Low dose methods for treating disorders in which tnf-alpha activity is detrimental

<u>Background of the Invention</u>

0001Tumor necrosis factor a (TNFα) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its capacity to induce the necrosis of certain mouse tumors (see <i>e.g</i>., <nplcit id="ncit0001" npl-type="s"><text>Old, L. (1985) Science 230:630-632</text></nplcit>). Subsequently, a factor termed cachectin, associated with cachexia, was shown to be the same molecule as TNFα. TNFα has been implicated in mediating shock (see <i>e.g</i>., <nplcit id="ncit0002" npl-type="s"><text>Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518</text></nplcit>; <nplcit id="ncit0003" npl-type="s"><text>Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655</text></nplcit>). Furthermore, TNFα has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease (see <i>e.g</i>., <nplcit id="ncit0004" npl-type="s"><text>Moeller, A., et al. (1990) Cytokine 2:162-169</text></nplcit>; <patcit id="pcit0001" dnum="US5231024A"><text>U.S. Patent No. 5,231,024 to Moeller et al</text></patcit><i>.;</i> European Patent Publication No. <patcit id="pcit0002" dnum="EP260610B1"><text>260 610 B1 by Moeller, A., et al</text></patcit>.<nplcit id="ncit0005" npl-type="s"><text>Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452</text></nplcit>; <nplcit id="ncit0006" npl-type="s"><text>Tracey, K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503</text></nplcit>).

0002Because of the harmful role of human TNFα (hTNFα) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNFα activity. In particular, antibodies that bind to, and neutralize, hTNFα have been sought as a means to inhibit hTNFα activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFα (see <i>e.g</i>., <nplcit id="ncit0007" npl-type="s"><text>Hahn T; et al., (1985) Proc Natl Acad Sci USA 82: 3814-3818</text></nplcit>; <nplcit id="ncit0008" npl-type="s"><text>Liang, C-M., et al. (1986) Biochem. Biophys. Res. Commun. 137:847-854</text></nplcit>; <nplcit id="ncit0009" npl-type="s"><text>Hirai, M., et al. (1987) J. Immunol. Methods 96:57-62</text></nplcit><u>;</u><nplcit id="ncit0010" npl-type="s"><text>Fendly, B.M., et al. (1987) Hybridoma 6:359-370</text></nplcit>; <nplcit id="ncit0011" npl-type="s"><text>Moeller, A., et al. (1990) Cytokine 2:162-169</text></nplcit>; <patcit id="pcit0003" dnum="US5231024A"><text>U.S. Patent No. 5,231,024 to Moeller et al</text></patcit><i>.;</i> European Patent Publication No. <patcit id="pcit0004" dnum="EP186833B1"><text>186 833 B1 by Wallach, D.</text></patcit>; European Patent Application Publication No. <patcit id="pcit0005" dnum="EP218868A1"><text>218 868 A1 by Old et al</text></patcit><i>.;</i> European Patent Publication No. <patcit id="pcit0006" dnum="EP260610B1"><text>260 610 B1 by Moeller, A., et al</text></patcit><i>.</i>)<i>.</i> While these mouse anti-hTNFα antibodies often displayed high affinity for hTNFα (<i>e.g.,</i> Kid ≤ 10^-9M) and were able to neutralize hTNFα activity, their use <i>in vivo</i> may be limited by problems associated with administration of mouse antibodies to humans, such as short serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the "human anti-mouse antibody" (HAMA) reaction).

0003In an attempt to overcome the problems associated with use of fully-murine antibodies in humans, murine anti-hTNFα antibodies have been genetically engineered to be more "human-like." For example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (<nplcit id="ncit0012" npl-type="s"><text>Knight, D.M, et al. (1993) Mol. Immunol. 30:1443-1453</text></nplcit>; <patcit id="pcit0007" dnum="WO9216553A"><text>PCT Publication No. WO 92/16553 by Daddona, P.E., et al</text></patcit><i>.</i>). Additionally, humanized antibodies, in which the hypervariable domains of the antibody variable regions are murine-derived but the remainder of the variable regions and the antibody constant regions are human-derived, have also been prepared (<patcit id="pcit0008" dnum="WO9211383A"><text>PCT Publication No. WO 92/11383 by Adair, J.R., et al</text></patcit>.). However, because these chimeric and humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged period<u>s</u>, <i>e.g</i>., for chronic indications, such as rheumatoid arthritis (see <i>e.g</i>., <nplcit id="ncit0013" npl-type="s"><text>Elliott, M.J., et al. (1994) Lancet 344:1125-1127</text></nplcit>; <nplcit id="ncit0014" npl-type="s"><text>Elliot, M.J., et al. (1994) Lancet 344:1105-1110</text></nplcit>).

