immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds. Each heavy chain is made up of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a CL domain. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), separated with regions that are more conserved, termed framework regions (FR). Each VH and VL is made up of three CDRs and four FRs, arranged from the amino end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen ( for example, hTNFa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond to the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), consisting of a domain VH; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be obtained as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Aca. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. Also included are other forms of single chain antibodies, such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed in a single polypeptide chain, but using a linker that is too short to allow the formation of pairs between the two domains in

ES 2 198 552 T3 the same chain, thereby forcing the domains to pair with complementary domains from another chain and creating two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc Natl Acad Sci.

USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123).

Furthermore, an antibody or an antigen-binding portion thereof can be part of larger immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or the antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to obtain a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and the use of a cysteine ​​residue, a marker peptide and a C-terminal polyhistidine tail to obtain bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al., (1994) Mol. Immunol. 31: 1047-1058). Antibody portions, such as Fab and F (ab ') fragments, can be prepared₂ from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. In addition, antibodies, antibody moieties, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

The term "human antibody", as used herein, is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo), for example, in the CDRs and in particular the CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies into which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences. .

The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell. (further described in Section II below), antibodies isolated from a recombinant combinatorial human antibody library (described further in Section III, shown below), antibodies isolated from an animal (e.g., mouse) that is transgenic for human immunoglobulin genes (see, for For example, Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, created or isolated by any other means involving the splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL of recombinant antibodies are sequences that, although they originate from and are related to the VH and VL sequences of the human general line, they may not exist naturally within the repertoire of the general human antibody line in vivo.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies that have different antigenic specificities (eg, an isolated antibody that specifically binds to hTNFa is substantially free of antibodies that specifically bind to antigens other than hTNFa). However, an isolated antibody that specifically binds hTNFa can be cross-reactive with other antigens, such as TNFa molecules from other species (as discussed in more detail below). Furthermore, an isolated antibody can be substantially free of other cellular materials and / or chemical agents.

A "neutralizing antibody", as used herein (or an "antibody that neutralizes the activity of hTNFa"), is intended to refer to an antibody whose binding to hTNFa causes inhibition of the biological activity of hTNFa. This inhibition of hTNFa biological activity can be assessed by measuring one or more indicators of hTNFa biological activity, such as hTNFa-induced cytotoxicity (in vitro or in vivo), hTNFa-induced cell activation, and hTNFa binding to receptors. by hTNFa. These indicators of hTNFa biological activity can be evaluated by one or more of several standard in vitro or in vivo assays known in the art (see Example 4). Preferably, the ability of an antibody to neutralize hTNFa activity is assessed by inhibiting hTNFa-induced cytotoxicity of L929 cells. As an additional or alternative parameter of hTNFa activity, the ability of an antibody to inhibit hTNFa-induced ELAM1 expression in HUVEC can be evaluated as a measure of hTNFa-induced cellular activation.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that enables the analysis of biospecific interactions in real time by detecting alterations in protein concentrations within a biosensor array, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweeden and Piscataway, NJ). For additional descriptions, see Example 1 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 286-277.

The term "Koff", as used herein, is intended to refer to the rate constant for dissociation of an antibody from the antibody / antigen complex.

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The term "Kd", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.

The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA.

The term "isolated nucleic acid molecule", as used herein in reference to nucleic acids encoding antibodies or antibody portions (eg, VH, VL, CDR3) that bind to hTNFa, is intended to refer to a nucleic acid molecule in which the nucleotide sequences that encode the antibody or the antibody portion lack other nucleotide sequences that encode antibodies or antibody portions that bind to antigens other than hTNFa, being able to flank said other naturally occurring nucleic acid sequences in human genomic DNA. Thus, for example, an isolated nucleic acid of the invention encoding a VH region of an anti-TNFa antibody does not contain any other sequence encoding another VH region that binds antigens other than TNFa.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be attached. Another type of vector is a viral vector, where additional DNA segments can be attached to the viral genome. Certain vectors are capable of autonomously replicating in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, and thus replicate along with the genome of the host. Furthermore, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably, since plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adenovirus-associated viruses), which have equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular target cell, but to the progeny of such cell. As certain modifications may occur in later generations due to mutations or environmental influences, in fact, such progeny may not be identical to the parent cell, but is included within the scope of the term "host cell" as used herein.

Various aspects of the invention are described in more detail in the following subsections.

I. Human antibodies that bind to human TNFa

This invention provides isolated human antibodies, or antigen-binding portions thereof, that bind to human TNFa with high affinity, a low rate of dissociation, and a high neutralization capacity. Preferably, the human antibodies of the invention are recombinant neutralizing human anti-hTNFa antibodies. The most preferred recombinant neutralizing antibody of the invention is referred to herein as D2E7 and has the VL and VH sequences shown in Figure 1A, 1B and Figure 2A, 2B, respectively (the amino acid sequence of the VL region of D2E7 also is shown in SEQ ID NO: 1; the amino acid sequence of the VH region of D2E7 is also shown in SEQ ID NO: 2). The binding properties of D2E7, compared to the murine anti-hTNFa mAb MAK 195 exhibiting high affinity and low dissociation kinetics and another anti-human hTNFa antibody related in sequence to D2E7, 2SD4, are summarized below:

<td>Antibody</td><td>Koff sec^-1</td><td>Kon M^-1 sec^-1</td><td>KD M</td><td>Stoichiometry</td>

<td>D2E7 IgG1</td><td>8.81 x 10^-5</td><td>1.91 x 10⁵</td><td>6.09 x 10^-10</td><td> 1,2</td>

<td>2SD4 IgG4</td><td>8.4 x 10^-3</td><td>4.20 x 10⁵</td><td>2.00 x 10^-8</td><td> 0,8</td>

<td>MAK 195 F (ab ') 2</td><td>8.70 x 10^-5</td><td>1.90 x 10⁵</td><td>4.60 x 10^-10</td><td> 1,4</td>

The D2E7 antibody, and related antibodies, also exhibit a strong ability to neutralize hTNFa activity, as assessed by various in vitro and in vivo assays (see Example 4). For example, these antibodies neutralize the hTNFa-induced cytotoxicity of L929 cells with IC50 values ​​in the range of

ES 2 198 552 T3 approximately 10^-7 M to about 10^-10 M. D2E7, when expressed as a full-length IgG1 antibody, neutralizes the hTNFa-induced cytotoxicity of L929 cells with an IC₅₀ about 1.25 x 10^-10 M. In addition, the neutralizing ability of D2E7 is maintained when the antibody is expressed as a Fab, F (ab ') 2 or scFv fragment. D2E7 also inhibits TNFa-induced cellular activation, as measured by hTNFa-induced ELAM-1 expression in HUVEC (IC5o = approximately 1.85 x 10^-10 M) and the binding of hTNFa to hTNFa receptors on U-937 cells (IC50 = approximately 1.56 x 10^-10 M). Regardless of the latter, D2E7 inhibits the binding of hTNFa to both p55-like and p75-like hTNFa receptors. Furthermore, the antibody inhibits hTNFa-induced lethality in vivo in mice (DE₅₀ = 1-2.5 µg / mouse).

With regard to the binding specificity of D2E7, this antibody binds to human TNFa in various ways, including soluble hTNFa, transmembrane hTNFa, and cellular receptor-bound hTNFa. D2E7 does not specifically bind to other cytokines such as lymphotoxin (TNFe), IL-1a, IL-ίβ, IL-2, IL-4, IL-6, IL-8, IFNy, and TGFje. However, D2E7 is cross-reactive with tumor necrosis factors from other species. For example, the antibody neutralizes the activity of at least five primate TNFa (chimpanzee, baboon, marmoset, cynomolgus monkey, and rhesus monkey), with IC5o values ​​approximately equivalent to neutralizing hTNFa (see Example 4 subsection E). D2E7 also neutralizes the activity of mouse TNFa, although approximately 1000 times worse than human TNFa (see Example 4, subsection E). D2E7 also binds to canine and porcine TNFa.

In one aspect, the invention relates to D2E7 antibodies and portions of antibodies, to D2E7-related antibodies and portions of antibodies, and to other human antibodies and antibody portions, with properties equivalent to D2E7, such as high binding affinity to hTNFa. with low dissociation kinetics and high neutralization capacity. In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFa with a Kd of 1 x 10.^-8 Mo less and a rate constant Koff of 1 x 10^-3 s^-1 lower, determined by surface plasmon resonance, and neutralizes the cytotoxicity of human TNFa in a standard in vitro L929 assay with an IC5o of 1 x 10^-7 M or less. More preferably, the isolated human antibody, or the antigen-binding portion thereof, dissociates from human TNFa with a Koff of 5 x 10^-4 s^-1 or less, or even more preferably with a K_off of 1 x 10^-4 s^-1 or less. More preferably, the isolated human antibody, or the antigen-binding portion thereof, neutralizes the cytotoxicity of human TNFa in a standard in vitro L929 assay with an IC5o of 1 x 10^-8 M or less, even more preferably with an IC5o of 1 x 10^-9 M or less, and even more preferably with an IC5o of 5 x 10^-1o M or less. In a preferred embodiment, the antibody is an isolated human recombinant antibody, or an antigen-binding portion thereof. In another preferred embodiment, the antibody also neutralizes TNFa-induced cellular activation, assessed using a standard in vitro assay for TNFa-induced ELAM-1 expression in human umbilical vein endothelial cells (HUVEC).

Surface plasmon resonance analysis to determine Kd and Koff can be performed as described in Example 1. In Example 4, subsection A, a standard in vitro L929 assay to determine IC5o values ​​is described. In Example 4, subsection C, a standard in vitro assay for TNFα-induced ELAM1 expression in human umbilical vein endothelial cells (HUVEC) is described. Examples of recombinant human antibodies that meet, or are expected to meet, the aforementioned kinetic and neutralization criteria include antibodies having the following [VH / VL] pairs, the sequences of which are shown in Figures 1A, 1B, 2A and 2B (see also Examples 2, 3 and 4 for kinetic and neutralization analyzes): [D2E7 VH / D2E7 VL]; [HD2E7 * .A1 / D2E7 VL], [HD2E7 * .A2 / D2E7 VL], [HD2E7 * .AE / D2E7 VL], [D2E7 * .A4 / D2E7 VL], [D2E7 * .A5 / D2E7 VL], [HD2E7 * .A6 / D2E7 VL], [HD2E7 * .A7 / D2E7 VL], [HD2E7 * .A8 / D2E7 VL], [HD2E7 *. A9 / D2E7 VL], [D2E7VH / LD2E7 * .A1], [D2E7 VH / LD2E7 * .A4], [D2E7 VH / LD2E7 * .A5], [D2E7 VH / LD2E7 * .A7], [D2E7 VH / LD2E7 * .A8], [D2E7 * .A9 / LD2E7 * .A1], [VH1-D2 / LOE7], [VH1-D2.N / LOE7.T], [VH1-D2.Y / LOE7.A], [ VH1-D2.N / LOE7.A], [VH1-D2 / EP B12] and [3C-H2 / LOE7].

It is well known in the art that the CDR3 domains of the heavy and light chains of antibodies play an important role in the specificity / affinity of binding of an antibody to an antigen. Accordingly, in another aspect, the invention relates to human antibodies that have low dissociation kinetics for association with hTNFa and that have heavy and light chain CDR3 domains that are structurally identical or related to those of D2E7. As demonstrated in Example 3, D2E7 VL CDR3 position 9 can be occupied by Ala or Thr without substantially affecting Koff. Accordingly, a consensus motif for D2E7 VL CDR3 comprises the amino acid sequence: QRYNRAPY- (T / A) (SEQ ID NO: 3). Furthermore, D2E7 VH CDR3 position 12 can be occupied by TyroAsn, without Koff being substantially affected. Accordingly, a consensus motif for D2E7 VH CDR3 comprises the amino acid sequence: V-SY-LSTASSLD- (Y / N) (SEQ ID NO: 4). Furthermore, as demonstrated in Example 2, the CDR3 domain of the D2E7 heavy and light chains is capable of substitution with a single alanine residue (at position 1, 4, 5, 7 or 8 within the CDR3 of VL or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without the Koff being substantially affected. Furthermore, one of skill in the art will appreciate that, given the susceptibility of the CDR3 domains of VL and VH of D2E7 to substitutions by alanine, the substitution of other amino acids within the CDR3 domains is possible while retaining the low dissociation rate constant. of the antibody, in particular conservative amino acid substitutions. A "conservative amino acid substitution", as used herein, is one in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine,

ES 2 198 552 T3 serine, threonine, tyrosine, cysteine), non-polar side chains (eg alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), side chains with beta branches (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Preferably, no more than one to five conservative acid substitutions are made within the CDR3 domains of VL and / or VH of D2E7. More preferably, no more than one to three conservative acid substitutions are made within the CDR3 domains of VL and / or VH of D2E7. Furthermore, conservative amino acid substitutions should not be made at critical amino acid positions for hTNFa binding. As shown in Example 3, D2E7 VL CDR3 positions 2 and 5 and D2E7 VH CDR3 positions 1 and 7 appear to be critical for interaction with hTNFa and thus preferably not they make conservative amino acid substitutions at these positions (although an alanine substitution at position 5 of the VL CDR3 of D2E7, as described above, is acceptable).

Accordingly, in another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, with the following characteristics:

a) dissociates from human TNFa with a Koff rate constant of 1 x 10^-3 s^-1 or less, determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4 , 5, 6, 8, 9, 10, or 11 or by one to five conservative amino acid substitutions at positions 2,3, 4,5, 6,8, 9, 10, 11, and / or 12.

More preferably, the antibody, or the antigen-binding portion thereof, dissociates from human TNFa with a Koff of 5 x 10^-4 s^-1 or less. Even more preferably, the antibody, or the antigen-binding portion thereof, dissociates from human TNFa with a Koff of 1 x 10^-4 s^-1 or less.