0004A preferred hTNFα inhibitory agent to murine mAbs or derivatives thereof (<i>e.g</i>., chimeric or humanized antibodies) would be an entirely human anti-hTNFα antibody, since such an agent should not elicit the HAMA reaction, even if used for prolonged periods. Human monoclonal autoantibodies against hTNFα have been prepared using human hybridoma techniques (<nplcit id="ncit0015" npl-type="s"><text>Boyle, P., et al. (1993) Cell. Immunol. 152:556-568</text></nplcit>; <nplcit id="ncit0016" npl-type="s"><text>Boyle, P., et al. (1993) Cell. Immunol. 152:569-581</text></nplcit>; European Patent Application Publication No. <patcit id="pcit0009" dnum="EP614984A2"><text>614 984 A2 by Boyle, et al</text></patcit> However, these hybridoma-derived monoclonal autoantibodies were reported to have an affinity for hTNFα that was too low to calculate by conventional methods, were unable to bind soluble hTNFα and were unable to neutralize hTNFα-induced cytotoxicity (see Boyle, <i>et al.; supra</i>)<i>.</i> Moreover, the success of the human hybridoma technique depends upon the natural presence in human peripheral blood of lymphocytes producing autoantibodies specific for hTNFα. Certain studies have detected serum autoantibodies against hTNFα in human subjects (<nplcit id="ncit0017" npl-type="s"><text>Fomsgaard; A., et al. (1989) Scand. J. Immunol. 30:219-223</text></nplcit>; <nplcit id="ncit0018" npl-type="s"><text>Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B:447-452</text></nplcit>), whereas others have not (<nplcit id="ncit0019" npl-type="s"><text>Leusch, H-G., et al. (1991) J. Immunol. Methods 139:145-147</text></nplcit>).

0005Alternative to naturally-occurring human anti-hTNFα antibodies would be a recombinant hTNFα antibody. Recombinant human antibodies that bind hTNFα with relatively low affinity (<i>i.e</i>., K_d ∼10^-7M) and a fast off rate (<i>i.e</i>., K_off ∼ 10^-2 sec^-1) have been described (<nplcit id="ncit0020" npl-type="s"><text>Griffiths, A.D., et al. (1993) EMBO J. 12:725-734</text></nplcit>). However, because of their relatively fast dissociation kinetics, these antibodies may not be suitable for therapeutic use. Additionally, a recombinant human anti-hTNFα has been described that does not neutralize hTNFα activity, but rather enhances binding of hTNFα to the surface of cells and enhances internalization of hTNFα (<nplcit id="ncit0021" npl-type="s"><text>Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45</text></nplcit>; <patcit id="pcit0010" dnum="WO9203145A"><text>PCT Publication No. WO 92/03145 by Aston, R. et al</text></patcit><i>.)</i>

0006Accordingly, human antibodies, such as recombinant human antibodies, that bind soluble hTNFα with high affinity and slow dissociation kinetics and that have the capacity to treat disorders in which TNFα activity is detrimental, are still needed.

<u>Summary of the Invention</u>

0007The invention pertains to a human anti-TNFa antibody which is either D2E7, or a human anti-TNFa antibody with equivalent properties to D2E7 which dissociates from human TNFa with a K_d of 1 x 10^-8 M or less and a <i>k_off</i> rate constant of 1x10^-3 s^-1 or less, both determined by surface plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard <i>in vitro</i> L929 assay with an IC₅₀ of 1 x 10^-7 M or less, for use in the treatment or alleviation of arthritis, wherein the anti-TNFa antibody is to be administered in a low dose of 0.01-0.11 mg/kg, such that the arthritis is treated or alleviated.

<u>Brief Description of the Drawings</u>

0008<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">Figure 1</figref> shows arthritic scores of each mouse in the treatment groups receiving different doses of D2E7. Arthritic scores were recorded weekly starting at 1 week of age. For each treatment group, mean ± standard error of arthritis scores are indicated in the graph. The treatment groups were as follows: Control group: 11 female, 9 male mice (n=20); 10 mg/kg dose group: 2 female, 2 male mice (n=4); 5 mg/kg dose group: 6 female, 1 male mice (n=7); 1 mg/kg dose group: 5 female, 3 male mice (n=8); 0.5 mg/kg dose group: 3 female, 2 male mice (n=5); 0.1 mg/kg dose group: 3 female, 3 male mice (n=6); 0.01 mg/kg dose group: 4 female, 2 male mice (n=6).</li><li><figref idref="f0002">Figure 2</figref> shows arthritic scores of each mouse in the treatment groups receiving different doses of Remicade. Arthritic scores were recorded weekly starting at 1 week of age. For each treatment group, mean ± standard error of arthritis scores are indicated in the graph. The treatment groups were as follows: Control group: 11 female, 9 male mice (n=20); 10 mg/kg dose group: 4 female, 1 male mice (n=5); 5 mg/kg dose group: 3 female, 4 male mice (n=7); 1 mg/kg dose group: 6 female, 2 male mice (n=8); 0.5 mg/kg dose group: 4 female, 2 male mice (n=6); 0.1 mg/kg dose group: 1 female, 4 male mice (n=5); 0.01 mg/kg dose group: 2 female, 3 male mice (n=5).</li><li><figref idref="f0003">Figure 3</figref> shows arthritic scores of each mouse in the treatment groups receiving different doses of Enbrel. Arthritic scores were recorded weekly starting at 1 week of age. For each treatment group, mean ± standard error of arthritis scores are indicated in the graph. The treatment groups were as follows: Control group: 11 female, 9 male mice (n=20); 10 mg/kg dose group: 3 female, 2 male mice (n=5); 5 mg/kg dose group: 3 female, 3 male mice (n=6); 1 mg/kg dose group: 5 female, 1 male mice (n=6); 0.5 mg/kg dose group: 4 female, 3 male mice (n=7); 0.1 mg/kg dose group: 2 female, 3 male mice (n=5); 0.01 mg/kg dose group: 2 female, 4 male mice (n=6).</li><li><figref idref="f0004">Figure 4</figref> shows final arthritic scores in D2E7, Remicade, and Enbrel treated huTNF-Tg197 mice at 10 weeks of age.</li><li><figref idref="f0005">Figures 5A-D</figref> show a histopathological evaluation of various tissues taken from arthritic joints.</li><li><figref idref="f0006">Figures 6A, 6B, and 6C</figref> show circulating huTNF levels in D2E7, Remicade, and Enbrel treated huTNF-Tg197 mice.</li></ul>

<u>Detailed Description of the Invention</u>

0009The invention pertains to a human anti-TNFα antibody which is either D2E7, or a human anti-TNFα antibody with equivalent properties to D2E7 which dissociates from human TNFα with a K_d of 1 x 10^-8 M or less and a <i>k_off</i> rate constant of 1 x 10^-3 s^-1 or less, both determined by surface plasmon resonance, and neutralizes human TNFα cytotoxicity in a standard <i>in vitro</i> L929 assay with an IC₅₀ of 1 x 10^-7. M or less, for use in the treatment or alleviation of arthritis, wherein the anti-TNFα antibody is to be administered in a low dose of 0.01-0.11 mg/kg, such that the arthritis is treated or alleviated.

0010The term "human TNFα" (abbreviated herein as huTNF, hTNFα, or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a 17 kD secreted form and a 26 kD membrane associated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules. The structure of hTNFα is described further in, for example, <nplcit id="ncit0022" npl-type="s"><text>Pennica, D., et al. (1984) Nature 312:724-729</text></nplcit>; <nplcit id="ncit0023" npl-type="s"><text>Davis, J.M., et al. (1987) Biochemistry 26:1322-1326</text></nplcit>; and <nplcit id="ncit0024" npl-type="s"><text>Jones, E.Y., et al. (1989) Nature 338:225-228</text></nplcit>. The term human TNFα is intended to include recombinant human TNFα (rhTNFα), which can be prepared by standard recombinant expression methods or purchased commercially (R &amp; D Systems, Catalog No. 210-TA, Minneapolis, MN).

0011The term "TNFα inhibitor" includes agents which inhibit TNFα. Examples of TNFα inhibitors include etanercept (ENBREL, Immunex), infliximab (REMICADE, Johnson and Johnson), human anti-TNF monoclonal antibody (D2E7, Knoll Pharmaceuticals), CDP 571 (Cell tech), and CDP 870 (Celltech) and other compounds which inhibit TNFα activity, such that when administered to a subject suffering from or at risk of suffering from a disorder in which TNFα activity is detrimental, the disorder is treated. The term also includes each of the anti-TNFα human antibodies and antibody portions described herein as well as those described in <patcit id="pcit0011" dnum="US6090382A"><text>U.S. Patent Nos. 6,090,382</text></patcit> and <patcit id="pcit0012" dnum="US6258562B1"><text>6,258,562 B1</text></patcit>, in <patcit id="pcit0013" dnum="US6509015B"><text>U.S. Patent No. 6,509,015</text></patcit> and in <patcit id="pcit0014" dnum="US20030092059A1"><text>US 2003/0092059 A1</text></patcit>.

0012The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies are described in further detail in <patcit id="pcit0015" dnum="US6090382A"><text>U.S. Patent Nos. 6,090,382</text></patcit> and <patcit id="pcit0016" dnum="US6258562B1"><text>6,258,562 B1</text></patcit>, in <patcit id="pcit0017" dnum="US6509015B"><text>U.S. Patent No. 6,509,015</text></patcit> and in <patcit id="pcit0018" dnum="US20030092059A1"><text>US 2003/0092059 A1</text></patcit>.