In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10u11. Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., the VL CDR2 of D2E7) and the HCVR further has a CDR2 domain comprising the amino acid sequence of the SEQ ID NO: 6 (ie, the VH CDR2 of D2E7). Even more preferably, the LCVR further has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e., the VL CDR1 of D2E7) and the HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 (ie, the VH CDR1 of D2E7). The framework regions for VL, preferably are derived from the human germline family V_kI, more preferably from the A20 human germline Vk gene and even more preferably from the D2E7 VL structural sequences shown in Figures 1A and 1B. The framework regions for VH preferably are derived from the human germline VH3 family, more preferably from the human germline VH gene DP-31 and even more preferably from the VH framework sequences of D2E7 shown in Figures 2A and 2B.

In another embodiment the invention provides an isolated human antibody, or antigen-binding portion thereof, with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the VL of D2E7) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 (ie, the VH of D2E7). In certain embodiments, the antibody comprises a heavy chain constant region, such as a constant region from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody may comprise a light chain constant region, a kappa light chain constant region, or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.

In other embodiments, the invention provides an isolated human antibody, or antigen-binding portion thereof, having D2E7-related VL and VH CDR3 domains, eg, antibodies or antigen-binding portions thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: : 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19 SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a heavy chain variable region (HCVR) having a CDR3 domain comprising a selected amino acid sequence among the group consisting of SEQ ID No: 4, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

ES 2 198 552 T3

In another embodiment, the invention provides a recombinant human antibody, or an antigen-binding portion thereof, that neutralizes the activity of human TNFa but not human TNIye. Preferably, the antibody, or the antigen-binding portion thereof, also neutralizes the activity of chimpanzee TNFa and at least one additional primate TNFa selected from the group consisting of baboon TNFa, marmoset TNFa, cynomolgus monkey TNFa, and Rhesus monkey TNFa. Preferably, the antibody, or the antigen-binding portion thereof, neutralizes human, chimpanzee, and / or other primate TNFa in a standard in vitro L929 assay with an IC.₅₀of 1 x 10^-8 M or less, more preferably 1 x 10^-9 M or less and even more preferably 5 x 10^-10 M or less. In a sub-embodiment, the antibody also neutralizes canine TNFa activity, preferably in a standard in vitro L929 assay with an IC50 of 1 x 10^-7 M or less, more preferably 1 x 10^-8 M or less and even more preferably 5 x 10^-9 M or less. In another sub-embodiment, the antibody also neutralizes pig TNFα activity preferably in a standard in vitro L929 assay with an IC50 of 1 x 10^-5 M or less, more preferably 1 x 10^-6 M or less and even more preferably 5 x 10^-7 M or less. In another embodiment, the antibody also neutralizes mouse TNFa activity preferably in a standard in vitro L929 assay with an IC50 of 1 x 10^-4 M or less, more preferably 1 x 10^-5 M or less and even more preferably 5 x 10^-6 M or less.

An antibody or antibody portion of the invention may be modified or attached to another functional molecule (eg, another peptide or protein. Accordingly, antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the invention). human anti-hTNFa antibodies described herein, including immunoadhesion molecules. For example, an antibody or an antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (eg, an antibody bispecific or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent and / or a protein or peptide that can mediate the association of the antibody or the antibody portion with another molecule (such as the streptavidin core region or a polyhistidine signal).

A modified type of antibody is produced by the cross-linking of two or more antibodies (of the same type or of different types, for example, to create bispecific antibodies). Suitable crosslinks include those that are heterobifunctional, having two groups with different reactivity separated by an appropriate spacer (eg, m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (eg, disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.

Useful detectable agents with which an antibody or antibody portion of the invention can be modified include fluorescent compounds. Illustrative detectable fluorescent agents include fluorescein, fluorescein isothiocyanate, rhodamine, zinc or methylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. An antibody can also be modified with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose, oxidase, and the like. When an antibody is modified with a detectable enzyme, it is detected by the addition of additional reagents so that the enzyme is used to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody can also be modified with biotin, and detected by indirect measurement of avidin or streptavidin binding.

II. Antibody expression

An antibody, or an antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin heavy and light chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors that carry DNA fragments encoding the immunoglobulin heavy and light chains of the antibody such that the heavy and light chains are expressed in the antibody. host cell and, preferably, are secreted into the medium in which the host cells are cultured, the antibodies can be recovered from said medium. 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, Fritsch, and Maniatis (eds), Molecular Cloning ; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in US Patent No. 4,816,397 to Boss et al.

To express D2E7 or a D2E7-related antibody, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplifying and modifying the germline heavy and light chain variable sequences using the polymerase chain reaction (PCR). Human heavy and light chain variable region genes are known in the art (see, for example, the human germline sequence database "Vbase"; see also Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) “The Repertoire of Human Germline V_H Sequences Reveals about Fifty Groups of V_H Segments with Different Hypervariable Loops ”J. Mol. Biol. 227: 776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V_K Segments Reveals a Strong Bials in ther Usage ”Eur. J. Immunol. 24: 827-839; whose contents are expressly incorporated in this document by reference. To obtain a DNA fragment encoding the D2E7 heavy chain variable region, or a D2E7-related antibody, a member of the VH3 family of human germline VH genes is amplified by standard PCR.

ES 2 198 552 T3

More preferably, the DP-31 VH germline sequence is amplified. To obtain a DNA fragment encoding the D2E7 light chain variable region, or an antibody related to D2E7, a member of the V family is amplified._KI from human germline VL genes by conventional PCR. More preferably, the A20 VL germline sequence is amplified. Suitable PCR primers for use in amplifying the A20 general line VL and DP-31 germline VH sequences can be designed based on the nucleotide sequences described in the references cited above, using standard methods.

Once germline VH and VL fragments are obtained, these sequences can be mutated to encode the D2E7 or D2E7-related amino acid sequences described herein. The amino acid sequences encoded by the germline VH and VL DNA sequences are first compared to the D2E7 or D2E7-related VH and VL amino acid sequences to identify amino acid residues in the D2E7 or D2E7-related sequence that differs from the germ line. The appropriate nucleotides of the germline DNA sequences are then mutated such that the mutated germline sequence encodes the D2E7 or D2E7-related amino acid sequence, using the genetic code to determine the nucleotide changes that must be done. Mutagenesis of germline sequences is performed by conventional methods, such as PCR-mediated mutagenesis (in which mutated nucleotides are incorporated into PCR primers such that the PCR product contains the mutations) or mutagenesis of targeted location.

In addition, it should be noted that if the "germline" sequences obtained by PCR amplification encode amino acid differences in framework regions from the true germline configuration (that is, differences in the amplified sequence compared to the sequence germline, for example, as a result of a somatic mutation), it may be desirable to change these amino acid differences back to the true germline sequences (ie, "back-mutation" of structural residues to the germline configuration).

Once DNA fragments encoding VH and VL segments of D2E7 or related to D2E7 have been obtained (by amplification and mutagenesis of the germline VH and VL genes, as described above), these DNA fragments can be further manipulated by conventional recombinant DNA techniques. , for example, to convert variable region genes to full-length antibody chain genes, Fab fragment genes, or a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", as used in this context, is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in phase.

Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the DNA encoding VH with another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NiH Publication No. 91-3242) and DNA fragments that include these regions can be obtained by standard PCR amplification. The heavy chain constant region may be a constant region from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but more preferably it is a constant region from IgG1 or IgG4. For a heavy chain gene of an Fab fragment, the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the DNA encoding VL to another DNA molecule encoding the constant region. light chain, CL. Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is more preferably a kappa constant region.

To create a scFv gene, the DNA fragments encoding VH and V1 are operably linked to another fragment encoding a flexible bond, for example, encoding the amino acid sequence (Gly4-Ser) 3, such that the sequences VH and VL can be expressed as a contiguous single chain protein, with the VL and VH regions linked by the flexible linker (see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc Natl Acad Sci. USA 85: 5879-5883; McCafferty et al., Nature (1990) 348: 552-554).

To express the antibodies or antibody portions of the invention, DNAs encoding full or partial length light and heavy chains, obtained as described above, are inserted into expression vectors in such a way that the genes are operably linked to sequences of control, transcription and translation. In this context, the term "operably linked" is intended to mean that an antibody gene is linked to a vector in such a way that the transcriptional and translational control sequences within the vector perform their desired function of regulating transcription and translation. of the antibody gene. The expression vector and expression control sequences are chosen so as to be compatible with the expression host cell used. The

ES 2 198 552 T3 antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors or, more typically, the two genes are inserted into the same expression vector. Antibody genes are inserted into the expression vector by conventional methods (eg, binding of complementary restriction sites on the antibody gene fragment and vector, or blunt-ended binding if no restriction sites are present). Prior to insertion of D2E7 or D2E7-related heavy or light chain sequences, the expression vector may carry antibody constant region sequences. For example, one strategy to convert the D2E7 or D2E7-related VH and VL sequences into full-length antibody genes is to insert them into expression vectors that already encode heavy chain and light chain constant regions, respectively, in such a way that segment VH is operably linked to segment (s) CH within the vector and segment VL is operably linked to segment CL within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in phase to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a protein that is not an immunoglobulin).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that drive high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer ), Simian Virus 40 (SV40) (such as SV40 promoter / enhancer), adenovirus (eg, adenovirus major late promoter (AdMLP)), and polyoma. For a further description of viral regulatory elements and their sequences, see, for example, US Patent No. 5,168,062 to Stinski, US Patent No. 4,510,245 to Bell et al. and US Patent No. 4,968,615 to Schaffner et al.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selective marker genes. The selective marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all to Axel et al. ). For example, the selective marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Preferred selective marker genes include the dihydrofolate reductase (DHFR) gene (for use in host cells dhfr^- with methotrexate selection / amplification) and the neo gene (for G418 selection).

For the expression of the light and heavy chains, the expression vector or vectors encoding the heavy and light chains are used to transfect a host cell by standard techniques. The various forms of the term "transfection" are intended to include a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, eg, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, the most preferred is the expression of antibodies in eukaryotic cells, and even more preferably in mammalian host cells, because such eukaryotic cells and in particular mammalian cells they are more likely than prokaryotic cells to mount and secrete a properly folded immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for the production of high yields of active antibody (Boss, MA and Wood, CR (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary cells (CHO cells) (including CHO dhfr- cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selective marker, for example, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody in the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to produce intact antibody portions, such as Fab fragments or scFv molecules. It will be understood that variations of the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. You can also use the

ES 2 198 552 T3 recombinant DNA technology to remove some or all of the DNAs encoding the light chain and heavy chain, or both, that are not required for hTNFa binding. Molecules expressed from such truncated DNA molecules are also included by the antibodies of the invention. In addition, bifunctional antibodies can be produced in which one heavy chain and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than hTNFa by crosslinking an antibody of the invention with a second. antibody by conventional chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into CHO dhfr cells. - by calcium phosphate mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain and light chain genes are operably linked to enhancer / promoter regulatory elements (e.g., derivatives of SV40, CMV, adenovirus, and the like, such as the CMV enhancer regulatory element / AdMLP promoter or an SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transforming host cells are cultured to allow expression of the antibody heavy and light chains and an intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, cultivate the host cells, and recover the antibody from the culture medium.

In view of the foregoing, another aspect of the invention relates to nucleic acid, vector, and host cell compositions that can be used for recombinant expression of the antibodies and antibody portions of the invention. The nucleotide sequence encoding the D2E7 light chain variable region is shown in Figure 7 and SEQ ID NO: 36. The CDR1 domain of LCVR includes nucleotides 70-102, the CDR2 domain includes nucleotides 148-168, and the CDR3 domain includes nucleotides 265-291. The nucleotide sequence encoding the D2E7 heavy chain variable region is shown in Figure 8 and SEQ ID NO: 37. The CDR1 domain of HCVR includes nucleotides 91-105, the CDR2 domain includes nucleotides 148- 198 and the CDR3 domain includes nucleotides 295-330. One skilled in the art will appreciate that nucleotide sequences encoding D2E7-related antibodies, or portions thereof (eg, a CDR domain such as a CDR3 domain) can be obtained from nucleotide sequences encoding LCVR and HCVR of D2E7 using the genetic code and conventional molecular biology techniques.

In one embodiment, the invention provides an isolated nucleic acid encoding a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 (i.e., the VL CDR3 of D2E7), or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9. This nucleic acid may encode only the CDR3 region or, more preferably, encodes a whole antibody light chain variable region (LCVR). For example, the nucleic acid may encode an LCVR rune having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., the VL CDR2 of D2E7) and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (ie, the VL CDR of D2E7).

In another embodiment, the invention provides an isolated nucleic acid encoding a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4 (i.e., the VH CDR3 of D2E7) or modified from the SEQ ID NO: 4 for a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10, or 11 or for five conservative amino acid substitutions at positions 2, 3, 4, 5, 6 , 8, 9, 10, 11 and / or 12. This nucleic acid may encode only the CDR3 region or, more preferably, encode an entire antibody heavy chain variable region (HCVR). For example, the nucleic acid may encode an HCVR having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (i.e., the VH CDR2 of D2E7) and a CDR1 domain comprising the sequence of amino acids of SEQ ID NO: 8 (ie, the VH CDR1 of D2E7).

In yet another embodiment, the invention provides isolated nucleic acids encoding a CDR3 domain related to D2E7, for example, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33 , SEQ ID NO: 34 and SEQ ID NO: 35.

In yet another embodiment, the invention provides an isolated nucleic acid encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 (ie, the LCVR of D2E7). Preferably, this nucleic acid comprises the nucleotide sequence of SEQ ID NO: 36, although the skilled person will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences may encode the amino acid sequence of SEQ ID NO. °: 1. The nucleic acid may encode only the LCVR or it may also encode an antibody light chain constant region, operably linked to the LCVR. In one embodiment, this nucleic acid is in a recombinant expression vector.

In another embodiment, the invention provides an isolated nucleic acid encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (ie, the HCVR of D2E7).