0013The 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 to an antigen (<i>e.g</i>., hTNFα). 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 encompassed 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) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (<nplcit id="ncit0025" npl-type="s"><text>Ward et al., (1989) Nature 341:544-546</text></nplcit>), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see <i>e.g.,</i><nplcit id="ncit0026" npl-type="s"><text>Bird et al. (1988) Science 242:423-426</text></nplcit>; and <nplcit id="ncit0027" npl-type="s"><text>Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883</text></nplcit>). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see <i>e.g</i>., <nplcit id="ncit0028" npl-type="s"><text>Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448</text></nplcit>; <nplcit id="ncit0029" npl-type="s"><text>Poljak, R.J., et al. (1994) Structure 2:1121-1123</text></nplcit>). Antibody portions are described in further detail in <patcit id="pcit0019" dnum="US6090382A"><text>U.S. Patent Nos. 6,090,382</text></patcit> and <patcit id="pcit0020" dnum="US6258562B1"><text>6,258,562 B1</text></patcit>, in <patcit id="pcit0021" dnum="US6509015B"><text>U.S. Patent No. 6,509,015</text></patcit> and in <patcit id="pcit0022" dnum="US20030092059A1"><text>US 2003/0092059 A1</text></patcit>.

0014The present invention pertains to a human anti-TNFα antibody which is either D2E7, or a human anti-TNFα antibody with equivalent properties to D2E7 which dissociates from human TNFα with a K_d of 1 x 10^-8 M or less and a <i>k_off</i> rate constant of 1 x 10^-3 s^-1 or less, both determined by surface plasmon resonance, and neutralizes human TNFα cytotoxicity in a standard <i>in vitro</i> L929 assay with an IC₅₀ of 1 x 10^-7 M or less, for use in the treatment or alleviation of arthritis, wherein the anti-TNFα antibody is to be administered in a low dose of 0.01-0.11 mg/kg, such that the arthritis is treated or alleviated.

0015According to one embodiment, such isolated human antibody dissociates from human TNF* with a K_off of 5 x 10^-4 s^-1 or less, or even more preferably, with a K_off 1 x 10^-4 s^-1 or less. More preferably, the isolated human antibody neutralizes human TNFα cytotoxicity in a standard <i>in vitro</i> L929 assay with an IC₅₀ of 1 x 10^-8 M or less, even more preferably with an IC₅₀ of 1 x 10^-9 M or less and still more preferably with an IC₅₀ of 5 x 10^-10 M or less.

0016The term "low dose" or "low dosage" as used herein, refers to an amount of TNFα inhibitor which is administered to a subject, wherein the amount is substantially lower than that ordinarily employed. A "low dose therapy" includes a treatment regiment which is based on administering a low dose of a TNFα inhibitor. In accordance with the invention, a low dose of D2E7 is considered 0.01-0.11 mg/kg. Such low dose may be used to treat rheumatoid arthritis and symptoms associated with the disease. For example, symptoms which can be treated using low dose therapy of D2E7 include bone erosion, cartilage erosion, inflammation, and vascularity. Low doses of a TNFα inhibitor are advantageous for a number of reasons, including the reduction in the frequency and severity of side effects which may be associated with the normal prescribed dose of TNFα inhibitor.

I. Uses of TNFα Inhibitors

0017TNFα has been implicated in the pathophysiology of a wide variety of disorders (see <i>e.g.,</i><nplcit id="ncit0030" npl-type="s"><text>Moeller, A., et al. (1990) Cytokine 2:162-169</text></nplcit>; U. S. Patent No. <patcit id="pcit0023" dnum="US5231024A"><text>5,231,024 to Moeller et al</text></patcit><i>.;</i> European Patent Publication No. <patcit id="pcit0024" dnum="EP260610B1"><text>260 610 B1 by Moeller, A</text></patcit>). In accordance with the invention as defined above, disclosed herein are methods for inhibiting TNFα activity in a subject suffering from such a disorder, which method comprises administering to the subject low dose of the antibody such that TNFα activity in the subject is inhibited. Preferably, the TNFα is human TNFα and the subject is a human subject. Alternatively, the subject can be a mammal expressing a TNFα with which an antibody of the invention cross-reacts. The antibody is to be administered to a human subject for therapeutic purposes in low doses of 0.01-0.11 mg/kg. Moreover, a low dose of the antibody can be administered to a non-human mammal expressing a TNFα with which the antibody cross-reacts (<i>e.g</i>., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies (<i>e.g</i>., testing of dosages and time courses of administration).

0018As used herein, the term "a disorder in which TNFα activity is detrimental" is intended to include diseases and other disorders in which the presence of TNFα in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. For the purposes of the invention, treating a disorder in which TNFα activity is detrimental relates to treating arthritis and includes, but is not limited to, alleviating symptoms associated with said disorder. Accordingly, a disorder in which TNFα activity is detrimental is a disorder in which inhibition of TNFα activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of TNFα in a biological fluid of a subject suffering from the disorder (e. g., an increase in the concentration of TNFα in serum, plasma, synovial fluid, <i>etc</i>. of the subject), which can be detected, for example, using an anti-TNFα antibody as described above. The use of a low dose of the antibodies in the treatment of arthritis, in particular rheumatoid arthritis, are discussed further below. In certain embodiments, a low dose of the antibody is administered to the subject in combination with another therapeutic agent, as described below.