ES 2 198 552 T3

Preferably, this nucleic acid comprises the nucleotide sequence of SEQ ID NO: 37, although the skilled person will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences may encode the amino acid sequence of SEQ ID NO. °: 2. The nucleic acid may encode only the HCVR or it may also encode a heavy chain constant region, operably linked to the HCVR. For example, the nucleic acid can comprise a constant region of IgG1 or IgG4. In one embodiment, this nucleic acid is in a recombinant expression vector.

The invention also provides recombinant expression vectors encoding both an antibody heavy chain and an antibody light chain. For example, in one embodiment, the invention provides a recombinant expression vector that encodes:

a) an antibody light chain having a variable region comprising the amino acid sequence of SEQ ID NO: 1 (ie, the LCVR of D2E7); Y

b) an antibody heavy chain having a variable region comprising the amino acid sequence of SEQ ID NO: 2 (ie, the D2E7 HCVR).

The invention also provides host cells into which one or more of the recombinant expression vectors of the invention have been introduced. Preferably the host cell is a mammalian host cell, more preferably the host cell is a CHO cell, an NS0 cell or a COS cell.

Furthermore, the invention provides a method of synthesizing a recombinant human antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant human antibody of the invention is synthesized. The method may further comprise isolating the recombinant human antibody from the culture medium.

III. Selection of recombinant human antibodies

The recombinant human antibodies of the invention, in addition to the D2E7 or D2E7-related antibodies described herein, can be isolated by selection from a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using VL cDNA and Human VH prepared from mRNA derived from human lymphocytes. Methodologies for preparing and selecting such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (for example, the Recombinant Phage Antibody System from Pharmacia, catalog # 27-9400-1; and the SurfZAP (tm) phage display kit from Stratagene, Catalog No. 240612), examples of particularly suitable methods and reagents for use in generating and screening antibody display libraries can be found in, for example, Ladner et al. US Patent No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271, Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281: McCafferty et al., Nature (1990) 348: 552-554; Griffiths et al. (1993) EMBO J 12: 725-734; hAWKINS et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982.

In a preferred embodiment, to isolate human antibodies with high affinity and a low separation rate constant for hTNFa, a murine anti-hTNFa antibody is first used that has high affinity and a low separation rate constant for hTNFa (e.g. example, MAK 195, the hybridoma for which it has the deposit number ECACC 87 050801) to select human heavy and light chain sequences that have similar binding activity towards hTNFa, using epitope imprinting, or guided selection, methods described in Hoogenboom et al. PCT Publication No. WO 93/06213. Antibody libraries used in this method are preferably scFv libraries prepared and selected as described in McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990) 348: 552-554; and Griffiths et al., (1993) EMBO J 12: 725-734. The scFv antibody libraries are preferably screened using recombinant human TNFa as the antigen.

Once the initial human VL and VH segments are selected, "mix and match" experiments are selected, in which different pairs of the initially selected VL and VH segments are selected for binding of hTNFa, to select preferred VL / VH pair combinations. . Furthermore, to further improve the affinity and / or reduce the separation rate constant for hTNFa binding, the VL and VH segments of the preferred VL / VH pair (s) may be randomly mutated, preferably within the CDR3 region of VH and / or VL, in a process analogous to the somatic mutation process in vivo responsible for the maturation of antibody affinity during a natural immune response. This in vitro affinity mutation can be carried out by amplifying VH and VL regions using PCR primers complementary to the VH CDR3 or VL CDR3, respectively, primers to which a random mixture of the four nucleotide bases has been "added" at certain positions. such that the resulting PCR products encode VH and VL segments into which random mutations have been introduced in the VH and / or VL CDR3 regions. These randomly mutated VH and VL segments can be reselected for hTNFa binding, and sequences exhibiting high affinity and low rate of separation can be selected for hTNFa binding.

ES 2 198 552 T3

The heavy and light chain amino acid sequences of selected antibodies can be compared to germline heavy and light chain amino acid sequences. In cases where certain structural residues of the selected VL and / or VH chains differ from the germline configuration (for example, as a result of somatic mutation of the immunoglobulin genes used to prepare the phage library), you can it is desirable to "back-mutate" the altered structural residues of the selected antibodies to the germline configuration (ie, change the structural amino acid sequences of the selected antibodies so that they are the same as the structural amino acid sequences of the germ line). Such "back-mutation" (or "germline alignment") of structural residues can be performed by standard molecular biology methods to introduce specific mutations (eg, site-directed mutagenesis; PCR-mediated mutagenesis and the like).

After selection and isolation of an anti-hTNFa antibody of the invention from a display library of recombinant immunoglobulins, the nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the genome of the phage) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other forms of antibody of the invention (eg, binding to a nucleic acid encoding additional immunoglobulin domains such as additional constant regions). To express a recombinant human antibody isolated by selection from a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described in more detail in section II above. .

IV. Pharmaceutical Compositions and Pharmaceutical Administration

The antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or an antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, 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 mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or efficacy of the antibody or the antibody portion.

The compositions of this invention can be in a variety of forms. These include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the desired mode of administration and the therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., an antibody or an antibody portion) in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, when necessary, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produces a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, through the use of a coating such as lecithin, through the maintenance of the required particle size in the case of dispersion and through the use of surfactants. Prolonged absorption of injectable compositions can be accomplished by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

The antibodies and antibody portions of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is injection or intravenous infusion. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. In certain embodiments, the active compound can be prepared with a carrier that protects the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. They can

ES 2 198 552 T3 to use biocompatible biodegradable polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those of skill in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody or an antibody portion of the invention can be administered orally, for example, with an inert diluent or assimilable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a soft or hard shell gelatin capsule. Compressed into tablets or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by administration other than parenteral, it may be necessary to coat the compound or co-administer the compound with a material to prevent its inactivation.

Supplemental active compounds may also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is co-formulated and / or co-administered with one or more additional therapeutic agents that are useful for treating disorders in which TNFα activity is detrimental. For example, an antibody a portion of anti-hTNFa antibody can be coformulated and / or co-administered with one or more additional antibodies that bind to other targets (e.g., antibodies that bind to other cytokines or that bind to surface molecules cell), one or more cytokines, soluble TNFα receptor (see, for example, PCT Publication No. WO 94/06476) and / or one or more chemical agents that inhibit hTNFa production or activity (such as cyclohexaneylidene derivatives as described in PCT Publication No. WO 93/19751). In addition, one or more antibodies of the invention can be used in combination with two or more of the above therapeutic agents. Such combination therapies can advantageously use lower doses of the administered therapeutic agent, thus avoiding the potential toxicities or complications associated with the various monotherapies.

Non-limiting examples of rheumatoid arthritis therapeutic agents with which an antibody or antibody portion of the invention may be combined include the following: non-steroidal anti-inflammatory drugs (NSAIDs); cytokine suppressive anti-inflammatory drugs (CSAIDs); CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody); Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see, for example, Arthritis &amp; Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44,235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1 / SB 210396 (Non-reducing primatized anti-CD4 antibody; IDEC / SmithKline; see, for example, Arthritis &amp; Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and / or DAB 389-IL-2 (IL2 fusion proteins; Seragen; see, for example, Arthritis &amp; Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanized anti-IL-2Ra; Protein Design Labs / Roche); IL-4 (anti-inflammatory cytokine; DNAX / Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX / Schering); IL-4; IL-10 and / or IL-4 agonists (eg, agonist antibodies); IL-1RA (IL-1 receptor antagonists; Synergen / Amgen); TNF-bp / s-TNFR (soluble TNF binding protein; see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39 No. 9 (supplement), S284; Amer. J. Physiol. - Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42); R973401 (Type IV phosphodiesterase inhibitor; see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2 inhibitor, see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S81); Iloprost (see, for example Arthritis &amp; Rheumatism (1996) Vol. 39 No. 9 (supplement), S82); methotrexate, thalidomide (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39 No. 9 (supplement), S282) and thalidomide-related drugs (eg, Celgen); leuflunomide (anti-inflammatory and cytokine inhibitor, see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39 No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid (plasminogen activation inhibitor; see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S284); T-614 (cytokine inhibitor, see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); prostaglandin E1 (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tendinap (non-steroidal anti-inflammatory drug; see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidal anti-inflammatory drug; see, for example, Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); Azathioprine (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S281), ICE inhibitor (interleukin-1β converting enzyme inhibitor); zap-70 and / or lck inhibitor (zap-70 tyrosine kinase inhibitor or lck); VEGF inhibitor and / or VEGF-R inhibitor (vascular endothelial cell growth factor or endothelial cell growth factor receptor inhibitors; angiogenesis inhibitors); corticosteroid anti-inflammatory drugs (eg SB203580); TNFconvertase inhibitors; anti-IL-12 antibodies, interleukin-11 (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement) S296); interleukin-13 (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold, penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5 toxins; orally administered peptides and collagen; disodium lobenzarite; Cytokine Regulatory Agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodexoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulfate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fatty acids from vegetable and fish seeds;

ES 2 198 552 T3 see, for example, DeLuca et al. (1995) Rheum. Dis. Clin. NortAm. 21: 759-777); auranofin; phenylbutazone, meclofenamic acid; flufenamic acid; intravenous immunoglobulin; zileuton; mycophenolic acid (RS-61443); tracrolimus (FK-506); sirolimus (rapamycin); amiprilose (terafectin); cladribine (2-chlorodeoxyadenosine); and Azaribine.

Non-limiting examples of therapeutic agents for inflammatory bowel disease with which an antibody or an antibody portion of the invention may be combined include the following: bidenoside; epidermal growth factor; corticosteroids; cyclosporine, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; monoclonal anti-IL-1β antibodies; monoclonal anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody; Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFRIgG (75 kD TNF receptor-IgG fusion protein; Immunex ,; see, for example, Arthritis &amp; Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFIgG receptor fusion protein; Hoffmann-LaRoche); interleukin-10 (sCh 52000; Schering Plow); IL-4; IL-10 and / or IL-4 agonists (eg, agonist antibodies); interleukin-11; glucuronide or dextran conjugated prodrugs of prednisolone, dexamethaxone or budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); slow release mesalazine; methotrexate; Platelet Activating Factor (PAF) antagonists; ciprofloxacin; and lignocaine.

Non-limiting examples of multiple sclerosis therapeutics with which an antibody or antibody portion of the invention may be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-e1a (Avonex (tm); Biogen); interferon- ^ 1b (Betaseron (tm); Chiron / Berlex); Copolymer 1 (Cop-1; Copaxone (tm); Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody; Celltech / Bayer); cA2 (Chimeric anti-TNFa antibody; Centocor); 75 kdTNFRIgG (75 kD TNF-IgG receptor defusion protein; Immunex; see, for example, Arthritis &amp; Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann, LaRoche); IL-1; IL-4; and IL-10 and / or IL-4 agonists (eg, agonist antibodies).

Non-limiting examples of sepsis therapeutics with which an antibody or antibody portion of the invention may be combined include the following: hypertonic saline solutions; antibiotics; intravenous gamma globulin; continuous hemofiltration; carbapenems (eg meropenem); cytokine antagonists such as TNFa, IL- ^ β, IL-6 and / or IL-8; CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody; Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF-IgG receptor fusion protein; Immunex; see, for example, Arthritis &amp; Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann, LaRoche); Cytokine Regulatory Agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&amp;F 107647 (low molecular weight peptide; SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); Tissue Factor Pathway Inhibitor (TFPI; Chiron; PHP (chemically modified hemoglobin; APEX Bioscience); iron chelators and chelates, including diethylenetriaminepentaacetic acid-iron (III) complex (DTPA iron (III); Molichem Medicines); lysophylline (synthetic small molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (water soluble e1,3-glucan; Alpha-Beta Technology); lipid-reconstituted polypoprotein A-1; chiral hydroxamic acids (synthetic antibacterials that inhibit lipid A biosynthesis); anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPI21 (Recombinant N-terminal fragment of human Bactericidal / Permeability Enhancing Protein); and Synthetic Anti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories).

Non-limiting examples of adult respiratory distress syndrome (ARDS) therapeutic agents with which an antibody or antibody portion of the invention may be combined include the following: anti-IL-8 antibodies; surfactant replacement therapy; CDP-571 / BAY-10- 3356 (humanized anti-TNFa antibody; Celltech / Bayer); cA2 (Chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see, for example, Arthritis &amp; Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann, LaRoche).

The use of the antibodies or antibody moieties of the invention in combination with other therapeutic agents is further described in subsection IV.

The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" refers to an effective amount, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease status, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to induce a desired response. in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or the antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at dosages and for periods necessary to achieve the desired prophylactic result. Typically, as a prophylactic dose is used in subjects prior to illness or in

In the early stages of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimal desired response (eg, a therapeutic or prophylactic response. For example, a single bolus injection can be given, or several divided doses can be given over time, or the dose can be reduced or be increased proportionally when indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit forms, as used herein, refer to physically discrete units suitable as unit dosages for mammalian subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention is dictated by and is directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An illustrative non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1 to 20 mg / kg, more preferably 1 to 10 mg / kg. It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated. It will be understood that for any particular subject, specific dosage regimens must be adjusted over time according to the needs of the individual and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges stated herein are illustrative only and are not intended to limit the scope or practice of the claimed composition.

IV. Uses of the Antibodies of the Invention

Given their ability to bind to hTNFa, the anti-hTNFa antibodies, or portions thereof, of the invention can be used to detect hTNFa (for example, in a biological sample, such as serum or plasma) using a standard immunoassay, such as such as enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay (RIA), or tissue immunohistochemistry. The invention provides a method for detecting hTNFa in a biological sample which comprises contacting the biological sample with an antibody, or an antibody portion, of the invention and detecting the antibody , or antibody portion bound to the hTNFa or non-antibody. bound (or antibody portion) to thereby detect hTNFa in the biological sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; An example of a luminescent material includes luminol; and examples of radioactive materials include ¹²⁵I, ¹³¹I, ³⁵Yes ³H.

As an alternative to antibody labeling, hTNFa can be assayed in biological fluids by competitive immunoassays using rhTNFa standards labeled with a detectable substance and an unlabeled anti-hTNFa antibody. In this assay, the biological sample, the labeled rhTNFa standards, and the anti-hTNFa antibody are combined and the amount of the labeled rhTNFa standard bound to the unlabeled antibody is determined. The amount of hTNFa in the biological sample is inversely proportional to the amount of labeled rhTNFa standard bound to the anti-hTNFa antibody.

A D2E7 antibody of the invention can also be used to detect TNFa from species other than humans, in particular TNFa from primates (eg, chimpanzee, baboon, marmoset, cynomolgus monkey and rhesus monkey), pig and mouse, since D2E7 can bind to each of these TNFa (as further described in Example 4, subsection E).

The antibodies and antibody portions of the invention can neutralize hTNFa activity both in vitro and in vivo (see Example 4). Furthermore, some of the antibodies of the invention, such as D2E7, can neutralize the TNFα activity of other species. Accordingly, the antibodies and antibody portions of the invention can be used to inhibit TNFa activity, for example, in culture of hTNFa-containing cells, in humans, or in other mammals that have TNFa cross-reactively with a antibody of the invention (eg, chimpanzee, baboon, marmoset, cynomolgus and rhesus monkey, pig or mouse). In one embodiment, the invention provides a method of inhibiting TNFa activity which comprises contacting TNFa with an antibody or antibody portion of the invention in such a way as to inhibit TNFa activity. Preferably, the TNFa is human TNFa. For example, in a cell culture containing or suspected of containing hTNFa, an antibody or an antibody portion of the invention can be added to the culture medium to inhibit the hTNFa activity of the culture.

In another embodiment, the invention provides a method of inhibiting TNFa activity in a subject suffering from a disorder in which TNFa activity is detrimental. TNFa has been implicated in the pathophysiology of a wide

ES 2 198 552 T3 diversity of disorders (see, for example, Moeller, A., et al. (1990) Cytokine 2: 162-169; US Patent No. 5,231,024 to Moeller et al .; Patent Publication European No. 260 610 B1 of Moeler, A.). The invention provides methods for monitoring TNFa activity in a subject suffering from such a disorder, the method comprising administering to the subject an antibody or antibody portion of the invention in such a way as to inhibit TNFa activity in the subject. Preferably the TNFa is human TNFa and the subject is a human. Alternatively, the subject can be a mammal that expresses a TNFa to which an antibody of the invention crosslinks. Furthermore, the subject may be a mammal into which hTNFa has been introduced (eg, by administration of hTNFa or by expression of a hTNFa transgene). An antibody of the invention can be administered to a human for therapeutic purposes (discussed in more detail below). In addition, an antibody of the invention can be administered to a non-human mammal that expresses a TNFa to which the antibody is cross-linked (eg, a primate, pig, or mouse) for veterinary purposes or as an animal model of human disease. With regard to the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (eg, for testing dosages and time courses of administration).

As used herein, the term "a disorder in which TNFa activity is deleterious" is intended to include diseases and other disorders in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected which is responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which TNFa activity is deleterious is a disorder in which inhibition of TNFa activity is expected to alleviate the symptoms and / or progression of the disorder. Such disorders can be evidenced, for example, by an increase in the concentration of TNFa in a biological fluid of a subject suffering from the disorder (eg, an increase in the concentration of TNFa in serum, plasma, synovial fluid, etc. of the subject, which can be detected, for example, using an anti-TNFa antibody as described above. There are numerous examples of disorders in which TNFa activity is detrimental. The use of antibodies and antibody portions of the invention in the treatment of specific disorders is described in more detail below.

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology of sepsis, with biological effects including hypotension, myocardial suppression, vascular leak syndrome, organ necrosis, stimulation of the release of secondary toxic mediators and activations of the cascade of coagulation (see, for example, Moeller, A., et al. (1990) Cytokine 2: 162-169; US Patent No. 5,231,024 to Moeller et al .; European Patent Publication No. 260 610 B1 by Moeller, A .; Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45: 491-503; Russell, D and Thompson, RC (1993) Curr. Opin. Biotech. 4: 714-721). Accordingly, human antibodies and antibody portions of the invention can be used to treat sepsis in any of its clinical situations, including septic shock, endotoxic shock, Gram negative sepsis, and toxic shock syndrome.

In addition, to treat sepsis, an anti-hTNFa antibody, or portion of the antibody, of the invention can be co-administered with one or more additional therapeutic agents that can further alleviate sepsis, such as an interleukin-1 inhibitor (such as those described in PCT Publication No. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see, for example, PCT Publication No. WO 93/11793) or a platelet activating factor antagonist (see, for example, European Patent Application Publication No. EP 374 510). Other combination therapies for the treatment of sepsis are discussed further in subsection III.

Furthermore, in a preferred embodiment, an anti-TNFa antibody or antibody portion of the invention is administered to a human within a subgroup of patients with sepsis who have a serum or plasma concentration of IL-6 greater than 500 pg. / ml, and more preferably 1000 pg / ml, at the time of treatment (see PCT Publication No. WO 95/20978 by Dauman, L., et al.).

B. Autoimmune diseases

Tumor necrosis factor has been implicated in the pathophysiology of a variety of autoimmune diseases. For example, TNFa has been implicated in the activation of tissue inflammation and in the production of joint destruction in rheumatoid arthritis (see, for example, Moeller, A., et al. (1990) Cytokine 2: 162- 169; US Patent No. 5,231,024 to Moeller et al .; European Patent Publication No. 260 610 B1 by Moeller, A .; Tracey and Cerami, supra; Arend, WP and Dayer, JM. (1995) Arth. Rheum. 38: 151-160; Fava, RA, et al. (1993) Clin. Exp. Immunol. 94: 261-266). TNFa has also been implicated in promoting islet cell death and mediating insulin resistance in diabetes (see, for example, Tracey and Cerami, supra; PCT Publication No. WO 94 / 08609). TNFa has also been implicated in mediating oligodendrocyte cytotoxicity and induction of inflammatory plaques in multiple sclerosis (see, eg, Tracey and Cerami, supra). Humanized murine and chimeric anti-hTNFa antibodies have undergone a clinical trial for the treatment of rheumatoid arthritis (see, for example, Elliott, MJ, et al. (1994) Lancet 344: 1125-1127; Elliot, MJ, et al. (1994) Lancet 344: 1105-1110; Rankin, EC, et al. (1995) Br. J. Rheumatol. 34: 334-342).

Human antibodies and antibody portions of the invention can be used to treat autoimmune diseases, particularly those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome. Typically, the antibody or antibody portion is administered systemically, although for certain

ES 2 198 552 T3 disorders, local administration of the antibody or the antibody portion at a site of inflammation (for example, local administration to the joints in rheumatoid arthritis or topical application in diabetic ulcers, alone or in combination with cyclohexane-ylidene derivatives as described in PCT Publication No. WO 93/19751). An antibody, or antibody portion, of the invention may also be administered with one or more additional therapeutic agents useful in the treatment of autoimmune diseases, as further described in subsection III.

C. Infectious diseases

Tumor necrosis factor has been implicated in mediating biological effects observed in a variety of infectious diseases. For example, TNFa has been implicated in mediating brain inflammation and capillary thrombosis and stroke in malaria. TNFa has also been implicated in mediating brain inflammation, inducing the breakdown of the blood-brain barrier, inducing septic shock syndrome, and activating venous infarction in meningitis. TNFa has also been implicated in the induction of cachexia, the stimulation of viral proliferation, and the mediation of central nervous system damage in acquired immunodeficiency syndrome (AIDS). Accordingly, the antibodies and antibody portions of the invention can be used in the treatment of infectious diseases, including bacterial meningitis (see, for example, European Patent Application Publication No. EP 585 705), cerebral malaria, AIDS and AIDS-related complex (ARC) (see, for example, European Patent Application Publication No. EP 230 574), as well as cytomegalovirus infection secondary to transplantation (see, for example, Fietze, E., et al. (1994) Transplantation 58: 675680). The antibodies and antibody portions of the invention can also be used to alleviate symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza) and cachexia secondary to infection (eg, secondary to AIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograft rejection and host versus graft disease (GVHD) and in mediating an adverse reaction that has been observed when the rat antibody OKT3, directed against the complex CD3 T cell receptor is used to inhibit kidney transplant rejection (see, for example, Eason, JD, et al. (1995) Transplantation 59: 300-305; Suthanthiran, M. and Strom, TB (1994) New Engl J. Med. 331: 365-375). Accordingly, the antibodies, and antibody portions, of the invention can be used to inhibit transplant rejection, including allograft and xenograft rejection, and to inhibit GVHD. Although the antibody or antibody portion can be used alone, more preferably it is used in combination with one or more other agents that inhibit the immune response against the allograft or inhibit GVHD. For example, in one embodiment, an antibody or antibody portion of the invention is used in combination with OKT3 to inhibit OKT3-induced reactions. In another embodiment, an antibody or antibody portion of the invention is used in combination with one or more antibodies directed to other targets involved in the regulation of immune responses, such as cell surface molecules CD25 (interleukin-receptor a). 2), CD1 1a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 (B7-1) and / or CD86 (B7-2). In another embodiment, an antibody or antibody portion of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.

E. Malignancy

Tumor necrosis factor has been implicated in the induction of cachexia, in the stimulation of tumor growth, in the increase of the metastatic potential and in the mediation of cytotoxicity in malignancies. Accordingly, the antibodies and antibody portions of the invention can be used in the treatment of malignancies to inhibit tumor growth or metastasis and / or to alleviate cachexia secondary to malignancy. The antibody or antibody portion can be administered systemically or locally to the tumor site.

F. Pulmonary disorders

Tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome (ARDS), including stimulating endothelial leukocyte activation, directing cytotoxicity to pneumocytes, and inducing vascular leak syndrome. Accordingly, the antibodies and antibody portions of the invention can be used to treat various lung disorders, including respiratory distress syndrome in adults (see, for example, PCT Publication No. WO 91/04054), pulmonary shock, inflammatory disease. chronic lung, pulmonary sarcoidosis, pulmonary fibrosis, and silicosis. The antibody, or the antibody portion, can be delivered systemically or locally to the surface of the lung, for example with an aerosol. An antibody, or an antibody portion of the invention, may also be administered with one or more additional therapeutic agents useful in the treatment of pulmonary disorders, as further described in subsection III.

G. Intestinal disorders

Tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel disorders (see, for example, Tracy, KJ, et al. (1986) Science 234: 470-474; Sun, XM, et al. (1988) J Clin. Invest. 81: 13281331; MacDonald, TT, et al. (1990) Clin. Exp. Immunol. 81: 301-305). Have undergone an anti-20 clinical trial

ES 2 198 552 T3 chimeric murine anti-hTNFa bodies for the treatment of Crohn's disease (van Dullemen, HM, et al. (1995) Gastroenterology 109: 129-135). The human antibodies, and antibody portions, of the invention can also be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndromes. Crohn's disease and ulcerative colitis. An antibody, or antibody portion of the invention may also be administered with one or more additional therapeutic agents useful in treating intestinal disorders, as further described in subsection III.

H. Cardiac disorders

The antibodies and antibody portions of the invention can also be used to treat various cardiac disorders, including ischemia of the heart (see, for example, European Patent Application Publication No. EP 453 898) and heart failure (weakness of the heart). heart muscle) (see, for example, PCT Publication No. WO 94/20139).

I. Others

The antibodies and antibody portions of the invention can also be used to treat various other disorders in which TNFa activity is detrimental. Examples of other diseases and disorders in which TNFa activity has been implicated in pathophysiology and thus can be treated using an antibody and an antibody portion of the invention include inflammatory bone disorders and resorption diseases (see, for example, Bertolini, et al. (1986) Nature 319: 516-518; Konig, A., et al. (1988) J. Bone Miner. Res. 3: 621-627; Lerner, U. H and Ohlin, A. (1993) J. Bone Miner. Res. 8: 147-155; and Shankar, G. and Stern, PH (1993) Bone 14: 871876), hepatitis, including alcoholic hepatitis (see, for example, McClain, CJ and Cohen, DA (1989) Hepatology 9: 349-351; Felver, ME, et al. (1990) Alcohol. Clin. Exp. Res. 14: 255-259; and Hansen, J., et al. 81994) Hepatology 20: 461-474), viral hepatitis (Sheron N., et al. ( 1991) J. Hepatol. 12: 241-245; and Hussain, MJ, et al. (1994) J. Clin. Pathol. 47¿: 1112-1115) and fulminant hepatitis; coagulation disturbances (see, for example, van der Poll, T., et al. (1990) N. Engl. J. Med. 322: 1622-1627; and van der Poll, T., et al. (1991) ) Prog. Clin. Biol. Res. 367: 55-60), burns (see, for example, Giroir, BP, et al. (1994) Am. J. Physiol. 267: H118-124; and Liu XS, et al. al. 81994) Burns 20: 40-44), reperfusion injury (see, eg, Scales, WE, et al. (1994) Am. J. Physiol. 267: G11221127; Serrick, C., et al. (1994) Transplantation 58: 1158-1162; and Yao, YM, et al. (1995) Rhesuscitation 29: 157168), keloid formation (see, eg, McCauley, RL, et al. (1992) J. Clin. Immunol. 12: 300-308), scar tissue formation; pyrexia; periodontal disease; obesity and radiation toxicity.

This invention is further illustrated by the following examples which are not to be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this specification are incorporated herein by reference.

Example 1

Kinetic Analysis of Human Antibody Binding to hTNFa

Real-time binding interactions between a ligand (biotinylated recombinant human TNFa (rhTNFa) immobilized on a biosensor matrix) and analyte (antibodies in solution) were measured by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway , NJ). The system uses the optical properties of SPR to detect alterations in protein concentrations within a dextran biosensor matrix. Proteins are covalently bound to the dextran matrix at known concentrations. Antibodies are injected through the dextran matrix and the specific binding between the injected antibodies and the immobilized ligand results in an increase in the concentration of matrix protein and a resulting change in the SPR signal. These changes in the SPR signal are recorded as resonance units (RU) and plotted with respect to time along the y-axis of a sensorgram.