Rheumatoid Arthritis

0019TNFα has been implicated in activating tissue inflammation and causing joint destruction in rheumatoid arthritis (see <i>e.g</i>., <nplcit id="ncit0031" npl-type="s"><text>Moeller, A., et al. (1990) Cytokine 2:162-169</text></nplcit>; U. S. Patent No. <patcit id="pcit0025" dnum="US5231024A"><text>5,231,024 to Moeller et al</text></patcit><i>.;</i> European Patent Publication No. <patcit id="pcit0026" dnum="EP260610B1"><text>260 610 B 1 by Moeller, A</text></patcit>.; Tracey and Cerami, <i>supra;</i><nplcit id="ncit0032" npl-type="s"><text>Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum. 38:151-160</text></nplcit>; <nplcit id="ncit0033" npl-type="s"><text>Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266</text></nplcit>). Chimeric and humanized murine anti-hTNFα antibodies have undergone clinical testing for treatment of rheumatoid arthritis (see <i>e</i>.<i>g</i>., <nplcit id="ncit0034" npl-type="s"><text>Elliott, M. J., et al. (1994) Lancet 344:1125-1127</text></nplcit><u>;</u><nplcit id="ncit0035" npl-type="s"><text>Elliot, M. J., et al. (1994) Lancet 344:1105-1110</text></nplcit>; <nplcit id="ncit0036" npl-type="s"><text>Rankin, E. C., et al. (1995) Br.J. Rheumatol. 34:334-342</text></nplcit>).

0020Low doses of anti-TNFα antibodies in accordance with the invention as defined above can be used to treat rheumatoid arthritis. Low doses of anti-TNFα antibodies can be used to treat rheumatoid arthritis by alleviating symptoms associated with said disorder. Examples of symptoms or signs commonly associated with rheumatoid arthritis include, but are not limited to, bone erosion in the joints, cartilage erosion in the joints, inflammation in the joints, vascularity in the joints, and combinations thereof. Other examples of symptoms associated with rheumatoid arthritis include weight gain, joint distortion, swelling of the joints, joint deformation, ankylosis on felxion, severely impaired movement, and combinations thereof.

0021The human antibodies in accordance with the invention can be used to treat rheumatoid arthritis, osteoarthritis and gouty arthritis. Typically, the antibody is administered systemically, although for certain disorders, local administration of the antibody at a site of inflammation may be beneficial (e. g., local administration in the joints in rheumatoid arthritis, alone or in combination with a cyclohexane-ylidene derivative as described in <patcit id="pcit0027" dnum="WO9319751A"><text>PCT Publication No. WO 93/19751</text></patcit>). The antibody also can be administered with one or more additional therapeutic agents useful in the treatment of autoimmune diseases, as discussed further in subsection II.

0022The antibodies can also be used to treat juvenile rheumatoid arthritis, psoriatic arthritis, and ostoarthritis.

II.

Pharmaceutical Compositions and Pharmaceutical Administration

0023The antibodies disclosed herein can be incorporated into pharmaceutical compositions suitable for low dose administration to a subject. Typically, the pharmaceutical composition comprises an antibody 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 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 comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody, antibody portion, or other TNFα inhibitor.

0024The compositions may be in a variety of forms suitable for low dose administration. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (<i>e.g</i>., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and 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 or other TNFα inhibitors. The preferred mode of administration is parenteral (<i>e.g</i>., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, a low dose of the antibody is administered by intravenous infusion or injection. In another preferred embodiment, a low dose of the antibody is administered by intramuscular or subcutaneous injection.

0025Therapeutic 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 to high drug concentration. Sterile injectable solutions can be prepared by incorporating a low dose of the active compound (<i>i.e</i>., antibody, antibody portion, or other TNFα inhibitor) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

0026Also disclosed are packaged pharmaceutical compositions which comprise a low dose of the antibody and instructioris for using the inhibitor to treat a particular disorder in which TNFα activity is detrimental, as described above.

0027The pharmaceutical compositions may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody. A "therapeutically effective amount" is an amount which is determined to be required to eliminate said disorder or to reduce and/or alleviate the symptoms of said disorder, as long as a "therapeutically effective amount" refers to an amount which is effective, at low doses of 0.01-0.11 mg/kg and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.

0028Dosage regimens may be adjusted to provide the optimum desired response (<i>e.g.,</i> a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided low doses may be administered over time or the low dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in low dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and 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.

0029The range in accordance with the invention for a therapeutically or prophylactically effective amount of the antibody is 0.01-0.11 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

0030The low dose of the antibody administered to a subject is between about 0.01-0.11 mg/kg, such as 0.06-0.11 mg/kg. In a preferred embodiment, the antibody is D2E7. Ranges intermediate to the above recited dosages are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.

0031A low dose of the antibodies can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, 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 skilled in the art. See, <i>e.g.,</i><nplcit id="ncit0037" npl-type="b"><text>Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978</text></nplcit>.

0032In certain embodiments, a low dose of an antibody may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may 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 low dose of the compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

0033Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, a low dose of the antibody is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating arthritis. For example, a low dose of the anti-hTNFα antibody may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (<i>e.g</i>., antibodies that bind other cytokines or that bind cell surface molecules), one or more cytokines, soluble TNFα receptor (see <i>e.g</i>., <patcit id="pcit0028" dnum="WO9406476A"><text>PCT Publication No. WO 94/06476</text></patcit>) and/or one or more chemical agents that inhibit hTNFα production or activity (such as cyclohexane-ylidene derivatives as described in <patcit id="pcit0029" dnum="WO9319751A"><text>PCT Publication No. WO 93/19751</text></patcit>). Furthermore, a low dose of one or more antibodies may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize even lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

III. Other Therapeutic Agents

0034The language "in combination with"a therapeutic agent includes coadministration of a low dose of the antibody with a therapeutic agent, administration of a low dose of the antibody first, followed by the therapeutic agent and administration of the therapeutic agent first, followed by the low dose of the antibody. Specific therapeutic agent (s) are generally selected based on the particular disorder being treated, as discussed below.

0035Nonlimiting examples of therapeutic agents for rheumatoid arthritis with which a low dose of the antibody can be combined include the following: non-steroidal anti-inflammatory drug (s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP- 571/BAY-10-3356 (humanized anti-TNFα antibody; CelltechBayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g., <nplcit id="ncit0038" npl-type="s"><text>Arthritis &amp; Rheumatism (1994) Vol. 37, S295</text></nplcit>; <nplcit id="ncit0039" npl-type="s"><text>J. Invest. Med. (1996) Vol. 44, 235A</text></nplcit>); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see <i>e.g.,</i><nplcit id="ncit0040" npl-type="s"><text>Arthritis &amp; Rheumatism (1995) Vol. 38, S185</text></nplcit>); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see <i>e.g.,</i><nplcit id="ncit0041" npl-type="s"><text>Arthritis &amp; Rheumatism (1993) Vol. 36, 1223</text></nplcit>); Anti-Tac (humanized anti-IL-2Rα; 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 (<i>e.g</i>., agonist antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF binding protein; see <i>e.g.,</i><nplcit id="ncit0042" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S284</text></nplcit>; <nplcit id="ncit0043" npl-type="s"><text>Amer. J. Physiol. - Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42</text></nplcit>); R973401 (phosphodiesterase Type IV inhibitor; see <i>e.g.,</i><nplcit id="ncit0044" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282</text></nplcit>); MK-966 (COX-2 Inhibitor; see <i>e.g</i>., <nplcit id="ncit0045" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S81</text></nplcit>); Iloprost (see <i>e.g.,</i><nplcit id="ncit0046" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S82</text></nplcit>); methotrexate; thalidomide (see <i>e.g.,</i><nplcit id="ncit0047" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282</text></nplcit>) and thalidomide-related drugs (<i>e.g</i>., Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; see <i>e.g.,</i><nplcit id="ncit0048" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S131</text></nplcit>; <nplcit id="ncit0049" npl-type="s"><text>Inflammation Research (1996) Vol. 45, pp. 103-107</text></nplcit>); tranexamic acid (inhibitor of plasminogen activation; see <i>e.g.,</i><nplcit id="ncit0050" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S284</text></nplcit>); T-614 (cytokine inhibitor; see <i>e.g.,</i><nplcit id="ncit0051" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282</text></nplcit>); prostaglandin E1 (see <i>e.g.,</i><nplcit id="ncit0052" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282</text></nplcit>); Tenidap (non-steroidal anti-inflammatory drug; see <i>e.g.,</i><nplcit id="ncit0053" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S280</text></nplcit>); Naproxen (non-steroidal anti-inflammatory drug; see <i>e.g.,</i><nplcit id="ncit0054" npl-type="s"><text>Neuro Report (1996) Vol. 7, pp. 1209-1213</text></nplcit>); 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 <i>e.g.,</i><nplcit id="ncit0055" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S281</text></nplcit>); Azathioprine (see <i>e.g.,</i><nplcit id="ncit0056" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S281</text></nplcit>); ICE inhibitor (inhibitor of the enzyme interleukin-1β converting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitos of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs (<i>e.g</i>., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; interleukin-11 (see <i>e.g.,</i><nplcit id="ncit0057" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S296</text></nplcit>); interleukin-13 (see <i>e.g.,</i><nplcit id="ncit0058" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S308</text></nplcit>); interleukin-17 inhibitors (see <i>e.g.,</i><nplcit id="ncit0059" npl-type="s"><text>Arthritis &amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S120</text></nplcit>); gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see <i>e.g</i>., <nplcit id="ncit0060" npl-type="s"><text>DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777</text></nplcit>); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.

0036This invention is further illustrated by the following examples which should not be construed as limiting.