To facilitate immobilization of biotinylated rhTNFa on the biosensor matrix, streptavidin is covalently attached through free amine groups to the dextran matrix by first activating the carboxyl groups on the matrix with 100 mM N-hydroxysuccinimide (NHS) and N- hydrochloride. ethyl-N '- (3-diethylaminopropyl) carbodiimide (EDC) 400 mM. Streptavidin is then injected through the activated matrix. 35 µl of streptavidin (25 µg / ml), diluted in sodium acetate, pH 4.5, is injected through the activated biosensor and the free amines of the protein are directly attached to the activated carboxyl groups. The EDC esters of the unreacted matrix are deactivated by an injection of 1 M ethanolamine. Streptavidin coupled biosensor chips are also commercially available (Pharmacia BR-1000-16, Pharmacia Biosensor, Piscataway, NJ).

Biotinylated rhTNFa was prepared by first dissolving 5.0 mg of biotin (D-biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester; Boehringer Mannheim Cat. No. 1008 960) in 500 μl of dimethylsulfoxide to obtain a 10 mg solution. / ml. 10 µl of biotin was added per ml of rhTNFa (at 2.65 mg / ml) for a 2: 1 molar ratio between biotin and rhTNFa. The reaction was mixed gently and incubated for two hours at room temperature in the dark. A PD-10 column, Sephadex G-25M (Pharmacia Cat. No. 17-0851-01) was equilibrated with 25 ml of cold PBS and loaded with 2 ml of rhTNFa-biotin per column. The column was eluted with 10 x 1 ml of cold PBS. Fractions were collected and read at OD 280 (1.0 OD = 1.25 ml / ml). The appropriate fractions were pooled and stored at -80 ° C until use. Biotinylated rhTNFa is also commercially available (R&amp;D Systems No.

ES 2 198 552 T3 of catalog FTA00, Minneapolis, MN).

Biotinylated rhTNFa, to be immobilized on the matrix via streptavidin, was diluted with PBS assay buffer (Gibco Catalog No. 14190-144, Gibco BRL, Grand Island, NY) supplemented with 0.05% (BIAcore) of P20 surfactant (Pharmacia BR-1000-54, Pharmacia Biosensor, Piscataway, NJ). To determine the ability of rhTNFa-specific antibodies to bind immobilized rhTNFa, a binding assay was performed as follows. Aliquots of biotinylated rhTNFa (25 nM; 10 μό aliquots were injected through the streptavidin-coupled dextran matrix at a flow rate of 5 μ ^ ώ. Before protein injection and immediately thereafter, PBS buffer alone flowed through from each flow cell The net difference in signal between the initial value and approximately 30 seconds after completion of the injection and rhTNFa was taken to represent the binding value (approximately 500 RU). Direct binding of rhTNFa specific antibody to biotinylated rhTNFa was measured. Antibodies (20 µg / ml) were diluted in PBS and 25 µl aliquots were injected through the immobilized protein matrices at a flow rate of 5 µ ^ ώ. Before antibody injection, and immediately thereafter, PBS buffer alone flowed through each flow cell. The net difference in the initial signal and in the signal after completion of the antibody injection was taken to represent the binding value of the particular sample. Arrays of biosensors were generated using 100 mM HCl prior to injection of the next sample. To determine the deactivation rate (Koff), the activation rate (Kon), the association rate (Ka) and the dissociation rate (Kd), a kinetic evaluation software BIAcore (version 2.1) was used.

Representative results for D2E7 (full-length IgG4 antibody) binding to biotinylated rhTNFa, compared to mouse mAb MAK 195 (F (ab ') 2 fragment), are shown in Table 1 below.

TABLE 1

Binding of IgG4 D2E7 or MAK 195 to biotinylated rhTNFa

<td>Antibody</td><td>[Ab], nM</td><td>rhTNFa, United, UK</td><td>Ab, United, UK</td><td>rhTNFa / Ab</td><td>Koff, sec^-1, (average)</td>

<td></td><td></td><td></td><td></td><td></td><td></td>

<td>D2E7</td><td> 267</td><td> 373</td><td> 1215</td><td> 1,14</td><td>8.45 x 10^-5</td>

<td></td><td> 133</td><td> 420</td><td> 1569</td><td> 1,30</td><td>5.42 x 10^-5</td>

<td></td><td> 67</td><td> 434</td><td> 1633</td><td> 1,31</td><td>4.75 x 10^-5</td>

<td></td><td> 33</td><td> 450</td><td> 1532</td><td> 1,19</td><td>4.46 x 10^-5</td>

<td></td><td> 17</td><td> 460</td><td> 1296</td><td> 0,98</td><td>3.47 x 10^-5</td>

<td></td><td> 8</td><td> 486</td><td> 936</td><td> 0,67</td><td>2.63 x 10^-5</td>

<td></td><td> 4</td><td> 489</td><td> 536</td><td> 0,38</td><td>2.17 x 10^-5</td>

<td></td><td> 2</td><td> 470</td><td> 244</td><td> 0,18</td><td>3.68 x 10^-5</td>

<td></td><td></td><td></td><td></td><td></td><td>(4.38 x 10^-5)</td>

<td></td><td></td><td></td><td></td><td></td><td></td>

<td>MAK 195</td><td> 400</td><td> 375</td><td> 881</td><td> 1,20</td><td>5.38 x 10^-5</td>

<td></td><td> 200</td><td> 400</td><td> 1080</td><td> 1,38</td><td>4.54 x 10^-5</td>

<td></td><td> 100</td><td> 419</td><td> 1141</td><td> 1,39</td><td>3.54 x 10^-5</td>

<td></td><td> 50</td><td> 427</td><td> 1106</td><td> 1,32</td><td>3.67 x 10^-5</td>

ES 2 198 552 T3

TABLE 1 (continued)

<td>Antibody</td><td>[Ab], nM</td><td>rhTNFa, United, UK</td><td>Ab, United, UK</td><td>rhTNFa / Ab</td><td>Koff, sec^-1, (average)</td>

<td></td><td> 25</td><td> 446</td><td> 957</td><td> 1,09</td><td>4.41 x 10^-5</td>

<td></td><td> 13</td><td> 464</td><td> 708</td><td> 0,78</td><td>3.66 x 10^-5</td>

<td></td><td> 6</td><td> 474</td><td> 433</td><td> 0,47</td><td>7.37 x 10^-5</td>

<td></td><td> 3</td><td> 451</td><td> 231</td><td> 0,26</td><td>6.95 x 10^-5</td>

<td></td><td></td><td></td><td></td><td></td><td>(4.94 x 10^-5)</td>

In a second series of experiments, the molecular kinetic interactions between a full-length form of D2E7 IgG1 and biotinylated rhTNFa were quantitatively analyzed, using BIAcore technology, as described above, and the kinetic rate constants, summarized further, were obtained. below in Tables 2, 3 and 4.

TABLE 2

Apparent dissociation rate constants of the interaction between D2E7 and biotinylated rhTNFa

<td>Experiment</td><td>Kd (s^-1)</td>

<td> 1</td><td>9.58 x 10^-5</td>

<td> 2</td><td>9.26 x 10^-5</td>

<td> 3</td><td>7.60 x 10^-5</td>

<td>Average</td><td>8.81 ± 1.06x 10^-5</td>

TABLE 3

Rate constants of apparent association of the interaction between D2E7 and biotinylated rhTNFa

<td>Experiment</td><td>Ka (M^-1, s^-1)</td>

<td> 1</td><td>1.33 x 10⁵</td>

<td> 2</td><td>1.05 x 10⁵</td>

<td> 3</td><td>3.36 x 10⁵</td>

<td>Average</td><td>1.91 ± 1.26x 10⁵</td>

TABLE 4

Apparent kinetic speed and activity constants of biotinylated D2E7 and rhTNF

<td>Experiment</td><td>Ka (M^-1, s^-1)</td><td>Kd (s^-1)</td><td>Kd (M)</td>

<td> 1</td><td>1.33 x 10⁵</td><td>9.58 x 10^-5</td><td>7.20 x 10^-10</td>

<td> 2</td><td>1.05 x 10⁵</td><td>9.26 x 10^-5</td><td>8.82 x 10^-10</td>

<td> 3</td><td>3.36 x 10⁵</td><td>7.60 x 10^-5</td><td>2.26 x 10^-10</td>

<td>Average</td><td>1.91 ± 1.26x 10⁵</td><td>8.81 ± 1.06x 10^-5</td><td>6.09 ± 3.42 x 10^-10</td>

ES 2 198 552 T3

The dissociation and association rate constants were calculated by analyzing the dissociation and association regions of the sensorgrams by a BIA analysis software. Standard chemical reaction kinetics were assumed for the interaction between D2E7 and the biotinylated rhTNF molecule: a zero order dissociation and a first order association kinetics. For the analysis, the interaction between only one arm of the bivalent D2E7 antibody and one unit of the trimeric biotinylated rhTNFa was considered. In the choice of molecular models for the analysis of kinetic data. Three independent experiments were performed and the results were analyzed separately. The mean apparent dissociation rate constant (kd) of the interaction between D2E7 and biotinylated rhTNFa was 8.81 ± 1.06 x 10^-5 s^-1, and the mean apparent association rate constant, ka, was 1.91 ± 1.26 x 10⁵ M^-1 s^-1. The apparent intrinsic dissociation constant (Kd) was then calculated by the formula: Kd = kd / ka. Thus, the mean Kd of the D2E7 antibody to rhTNF obtained from the kinetic parameters was 6.09 ± 3.42 x 10^-10 M. Minor differences in kinetic values ​​for the IgG1 form of D2E7 (presented in Tables 2, 3 and 4) and the IgG4 form of D2E7 (presented in Table 1 and in Examples 2 and 3) are not consider true differences resulting from the presence of none of the IgG1 or IgG4 constant regions, but rather are considered attributable to the more precise antibody concentration measurements used for IgG1 kinetic analysis. Accordingly, the kinetic values ​​for the IgG1 form of D2E7 presented herein are considered the most accurate kinetic parameters for the D2E7 antibody.

Example 2

Alanine scanning mutagenesis of D2E7 CDR3 domains

A series of individual alanine mutations were introduced by standard methods along the CDR3 domain of the D2E7 VL and D2E7 VH regions. Light chain mutations are illustrated in Figure 1B (LD2E7 * .A1, LD2E7 * .A3, LD2E7 * .A4, LD2E7 * .A5, LD2E7 * .A7, and LD2E7 * .A8, which had an alanine mutation in position 1, 3, 4, 5, 7 or 8, respectively, of the VL CDR3 domain of D2E7). Heavy chain mutations are illustrated in Figure 2B (HD2E7 * .A1, HD2E7 * .A2, HD2E7 * .A3, HD2E7 * .A4, HD2E7 * .A5, HD2E7 * .A6, HD2E7 * .A7, and HD2E7 *. A9, which have an alanine mutation at position 2, 3, 4, 5, 6, 8, 9, 10, or 11, respectively, of the VH CDR3 domain of D2E7). The kinetics of the interaction of rhTNFa with an antibody composed of wild-type D2E7 VL and VH was compared to that of antibodies composed of 1) a wild-type D2E7 VL paired with alanine-substituted D2E7 VH; 2) a wild-type D2E7 VH paired with alanine substituted D2E7 VL; or 3) an alanine substituted D2E7 VL paired with a alanine substituted D2E7 VH. All antibodies were tested as full length IgG4 molecules.

The kinetics of antibody interaction with rhTNFa was determined by surface plasmon resonance as described in Example 1. The Koff rates for the different VH / VL pairs are summarized below in Table 5.

TABLE 5

Binding of D2E7 alanine scanning mutants to biotinylated rhTNFa

<td>VH</td><td>VL</td><td>Koff (sec^-1)</td>

<td>D2E7 VH</td><td>D2E7 VL</td><td>9.65 x 10^-5</td>

<td></td><td></td><td></td>

<td>HD2E7 * .A1</td><td>D2E7 VL</td><td>1.4 x 10^-4</td>

<td>HD2E7 * .A2</td><td>D2E7 VL</td><td>4.6 x 10^-4</td>

<td>HD2E7 * .A3</td><td>D2E7 VL</td><td>8.15 x 10^-4</td>

<td>HD2E7 * .A4</td><td>D2E7 VL</td><td>1.8 x 10^-4</td>

<td>HD2E7 * .A5</td><td>D2E7 VL</td><td>2.35 x 10^-4</td>

<td>HD2E7 * .A6</td><td>D2E7 VL</td><td>2.9 x 10^-4</td>

<td>HD2E7 * .A7</td><td>D2E7 VL</td><td>1.0 x 10^-4</td>

<td>HD2E7 * .A8</td><td>D2E7 VL</td><td>3.1 x 10^-4</td>

<td>HD2E7 * .A9</td><td>D2E7 VL</td><td>8.1 x 10^-4</td>

ES 2 198 552 T3

TABLE 5 (continued)

<td>VH</td><td>VL</td><td>Koff (sec^-1)</td>

<td>D2E7 VH</td><td>LD2E7 * .A1</td><td>6.6 x 10^-5</td>

<td>D2E7 VH</td><td>LD2E7 * .A3</td><td>NOT DETECTABLE</td>

<td>D2E7 VH</td><td>LD2E7 * .A4</td><td>1.75 x 10^-4</td>

<td>D2E7 VH</td><td>LD2E7 * .A5</td><td>1.8 x 10^-4</td>

<td>D2E7 VH</td><td>LD2E7 * .A7</td><td>1.4 x 10^-4</td>

<td>D2E7 VH</td><td>LD2E7 * .A8</td><td>3.65 x 10^-4</td>

<td></td><td></td><td></td>

<td>HD2E7 * .A9</td><td>LD2E7 * .A1</td><td>1.05 x 10^-4</td>

These results demonstrate that most of the positions of the CDR3 domains of the VL region and the VH region of D2E7 are capable of substitution with a single alanine residue. Substitution of a single alanine residue at position 1, 4, 5, or 7 of the CDR3 domain of VL of D2E7 or at position 2, 5, 6, 8, 9, or 10 of the CDR3 domain of VH of D2E7 does not significantly affect to the rate of inactivation of hTNFa binding compared to the parental wild-type D2E7 antibody. Alanine substitution at position 8 of VL CDR3 of D2E7 or at position 3 of D2E7 VH CDR3 provides 4-fold faster Koff and alanine substitution at position 4 or 11 of VH CDR3 of D2E7 provides 8 times faster Koff, indicating that these positions are more critical for binding to hTNFa. However, a single alanine substitution at position 1, 4, 5, 7, or 8 of the VL CDR3 domain of D2E7 or at position 2, 3, 4, 5, 6, 8, 9, 10, or 11 of the domain D2E7 VH CDR3 results in an anti-hTNFa antibody having a Koff of 1 x 10^-3 sec^-1 or less.