<u>EXAMPLE 1: Study of Efficacy of D2E7 Administered in Low Doses</u>

0037Studies were performed to determine the efficacy of D2E7, infliximab, and etanercept at low doses in preventing polyarthritis using the transgenic (Tg197) murine model of rheumatoid arthritis (RA). Infliximab is a human-mouse chimeric antibody, and etanercept is a p75 TNF receptor construct. D2E7 is a fully human antibody derived from a human immunoglobulin gene library. Transgenic mice, Tg197 carry the human TNF □gene and spontaneously develop a disease similar to human rheumatoid arthritis (<nplcit id="ncit0061" npl-type="s"><text>Keffer, J. et al, 1991, EMBO J. 10:4025</text></nplcit>). Signs of arthritic disease, including rheumatoid arthritis, include slower weight gain, joint distortion and swelling, joint deformation and ankylosis and impaired movement. Histopathological findings include hyperplasia of the synovial membrane, leukocyte infiltration, pannus formation, articular cartilage and bone destruction. Administration of anti-TNF agents prevents the development of polyarthritis in a dose dependent manner.

A. Comparison of Binding Characteristics of D2E7, Remicade, and Enbrel

0038Infliximab (Remicade) and Etanercept (Enbrel) are two anti-TNF drugs approved for rheumatoid arthritis. Remicade is a human-mouse chimeric IgG₁ antibody and Enbrel is a fusion protein made up of extra-cellular domain of the p75 TNF receptor and the constant region of IgG₁ molecule. D2E7 is a fully human antibody of the IgG₁,kappa class selected from human immunoglobulin gene libraries. All three anti-TNF agents bind to human TNF with relatively similar potency. The intrinsic affinities of D2E7, Remicade and Enbrel for TNF are 8.6x10-¹¹, 9.5x10^-11 and 15.7x10^-11 M (Kd values), respectively. The kinetics of binding to TNF are similar for the antibodies D2E7 and Remicade. Enbrel, on the other hand, binds to and dissociates from TNF fast. Thus, the 16 minute half-life of Enbrel:TNF complex is considerably shorter than the 184 and 255 minute half-lives for Remicade and D2E7:TNF complexes, respectively.

0039A BIAcore 3000 instrument was used to derive kinetic parameters of binding between human TNF and anti-TNF agents. Biosensor chips were covalently coupled with a goat anti-human Fc antibody. Anti-TNF agents (D2E7, Remicade and Enbrel) were then captured on the chips and varying concentrations of huTNF were added. Binding data were analyzed to derive the kinetic parameters, which are described in Table 1. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1: Binding of D2E7, Remicade, or Enbrel to human TNF</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="24mm" /><colspec colnum="4" colname="col4" colwidth="24mm" /><thead><row><entry valign="top"><b>Agent</b></entry><entry valign="top"><b>On-rate (M^-1s^-1)</b></entry><entry valign="top"><b>Off-rate (s^-1)</b></entry><entry valign="top"><b>Kd (M)</b></entry></row></thead><tbody><row><entry>D2E7</entry><entry>5.37x10⁵</entry><entry>4.53x10^-5</entry><entry>8.56 x 10^-11</entry></row><row><entry>Remicade</entry><entry>6.71x10⁵</entry><entry>6.29 x 10^-5</entry><entry>9.45 x 10^-11</entry></row><row><entry>Enbrel</entry><entry>4.47x10⁶</entry><entry>7.02x10^-4</entry><entry>1.57x10-¹⁰</entry></row></tbody></tgroup></table></tables>

B. Prevention of Arthritic Symptoms

0040Tg197 Mice were used as a model for studying the effects of a low dose regiment of D2E7, Remicade, and Enbrel on relieving symptoms commonly associated with rheumatoid arthritis. Human TNF transgenic mice were identified and verified by PCR. From the first week of age, separate litters of Tg197 mice were assigned to different study groups. Tg197 mice heterozygous for the human TNF gene received weekly intraperitoneal doses of D2E7, Remicade or Enbrel. Each drug treatment dose group consisted of mice from a single litter. The control group received the phosphate buffered saline diluent and consisted of mice from 4 litters. Weights of animals in each group were recorded weekly prior to dosing. Each group received one i.p. injection per week as follows:

Vehicle control

0041<tables id="tabl0002" num="0002"><table frame="none"><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="30mm" /><colspec colnum="2" colname="col2" colwidth="36mm" /><colspec colnum="3" colname="col3" colwidth="31mm" /><tbody><row><entry>D2E7, 10 mg/kg</entry><entry>Remicade, 10 mg/kg</entry><entry>Enbrel, 10 mg/kg</entry></row><row><entry>D2E7, 5 mg/kg</entry><entry>Remicade, 5 mg/kg</entry><entry>Enbrel, 5 mg/kg</entry></row><row><entry>D2E7, 1 mg/kg</entry><entry>Remicade, 1 mg/kg</entry><entry>Enbrel, 1 mg/kg</entry></row><row><entry>D2E7, 0.5 mg/kg</entry><entry>Remicade, 0.5 mg/kg</entry><entry>Enbrel, 0.5 mg/kg</entry></row><row><entry>D2E7, 0.1 mg/kg</entry><entry>Remicade, 0.1 mg/kg</entry><entry>Enbrel, 0.1 mg/kg</entry></row><row><entry>D2E7, 0.01 mg/kg</entry><entry>Remicade, 0.01 mg/kg</entry><entry>Enbrel, 0.01 mg/kg</entry></row></tbody></tgroup></table></tables>