Example 3

D2E7-related antibody binding assay

A series of antibodies related in sequence to D2E7 were analyzed for their binding to rhTNFa, compared to D2E7, by surface plasmon resonance as described in Example 1. The amino acid sequences of the VL regions tested are shown in the Figures 1A and 1B. The amino acid sequences of the VH regions tested are shown in Figures 2A and 2B. The Koff rates for various VH / VL pairs (in the indicated format, as full-length IgG1 or IgG4 antibody or as a scFv) are summarized below in Table 6.

TABLE 6

Binding of D2E7-related antibodies to biotinylated rhTNFa

<td>VH</td><td>VL</td><td>Format</td><td>Koff (sec^-1)</td>

<td>D2E7 VH</td><td>D2E7 VL</td><td>IgG1 / IgG4</td><td>9.65 x 10^-5</td>

<td>VH1-D2</td><td>LOE7</td><td>IgG1 / IgG4</td><td>7.7 x 10^-5</td>

<td>VH1-D2</td><td>LOE7</td><td>scFv</td><td>4.6 x 10^-4</td>

<td>VH1-D2.N</td><td>LOE7.T</td><td>IgG4</td><td>2.1 x 10^-5</td>

<td>VH1-D2.Y</td><td>LOE7.A</td><td>IgG4</td><td>2.7 x 10^-5</td>

<td>VH1-D2.N</td><td>LOE7.A</td><td>IgG4</td><td>3.2 x 10^-5</td>

ES 2 198 552 T3

TABLE 6 (continued)

<td>VH</td><td>VL</td><td>Format</td><td>Koff (sec^-1)</td>

<td>VH1-D2</td><td>EPB12</td><td>scFv</td><td>8.0 x 10^-4</td>

<td>VH1-D2</td><td>2SD4 VL</td><td>scFv</td><td>1.94 x 10^-3</td>

<td>3C-H2</td><td>LOE7</td><td>scFv</td><td>1.5 x 10^-3</td>

<td>2SD4 VH</td><td>LOE7</td><td>scFv</td><td>6.07 x 10^-3</td>

<td>2SD4 VH</td><td>2SD4 VL</td><td>scFv</td><td>1.37 x 10^-2</td>

<td>VH1A11</td><td>2SD4 VL</td><td>scFv</td><td>1.34 x 10^-2</td>

<td>VH1B12</td><td>2SD4 VL</td><td>scFv</td><td>1.01 x 10^-2</td>

<td>VH1B11</td><td>2SD4 VL</td><td>scFv</td><td>9.8 x 10^-3</td>

<td>VH1E4</td><td>2SD4 VL</td><td>scFv</td><td>1.59 x 10^-2</td>

<td>VH1F6</td><td>2SD4 VL</td><td>scFv</td><td>2.29 x 10^-2</td>

<td>VH1D8</td><td>2SD4 VL</td><td>scFv</td><td>9.5 x 10^-3</td>

<td>VH1G1</td><td>2SD4 VL</td><td>scFv</td><td>2.14 x 10^-2</td>

<td>2SD4 VH</td><td>EPB12</td><td>scFv</td><td>6.7 x 10^-3</td>

<td>2SD4 VH</td><td>VL10E4</td><td>scFv</td><td>9.6 x 10^-3</td>

<td>2SD4 VH</td><td>VL100A9</td><td>scFv</td><td>1.33 x 10^-2</td>

<td>2SD4 VH</td><td>VL100D2</td><td>scFv</td><td>1.41 x 10^-2</td>

<td>2SD4 VH</td><td>VL10F4</td><td>scFv</td><td>1.11 x 10^-2</td>

<td>2SD4 VH</td><td>VLLOE5</td><td>scFv</td><td>1.16 x 10^-2</td>

<td>2SD4 VH</td><td>VLLOF9</td><td>scFv</td><td>6.09 x 10^-3</td>

<td>2SD4 VH</td><td>VLLOF10</td><td>scFv</td><td>1.34 x 10^-2</td>

<td>2SD4 VH</td><td>VLLOG7</td><td>scFv</td><td>1.56 x 10^-2</td>

<td>2SD4 VH</td><td>VLLOG9</td><td>scFv</td><td>1.46 x 10^-2</td>

<td>2SD4 VH</td><td>VLLOH1</td><td>scFv</td><td>1.17 x 10^-2</td>

<td>2SD4 VH</td><td>VLLOH10</td><td>scFv</td><td>1.12 x 10^-2</td>

<td>2SD4 VH</td><td>VL1B7</td><td>scFv</td><td>1.3 x 10^-2</td>

<td>2SD4 VH</td><td>VL1C1</td><td>scFv</td><td>1.36 x 10^-2</td>

<td>2SD4 VH</td><td>VL1C7</td><td>scFv</td><td>2.0 x 10^-2</td>

<td>2SD4 VH</td><td>VL0.1F4</td><td>scFv</td><td>1.76 x 10^-2</td>

<td>2SD4 VH</td><td>VL0.1H8</td><td>scFv</td><td>1.14 x 10^-2</td>

ES 2 198 552 T3

The slow inactivation rates (i.e., K_off &lt;1 x 10^-4 sec^-1) for full-length antibodies (i.e., IgG format) that had a VL selected from D2E7, LOE7, LOE7.T, and LOE7.A, that have a threonine or alanine at position 9, indicate that position 9 of the VL CDR3 of D2E7 can be occupied by either of these two residues without substantially affecting Koff. Accordingly, a consensus motif for D2E7 VL CDR3 comprises the amino acid sequence: QRYNRAPY- (T / A) (SEQ ID NO: 3). Also, the slow inactivation rates (i.e., Koff &lt;1 x 10^-4 sec^-1) for antibodies that have a VH selected from D2E7, VH1-D2.N and VY1-D2.Y, that have a tyrosine or an asparagine at position 12, indicate that position 12 of the VH CDR3 of D2E7 can be occupied by either of these two remains without substantially affecting the Koff. Accordingly, a consensus motif for D2E7 VH CDR3 comprises the amino acid sequence: V-SY-LSTASSLD- (Y / N) (SEQ ID NO: 4).

The results shown in Table 6 demonstrate that, in the scFv format, antibodies containing the CDR3 region of VL or VH of 2SD4 exhibit a faster Koff (i.e., Koff &lt;1 x 10^-3 sec^-1) compared to antibodies containing the CDR3 region of VL or VH of D2E7. Within the CDR3 of VL, 2SD4 differs from D2E7 at positions 2, 5, and 9. As described above, however, position 9 can be occupied by Ala (as in 2SD4) or Thr (as in D2E7) without affecting substantially a la Koff. Thus, by comparing 2SD4 and D2E7, positions 2 and 5 of the VL CDR3 of D2E7, both arginines, can be identified as critical positions for the association of the antibody with hTNFa. These residues could be directly implicated as contact residues at the antibody binding site or could critically contribute to maintaining the scaffold architecture of the antibody molecule in this region. Regarding the importance of position 2, the replacement of Arg (in LOE7, which has the same VL CDR3 as D2E7) by Lys (in EP B12) accelerates the rate of inactivation by a factor of two. Regarding the importance of position 5, the replacement of Arg (in D2E7) by Ala (in LD2E7 * .A5), as described in Example 2, also accelerates the rate of inactivation twice. Also, without Arg at positions 2 and 5 (at 2SD4), the rate of inactivation is five times faster. However, it should be noted that although position 5 is important for enhancing hTNFa binding, a change in this position can be negated by changes in other positions, as seen in VLLOE4, VLLOH1 or VL0.1H8.

Within the CDR3 of VH, 2SD4 differs from D2E7 in positions 1,7 and 12. However, as previously described, position 12 can be filled by Asn (as in 2SD4) or Tyr (as in D2E7) without substantially affecting Koff. Thus, by comparing 2SD4 and D2E7, positions 1 and 7 of the VH CDR3 of D2E7 can be identified as critical positions for hTNFa binding. As described above, these residues could be directly implicated as contact residues at the antibody binding site or could critically contribute to maintaining the scaffold architecture of the antibody molecule in this region. Both positions are important for hTNFa binding, since when using the 3C-H2 VH CDR3 (which has a valine to alanine change at position 1 relative to the D2E7 VH CDR3), the scFv has a rate of inactivation 3 times faster than when using the VH CDR3 of D2E7, but this rate of inactivation is still four times slower than when using the VH CDR3 of 2SD4 (which has changes in positions 1 and 7 relative to the VH CDR3 of D2E7).

Example 4

D2E7 Functional Activity

To examine the functional activity of D2E7, the antibody was used in various assays that measure the ability of the antibody to inhibit hTNFa activity, in vitro or in vivo.

A. Neutralization of TNFa-induced cytotoxicity in L929 cells

Recombinant human TNFa (rhTNFa) produces cellular cytotoxicity in murine L929 cells after an incubation period of 18-24 hours. Human anti-hTNFa antibodies were evaluated in L929 assays by co-incubation of antibodies with rhTNFa and the cells listed below. A 96-well microtiter plate containing 100 µl of anti-hTNFa Ab was serially diluted 1/3 in duplicate using RPMI medium containing 10% fetal calf serum (FBS). 50 µl of rhTNFa was added for a final concentration of 500 pg / ml in each sample well. The plates were then incubated for 30 minutes at room temperature. Next, 50 μl of TNFα-sensitive L929 mouse fibroblast cells were added for a final concentration of 5 x 10⁴ cells per well, including 1 µg / ml Actinomycin-D. Controls included medium plus cells and rhTNFa plus cells. These controls, and a standard curve for TNFa, ranging from 2 ng / ml to 8.2 pg / ml, were used to determine the quality of the assay and provide a window of neutralization. The plates were then incubated overnight (18-24 hours) at 37 ° C in CO₂ at 5%.

100 μl of medium was removed from each well and 50 μl of 3- (4,4-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT; commercially available from Sigma Chemical Co., St. Louis, MO) at a concentration of 5 mg / ml in PBS. The plates were then incubated for 4 hours at 37 ° C. Then 50 µl of 20% sodium dodecyl sulfate (SDS) was added to each well and the plates were incubated overnight at 37 ° C. Optical density at 570/630 nm was measured, curves were plotted for each sample, and IC50 values ​​were determined by conventional methods.

ES 2 198 552 T3

Representative results for human antibodies having various VL and VH pairs are shown in Figure 3 and Table 7 below, compared to the murine mAb MAK 195.

TABLE 7

Neutralization of TNFa-induced L929 cytotoxicity

<td>VH</td><td>VL</td><td>Structure</td><td>IC50, M</td>

<td>D2E7</td><td>D2E7</td><td>scFv</td><td>1.1 x 10^-10</td>

<td>D2E7</td><td>D2E7</td><td>IgG4</td><td>4.7 x 10^-11</td>

<td>2SD4</td><td>2SD4</td><td>scFv / IgG1 / IgG4</td><td>3.0 x 10^-7</td>

<td>2SD4</td><td>LOE7</td><td>scFv</td><td>4.3 x 10^-8</td>

<td>VH1-D2</td><td>2SD4</td><td>scFv</td><td>1.0 x 10^-8</td>

<td>VH1-D2</td><td>LOE7</td><td>scFv / IgG1 / IgG4</td><td>3.4 x 10^-10</td>

<td>VH1-D2.Y</td><td>LOE7.T</td><td>IgG4</td><td>8.1 x 10^-11</td>

<td>VH1-D2.N</td><td>LOE7.T</td><td>IgG4</td><td>1.3 x 10^-10</td>

<td>VH1-D2.Y</td><td>LOE7.A</td><td>IgG4</td><td>2.8 x 10^-11</td>

<td>VH1-D2.N</td><td>LOE7.A</td><td>IgG4</td><td>6.2 x 10^-11</td>

<td>MAK 195</td><td>MAK 195</td><td>scFv</td><td>1.9 x 10^-8</td>

<td>MAK 195</td><td>MAK 195</td><td>F (ab ') 2</td><td>6.2 x 10^-11</td>

The results in Figure 3 and Table 7 demonstrate that the human anti-hTNFa antibody D2E7, and various D2E7-related antibodies, neutralize the TNFa-induced cytotoxicity of L929 with an ability roughly equivalent to that of the anti-hTNFa mAb MAK 195.

In another series of experiments, the ability of the IgG1 form of D2E7 to neutralize TNFα-induced cytotoxicity of L929 was examined as described above. The results of three independent experiments, and their average, are summarized below in Table 8:

TABLE 8

Neutralization of TNFa-induced cytotoxicity of L929 by D2E7 IgG1

<td>Experiment</td><td>IC50 [M]</td>

<td> 1</td><td>1.26 x 10^-10</td>

<td> 2</td><td>1.33 x 10^-10</td>

<td> 3</td><td>1.15 x 10^-10</td>

<td>Average</td><td>1.25 ± 0.01 x 10^-10</td>

This series of experiments confirmed that D2E7, in the form of full-length IgG1, neutralizes the cytotoxicity of L929 induced by TNFa with a mean IC5o [M] of 1.5 ± 0.01 x 10^-1o.

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B. Inhibition of TNFa Binding to TNFa Receptors in U-937 Cells

The ability of human anti-hTNFa antibodies to inhibit the binding of hTNFa to hTNFa receptors on the surface of cells was examined using the U-937 cell line (ATCC No. CRL 1593), a human histiocyte line that expresses receptors for hTNFa. U-937 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (HycloneA-1111, Hyclone Laboratories, Logan, UT), L-glutamine (4 nM), HEPES buffer (10 mM), penicillin ( 100 μg / ml) and streptomycin (100 μg / ml). To examine the activity of full-length IgG antibodies, U-937 cells were preincubated with PBS supplemented with 1 mg / ml human IgG (Sigma I-4506, Sigma Chemical Co., St. Louis, MO) for 45 minutes on ice and then the cells were washed 3 times with binding buffer. For the receptor binding assay, U-937 cells (5 x 10⁶ cells / well) were incubated in binding buffer (PBS supplemented with 0.2% bovine serum albumin) in 96-well microtiter plates (Costar 3799, Costar Corp., Cambridge, Ma) along with rhTNFa labeled with ¹²⁵I (3 x 10^-10 M; 25 µCi / ml; obtained from NEN Research Products, Wilmington, De), with or without anti-hTNFa antibodies, in a total volume of 0.2 ml. The plates were incubated on ice for 1.5 hours. Then, 75 μl of each sample was transferred to 1.0 ml test tubes (Sarstedt 72,700, Sarstedt Corp., Princeton, NJ) containing dibutyl phthalate (Sigma D-2270, Sigma Chemical Co., St. Louis, MO) and dinonyl phthalate (ICN 210733, ICN, Irvine, CA). The test tubes contained a 300 µl mixture of dibutyl phthalate and dinonyl phthalate, in a 2: 1 volume ratio, respectively. The rhTNFa marked with¹²⁵Free I (ie, unbound) was removed by microcentrifugation for 5 minutes. Then, the end of each test tube containing a cell pellet was cut off with the aid of a microtube scissors (Bel-Art 210180001, Bel-Art Products, Pequannock, NJ). The cell pellet contains rhTNFa labeled with¹²⁵I bound to the TNFa receptor p60 or p80, while the aqueous phase above the oil mixture contains an excess of rhTNFa labeled with ¹²⁵I free. All cell pellets were collected in a counting tube (Falcon 2052, Becton Dickinson Labware, Lincoln Park, NJ) and counted in a scintillation counter.

Representative results are shown in Figure 4. The IC50 value for D2E7 inhibition of hTNFa binding to hTNFa receptors in U-937 cells is approximately 3 x 10^-10 M in these experiments. These results demonstrate that the anti-human hTNFa antibody D2E7 inhibits the binding of rhTNFa to hTNFa receptors on U-937 cells at concentrations approximately equivalent to those of the anti-hTNFa mAb MAK 195.

In another series of experiments, the ability of the IgG1 form of D2E7 to inhibit the binding of rhTNFa to hTNFa receptors in U-937 cells was examined as described above. The results of three independent experiments, and the average of them, are summarized below in Table 9.

TABLE 9

Inhibition of TNF receptor binding in U-937 cells by IgG1 D2E7

<td>Experiment</td><td>IC50 [M]</td>

<td> 1</td><td>1.70 x 10^-10</td>

<td> 2</td><td>1.49 x 10^-10</td>

<td> 3</td><td>1.50 x 10^-10</td>

<td>Average</td><td>1.56 ± 0.12x 10^-10</td>

This series of experiments confirmed that D2E7, in the form of full-length IgG1, inhibits binding to the TNF receptor in U-937 cells with a mean IC50 [M] of 1.56 ± 0.12 x 10^-10.

To investigate the inhibitory potency of D2E7 on the binding of ¹²⁵I-rhTNF to individual p55 and p75 receptors, a solid phase radioimmunoassay was performed. To measure D2E7 IC50 values ​​for different TNF receptors, varying concentrations of the antibody were incubated at a concentration of 3 x 10^-10 from ¹²⁵I-rhTNF. The mixture was then assayed on separate plates containing the p55 or p75 TNF receptors in a dose dependent manner. The results are summarized below in Table 10.

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TABLE 10

Inhibition of TNF receptor binding to TNFR p55 and p75 by IgG1 D2E7

<td></td><td colspan="2">IC50 [M]</td>

<td>Reagent</td><td>p55 TNFR</td><td>p 75TNFR</td>

<td>D2E7</td><td>1.47 x 10^-9</td><td>1.26 x 10^-9</td>

<td>rhTNF</td><td>2.31 x 10^-9</td><td>2.70 x 10^-9</td>

Inhibition of the binding of ¹²⁵I-rhTNF to p55 and p75 TNF receptors in U937 cells by D2E7 followed a simple sigmoid curve, indicating similar IC50 values ​​for each receptor. In solid phase radioimmunoassay (RIA) experiments with recombinant TNF receptors, IC50 values ​​were calculated for inhibition of binding of¹²⁵I-rhTNF to p55 and p75 receptors by D2E7 of 1.47 x 10^-9 and 1.26 x 10^-9 M, respectively. The reduction in IC50 values ​​in the solid phase was probably due to the higher density of receptors in the RIA format, since unlabeled rhTNFa also inhibited with similar IC50 values. IC50 values ​​for inhibition of the binding of¹²⁵I-rhTNF to receptors p55 and p75 by unlabeled rhTNF were 2.31 x 10^-9 and 2.70 x 10^-9 M, respectively.

C. Inhibition of ELAM-1 expression in HUVEC

Human umbilical vein endothelial cells (HUVEC) can be induced to express endothelial cell leukocyte adhesion molecule 1 (ELAM-1) on their surface by rhTNFa treatment, which can be detected by reacting rhTNFa-treated HUVEC with an antibody to mouse anti-human HELAM-1. The ability of human anti-hTNFa antibodies to inhibit this TNFa-induced expression of ELAM-1 in HUVEC was examined as follows: HUVEC (ATCC No. CRL 1730) were cultured in 96-well plates (5 x 10⁴ cells / well) and incubated overnight at 37 ° C. The next day, serial dilutions of human anti-hTNFa antibody (1:10) were prepared in a microtiter plate, starting with 20-100 µg / ml of antibody. A stock solution of rhTNFa was prepared at 4.5 ng / ml, aliquots of rhTNFa were added to each well containing antibody, and the contents were mixed well. Controls included medium alone, medium plus antihTNFa antibody, and medium plus rhTNFa. The HUVEC plates were removed from their overnight incubation at 37 ° C and the medium was gently aspirated from each well. 200 µl of the antibody-rhTNFa mixture was transferred to each well of the HUVEC plates. The HUVEC plates were then further incubated at 37 ° C for 4 hours. Next, a solution of murine anti-ELAM-1 antibody was diluted 1: 1000 in RPMI. The medium was gently aspirated from each well of the HUVEC plate, 50 µl / well of the anti-ELAM-1 antibody solution was added, and the HUVEC plates were incubated for 60 minutes at room temperature. A solution of labeled anti-mouse Ig antibody was prepared.¹²⁵I in RPMI (approximately 50,000 cpm in 50 μΕ The medium was gently aspirated from each well of the HUVEC plates, the wells were washed twice with RPMI and 50 μl of the anti-mouse Ig solution labeled with ¹²⁵I. Plates were incubated for 1 hour at room temperature and then each well was washed 3 times with RPMI. One hundred and eighty microliters of 5% SDS was added to each well to lyse the cells. The cell lysate from each well was then transferred to a tube and counted in a scintillation counter.

Representative results are shown in Figure 5. The IC50 value for D2E7 inhibition of hTNFa-induced ELAM-1 expression in HUVEC is approximately 6 x 10^-11 M in these experiments. These results demonstrate that the anti-human hTNFa antibody D2D7 inhibits hTNFa-induced ELAM-1 expression in HUVEC at concentrations approximately equivalent to those of the murine anti-hTNFa mAb MAK 195.

In another series of experiments, the ability of the IgG1 form of D2E7 to inhibit hTNFα-induced expression of ELAM-1 in HUVEC was examined as described above. The results of three independent experiments, and their average, are summarized below in Table 11

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TABLE 11

Inhibition of TNFa-induced ELAM-1 expression by the IgG1 D2E7 receptor

<td>Experiment</td><td>IC50 [M]</td>

<td> 1</td><td>1.95 x 10^-10</td>

<td> 2</td><td>1.69 x 10^-10</td>

<td> 3</td><td>1.90 x 10^-10</td>

<td>Average</td><td>1.85 ± 0.14x 10^-10</td>

This series of experiments confirmed that D2E7, in the form of full-length IgG1, inhibits the expression of ELAM-1 induced by TNFa in HUVEC with a mean IC50 [M] of 1.85 ± 0.14 x 10^-10.

The neutralizing potency of IgG1 D2E7 for the expression of two other adhesion molecules, ICAM-1 and VCAM-1 induced by rhTNF, was also examined. Since the rhTNF trituration curve for ICAM-1 expression at 16 hours was very similar to the ELAM-1 expression curve, the same concentration of rhTNF was used in the antibody neutralization experiments. HUVECs were incubated with rhTNF in the presence of varying concentrations of D2E7 in a CO incubator.₂ at 37 ° C for 16 hours, and the expression of ICAM-1 was measured by mouse anti-ICAM-1 antibody followed by sheep anti-mouse labeled with ¹²⁵I. Two independent experiments were performed and IC50 values ​​were calculated. An unrelated human IgG1 antibody did not inhibit ICAM-1 expression.

The experimental procedure for testing inhibition of VCAM-1 expression was the same as the procedure for ELAM-1 expression, with the exception that the anti-VCAM-1 mAb was used in place of the anti-ELAM-1 mAb. 1. Three independent experiments were performed and IC50 values ​​were calculated. An unrelated human IgG1 antibody did not inhibit VCAM-1 expression.

The results are summarized below in Table 12.

TABLE 12

Inhibition of ICAM-1 and VCAM-1 expression by IgG1 D2E7

<td colspan="2">Inhibition of ICAM-1</td><td colspan="2">IC50 [M]</td>

<td>Experiment</td><td>IC50 [M]</td><td>Experiment</td><td>IC50 [M]</td>

<td> 1</td><td>1.84 x 10^-10</td><td> 1</td><td>1.03 x 10^-10</td>

<td> 2</td><td>2.49 x 10^-10</td><td> 2</td><td>9.26 x 10^-11</td>

<td></td><td></td><td> 3</td><td>1.06 x 10^-10</td>

<td>Average</td><td>2.17 ± 0.46x 10^-10</td><td>Average</td><td>1.01 ± 0.01 x 10^-10</td>

These experiments demonstrate that treatment of primary human endothelial vein endothelial cells with rhTNF led to optimal expression of adhesion molecules: ELAM-1 and VCAM-1 at four hours, and maximum positively regulated expression of ICAM-1 at 16 hours. D2E7 was able to inhibit the expression of all three adhesion molecules in a dose-dependent manner. IC50 values ​​for inhibition of ELAM-1, ICAM-1 and VCAM-1 were 1.85 x 10^-10, 2.17 x 10^-10 and 1.01 x 10^-10, respectively. These values ​​are very similar, indicating similar requirements for the dose of the rhTNF activation signal to induce the expression of ELAM-1, ICAM-1 and VCAM-1. Interestingly, D2E7 was similarly effective in the assay for major inhibition of ICAM-1 expression. The ICAM-1 inhibition assay required 16 hours of incubation of rhTNF and D2E7 with HUVEC instead of the 4 hours required for the ELAM-1 and VCAM-1 inhibition assays. As D2E7 has a slow inactivation rate for rhTNF, it is conceivable that during the 16 hour co-incubation period there was no significant competition for TNF receptors in HUVECs.

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D. In vivo neutralization of hTNFa

Three different in vivo systems were used to demonstrate that D2E7 is effective in inhibiting hTNFa in vivo.

I. Inhibition of TNF-induced lethality in mice sensitized with D-Galactosamine

Injection of recombinant human TNFa (rhTNFa) to D-galactosamine sensitized mice causes lethality within 24 hours. TNFa neutralizing agents have been shown to prevent lethality in this model. To examine the ability of human anti-hTNFa antibodies to neutralize hTNFa in vivo in this model, C57B1 / 6 mice were injected with varying concentrations of D2E7-IgG1 or a control protein in PBS intraperitoneally (ip). Mice were exposed 30 minutes later to 1 µg rhTNFa and 20 mg D-galactosamine in PBS ip, and observed 24 hours later. These amounts of rhTNFa and D-galactosamine, as previously determined, achieved a lethality of 80-90% in these mice.

Representative results are shown in Figure 6, represented as a bar graph of% survival versus antibody concentration. The black bars represent D2E7, while the hatched bars represent MAK 195. Injection of 2.5-25 µg of D2E7 antibody per mouse protected the animals from TNFα-induced lethality. The value of DE₅₀ it is approximately 1-2.5 µg / mouse. The positive control antibody, MAK 195, was similar in its protective ability. Injection of D2E7 in the absence of rhTNFa had no detrimental effect on mice. Injection of a non-specific human IgG1 antibody did not offer any protection from TNFα-induced lethality.

In a second experiment, forty-nine mice were divided into 7 equal groups. Each group received variable doses of D2E7 30 minutes before receiving a DL dose₈₀ of a mixture of rhTNF / D-galactosamine (1.0 µg of rhTNF and 20 mg of D-galactosamine per mouse). Control group 7 received the normal kappa IgG1 antibody at a dose of 25 µg / mouse. The mice were examined 24 hours later. The survival of each group is summarized below in Table 13.

TABLE 13

24-hour survival after D2E7 treatment

<td>Group</td><td>Survival (alive / total)</td><td>Survival (%)</td>

<td>1 (no antibody)</td><td> 0/7</td><td> 0</td>

<td>2 (1 Ag)</td><td> 1/7</td><td> 14</td>

<td>3 (2.6 μg)</td><td> 5/7</td><td> 71</td>

<td>4 (5.2 μg)</td><td> 6/7</td><td> 86</td>

<td>5 (26 μg)</td><td> 6/7</td><td> 86</td>

<td>6 (26 μg; no rhTNF)</td><td> 7/7</td><td> 100</td>

<td>7 (25 μg Hu IgG1)</td><td> 1/7</td><td> 14</td>

II. Inhibition of pyrexia in rabbits induced by TNF

The efficacy of D2E7 in inhibiting the rhTNF-induced pyrexia response in rabbits was examined. Groups of three female NZW rabbits weighing approximately 2.5 kg each were injected intravenously with D2E7, rhTNF, and immune complexes of D2E7 and rhTNF. Rectal temperatures were measured by thermistor probes on a Kaye temperature recorder every minute for approximately 4 hours. Recombinant human TNF in saline, injected at a dose of 5 µg / kg, induced a rise in temperature greater than 0.4 ° C at approximately 45 minutes after injection. The antibody preparation itself, in saline at a dose of 138 µg / kg, did not induce an increase in temperature in the rabbits until 140 minutes after administration. In all additional experiments, D2E7 or control reagents (human IgG1 or a saline vehicle) were injected iv to rabbits followed 15 minutes later by an injection of rhTNF in saline at 5 μg / kg iv. Representative results of various experiments are summarized in Table 14 below:

ES 2 198 552 T3

TABLE 14

Inhibition of rhTNF-induced pyrexia with D2E7 in rabbits

<td></td><td colspan="2">Temperature rise *, ° C</td><td></td><td>Relationship Cool</td><td>Temperature maximum</td>

<td>Dose of D2E7 Yg / kg)</td><td>rhTNF</td><td>rhTNF + D2E7</td><td>% from Inhib. **</td><td>D2E7: rhTNF</td><td>Minutes after rhTNF</td>

<td> 14</td><td> 0,53</td><td> 0,25</td><td> 53</td><td> 1</td><td> 60</td>

<td> 24</td><td> 0,43</td><td> 0,13</td><td> 70</td><td> 1,6</td><td> 40</td>

<td> 48</td><td> 0,53</td><td> 0,03</td><td> 94</td><td> 3,3</td><td> 50</td>

<td> 137</td><td> 0,53</td><td> 0,00</td><td> 100</td><td> 9,5</td><td> 60</td>

<td> 792</td><td> 0,80</td><td> 0,00</td><td> 100</td><td> 55</td><td> 60</td>

* = Maximum temperature ** =% inhibition = (1- {temperature increase with rhTNF and D2E7 / temperature increase with rhTNF alone}) x 100.

Intravenous pretreatment with D2E7 at a dose of 14 µg / kg partially inhibited the pyrogenic response, compared to rabbits pretreated with saline alone. D2E7 administered at 137 µg / kg totally suppressed the pyrogenic response of rhTNF in the same experiment. In a second experiment, D2E7 administered at 24 µg / kg also partially suppressed the pyrogenic response, compared to rabbits pretreated with saline alone. The molar ratio between D2E7 and rhTNF was 1/6: 1 in this experiment. In a third experiment, D2E7 injected iv at 48 μg / kg (D2E7: rhTNF molar ratio = 3.3: 1) totally suppressed the pyrogenic response compared to rabbits pretreated with control human IgG1 in saline at 30 μg / kg . In the final experiment, rabbits pretreated with D2E7 (792 μg / kg) at a very high ratio relative to rhTNF (55: 1) did not develop any increase in temperature at any time until 4 hours of observation. Treatment of rabbits with immune complexes generated from a mixture of D2E7 and rhTNF incubated at 37 ° C for 1 hour at a molar ratio of 55: 1, without the subsequent administration of rhTNF, also did not induce any increase in temperature in the same experiment.

III. Prevention of polyarthritis in Tg197 transgenic mice

The effect of D2E7 on disease development was investigated in a transgenic mouse model of arthritis. Transgenic mice (Tg197) expressing human wild-type TNF (modified in the 3 'region beyond the coding sequences) have been generated and these mice develop chronic polyarthritis with an incidence of 100% at 4-7 weeks of age (see EMBO J (1991) 10: 4025-4031 as a further description of the Tg197 model of polyarthritis).

Transgenic animals were identified by PCR at 3 days of age. Litters of transgenic mice were divided into six groups. Transgenic mice were verified by slot-blot hybridization analysis at 15 days of age. The treatment protocols for the six groups were as follows: Group 1 = no treatment; Group 2 = saline solution (vehicle); Group 3 = D2E7 at 1.5 µg / g; Group 4 = D2E7 at 15 Fg / g; Group 5 = D2E7 at 30 Fg / g; and Group 6 = IgG1 isotype control at 30 µg / g. A litter without transgenic mice was also included in the study to serve as a control (Group 7 - non-transgenic; no treatment). Each group received three ip injections per week of the indicated treatments. The injections were continued for 10 weeks. Every week, macroscopic changes in joint morphology were recorded for each animal. At 10 weeks, all mice were sacrificed and mouse tissue was harvested in formalin. A microscopic examination of the tissue was performed.

The weight in grams was taken for each mouse at the beginning of each week. At the same time joint size measurements (in mm) were also taken, as a measure of the severity of the disease. Joint size was established as an average of three measurements at the right ankle of the hind leg using a micrometer device. Arthritic scores were recorded weekly as follows: 0 = No arthritis (normal appearance and flexion); + = mild arthritis (distortion of the joint); ++ = moderate arthritis (swelling, joint deformity) and +++ = severe arthritis (ankylosis detected in flexion and severely impaired movement). Histopathological scores based on hematoxylin / eosin staining of sections of the joint were as follows; 0 = No detectable disease; 1 = proliferation of the synovial membrane; 2 = strong synovial thickening; 3 = destruction of cartilage and bone erosion.

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The effect of D2E7 treatment on the mean joint size of the Tg197 transgenic arthritic mouse is shown in the graph of Figure 9. The histopathological and arthritic scores of the Tg197 transgenic mouse, at 11 weeks of age, are summarized below in Table 15.

TABLE 15

Effect of D2E7 on histopathology and arthritic scoring in Tg197 mice:

<td>Group</td><td>Treatment</td><td>Punctuation Histopathological</td><td>Punctuation Arthritic</td>

<td> 1</td><td>none</td><td> 3 (7/70)</td><td> +++ (7/7)</td>

<td> 2</td><td>Saline solution</td><td> 3 (8/8)</td><td> +++ (8/8)</td>

<td> 6</td><td>IgG1 control</td><td> 3 (9/9)</td><td> +++ (7/9)</td>

<td> 3</td><td>D2E7 at 1.5 μg / g</td><td> 0 (6/8)</td><td> 0 (8/8)</td>

<td> 4</td><td>D2E7 at 15 μg / g</td><td> 0 (7/8)</td><td> 0 (8/8)</td>

<td> 5</td><td>D2E7 at 30 μg / g</td><td> 0 (8/8)</td><td> 0 (8/8)</td>

This experiment demonstrated that the D2E7 antibody has a definite beneficial effect on transgenic mice expressing wild-type human TNF (Tg197) with no apparent arthritis after the study period.

E. Neutralization of TNFa from other species by D2E7

The binding specificity of D2E7 was examined by measuring its ability to neutralize tumor necrosis factors from various primate and mouse species, using a L929 cytotoxicity assay (as described in Example 4, subsection A, above). The results are summarized in Table 16 presented below:

TABLE 16

Ability of D2E7 to neutralize TNF from different species in the L929 assay

<td>TNFa *</td><td>Fountain</td><td>CI₅₀ for the Neutralization by D2E7 (M) **</td>

<td>Human</td><td>Recombinant</td><td>7.8 x 10^-11</td>

<td>Chimpanzee</td><td>PBMC stimulated with LPS</td><td>5.5 x 10^-11</td>

<td>chuck</td><td>Recombinant</td><td>6.0 x 10^-11</td>

<td>marmoset</td><td>PBMC stimulated with LPS</td><td>4.0 x 10^-10</td>

<td>cynomolgus</td><td>PBMC stimulated with LPS</td><td>8.0 x 10^-11</td>

<td>rhesus</td><td>PBMC stimulated with LPS</td><td>3.0 x 10^-11</td>

<td>canine</td><td>WBC stimulated with LPS</td><td>2.2 x 10^-10</td>

<td>porcine</td><td>Recombinant</td><td>1.0 x 10^-7</td>

<td>murine</td><td>Recombinant</td><td>&gt; 1.0 x 10^-7</td>

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The results in Table 16 demonstrate that D2E7 can neutralize the activity of five primate TNFa roughly equivalent to human TNFa and, in addition, it can neutralize the activity of canine TNFa (approximately ten times worse than human TNFa) and porcine TNFa. and mouse (approximately ~ 1000 times worse than human TNFa). Furthermore, the binding of D2E7 to rhTNFa in solution phase was not inhibited by other cytokines, such as lymphotoxins (TNFe), IL-1a, IL-1e, IL-2, IL-4, IL-6, IL-8, IFNy and TGFe, indicating that D2E7 is highly specific for its ligand TNFa.

F. Absence of cytokine release by human whole blood incubated with D2E7

In this example, the ability of D2E7 to induce, by itself, normal human blood cells to secrete cytokines or surface molecules from the detached cell was examined. D2E7 was incubated with diluted whole blood from three different normal donors at varying concentrations for 24 hours. At the same time, a LPS positive control was processed, at a pre-determined concentration to stimulate immunocompetent blood cells to secrete cytokines. Supernatants were collected and tested in a panel of ten soluble cytokine, receptor and adhesion molecule ELISA kits: IL-1a, IL-1e, IL-1 receptor antagonist, IL-6, IL-8, TNFa, soluble TNF receptor 1, soluble TNF receptor 2, soluble ICAM-1 and soluble E-selectin. No significant amount of cytokine or detached cell surface molecules was measured as a result of co-incubation of D2E7 antibodies, at concentrations up to 343 µg / ml. Control cultures without the addition of the antibody also did not produce any measurable amount of cytokines, while the LPS co-culture control produced high values ​​in the range of many picograms to a few nanograms. These results indicate that D2E7 did not induce secretion of cell surface proteins or cytokines separated by whole blood cells above normal levels in ex vivo culture.

As part of this description, the attached Sequence List is presented, the content of which is summarized in the following table:

<td>SEQ ID NO:</td><td>CHAIN ​​OF ANTIBODY</td><td>REGION</td><td>KIND OF SEQUENCE</td>

<td> 1</td><td>D2E7</td><td>VL</td><td>amino acid</td>

<td> 2</td><td>D2E7</td><td>VH</td><td>amino acid</td>

<td> 3</td><td>D2E7</td><td>VL CDR3</td><td>amino acid</td>

<td> 4</td><td>D2E7</td><td>VH CDR3</td><td>amino acid</td>

<td> 5</td><td>D2E7</td><td>VL CDR2</td><td>amino acid</td>

<td> 6</td><td>D2E7</td><td>VH CDR2</td><td>amino acid</td>

<td> 7</td><td>D2E7</td><td>VL CDR1</td><td>amino acid</td>

<td> 8</td><td>D2E7</td><td>VH CDR1</td><td>amino acid</td>

<td> 9</td><td>2SD4</td><td>VL</td><td>amino acid</td>

<td> 10</td><td>2SD4</td><td>VH</td><td>amino acid</td>

<td> 11</td><td>2SD4</td><td>VL CDR3</td><td>amino acid</td>

<td> 12</td><td>EPB12</td><td>VL CDR3</td><td>amino acid</td>

<td> 13</td><td>VL10E4</td><td>VL CDR3</td><td>amino acid</td>

<td> 14</td><td>VL100A9</td><td>VL CDR3</td><td>amino acid</td>

<td> 15</td><td>VLL100D2</td><td>VL CDR3</td><td>amino acid</td>

ES 2 198 552 T3 (Continued)

<td>SEQ ID NO:</td><td>CHAIN ​​OF ANTIBODY</td><td>REGION</td><td>KIND OF SEQUENCE</td>

<td> 16</td><td>VLLOF4</td><td>VL CDR3</td><td>amino acid</td>

<td> 17</td><td>LOE5</td><td>VL CDR3</td><td>amino acid</td>

<td> 18</td><td>VLLOG7</td><td>VL CDR3</td><td>amino acid</td>

<td> 19</td><td>VLLOG9</td><td>VL CDR3</td><td>amino acid</td>

<td> 20</td><td>VLLOH1</td><td>VL CDR3</td><td>amino acid</td>

<td> 21</td><td>VLLOH10</td><td>VL CDR3</td><td>amino acid</td>

<td> 22</td><td>VL1B7</td><td>VL CDR3</td><td>amino acid</td>

<td> 23</td><td>VL1C1</td><td>VL CDR3</td><td>amino acid</td>

<td> 24</td><td>VL0.1F4</td><td>VL CDR3</td><td>amino acid</td>

<td> 25</td><td>VL0.1H8</td><td>VL CDR3</td><td>amino acid</td>

<td> 26</td><td>LOE7 .A</td><td>VL CDR3</td><td>amino acid</td>

<td> 27</td><td>2SD4</td><td>VL CDR3</td><td>amino acid</td>

<td> 28</td><td>VH1B11</td><td>VL CDR3</td><td>amino acid</td>

<td> 29</td><td>VH1D8</td><td>VL CDR3</td><td>amino acid</td>

<td> 30</td><td>VH1A11</td><td>VH CDR3</td><td>amino acid</td>

<td> 31</td><td>VH1B12</td><td>VH CDR3</td><td>amino acid</td>

<td> 32</td><td>VH1E4</td><td>VH CDR3</td><td>amino acid</td>

<td> 33</td><td>VH1F6</td><td>VH CDR3</td><td>amino acid</td>

<td> 34</td><td>3C-H2</td><td>VH CDR3</td><td>amino acid</td>

<td> 35</td><td>VH1-D2 .N</td><td>VH CDR3</td><td>amino acid</td>

<td> 36</td><td>D2E7</td><td>VL</td><td>nucleic acid</td>

<td> 37</td><td>D2E7</td><td>VH</td><td>nucleic acid</td>

Equivalents

Those skilled in the art will recognize, or be able to ascertain more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included in the following claims.