0042Arthritic scores in each group were recorded each week using the following scoring system: <tables id="tabl0003" num="0003"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="10mm" /><colspec colnum="2" colname="col2" colwidth="104mm" /><tbody><row><entry>0</entry><entry>no arthritis</entry></row><row><entry>1</entry><entry>mild arthritis (joint distortion)</entry></row><row><entry>2</entry><entry>moderate arthritis (swelling, joint deformation)</entry></row><row><entry>3</entry><entry>severe arthritis (ankylosis on flexion and severely impairment movement)</entry></row></tbody></tgroup></table></tables> Treatment continued for 10 weeks. Results from the scoring assay are shown in <figref idref="f0002">Figure. 2</figref>, and <figref idref="f0003">3</figref>. <figref idref="f0004">Figure 4</figref> shows the final arthritic score for the three antibodies in treated Tg197 mice at week 10. The results show that higher doses of Enbrel were needed to prevent the development of arthritic scores. The ED₅₀ value for Enbrel was close to 1mg/kg whereas the ED₅₀ value for D2E7 and Remicade was below 0.5 mg/kg. Between the two antibodies, D2E7 offered more protection than Remicade at the same doses. Furthermore, the onset of disease was delayed in mice treated with 0.5 mg/kg of D2E7 up to 5 weeks, and with mice treated with 0.1 mg/kg doses of D2E7 up to 4 weeks (<figref idref="f0001">Figure 1</figref>). In contrast, the onset of disease was delayed in mice treated with either 0.1 mg/kg or 0.5 mg/kg of Remicade for only 3 weeks (<figref idref="f0002">Figure 2</figref>).

0043In sum, all three agents, D2E7, Remicade, and Enbrel prevented the development of arthritis in Tg197 mice in a dose dependent fashion. Treated mice had lower arthritic scores and less inflammation and joint damage and gained more weight than the untreated mice. The pattern of response was similar for all three TNF antagonists, but the degree of protection varied among the three agents. Although the highest, saturating doses did not allow distinction between the agents, the potency of protection at intermediate doses was greatest for D2E7 treated mice, than for infliximab (Remicade), and the least for Etanercept (Enbrel) treated mice.

C. Analysis of Circulating huTNF levels in Treated Mice

0044Blood was collected at 5 and 10 weeks during the study. Serum was prepared and the levels of human TNF were determined by the Medgenix human TNF ELISA kit. TNF levels were measured for each mouse in treatment groups. Results from the study are shown in <figref idref="f0006">Figure 6</figref>. For each treatment group, mean ± standard error of TNF levels are indicated in the graph. A solid line at each graph is drawn at 2 ng/mL TNF level for orientation purpose. In the untreated group, serum huTNF levels were low, 0.1 and 0.2 ng/mL at 5 and 10 weeks, respectively. Weekly administration of anti-TNF agents resulted in sequestration of TNF in the serum. The levels of serum huTNF were similar for D2E7 or Remicade treated mice. The average huTNF levels decreased from 2 to 0.1 ng/mL as a function of administered dose. Enbrel treated mice, on the other hand, had much higher serum huTNF, reaching levels of 20 ng/mL.

0045In sum, measurement of human TNF by Medgenix ELISA, which detects both free and bound TNF, indicated that the anti-TNF agents were sequestering TNF into complexes. There were detectable levels of TNF in the serum of treated mice in contrast to very low levels in untreated mice. Interestingly, the level of noncleared TNF complexes for etanercept was 10-fold higher than in mice treated with infliximab or D2E7. Delayed TNF clearance with etanercept has been noted in published animal models and clinical studies.

D. Microscopic Analysis of Treated Mice

0046Following the 10 week treatment, all mice were sacrificed. Right and left hind limbs were harvested from two mice in each treatment group. Limbs were fixed in 10% neutral buffered formalin and then decalcified. Three consecutive sections from each limb sample were mounted on slides and the coded slides were sent for an independent evaluation by a pathologist.

0047Slides were stained with hematoxylin/eosin. The pathologist scored each slide with respect to severity of vascularity, inflammation, cartilage and bone erosion on a scale of 1-4. Results are shown in Table 2 below: <tables id="tabl0004" num="0004"><table frame="all"><title>Table 2: Approximate ED₅₀ (mg/kg) values of D2E7, Remicade or Enbrel for prevention of microscopic signs of arthritis in Tg197 mice</title><tgroup cols="4" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="42mm" /><colspec colnum="3" colname="col3" colwidth="42mm" /><colspec colnum="4" colname="col4" colwidth="43mm" colsep="1" /><thead><row valign="middle"><entry align="center" /><entry align="center"><b>D2E7 ED₅₀ , mg/kg</b></entry><entry align="center"><b>Remicade ED₅₀ , mg/kg</b></entry><entry align="center"><b>Enbrel ED₅₀ mg/kg</b></entry></row></thead><tbody><row valign="middle"><entry align="center">Inflammation</entry><entry align="center">0.1</entry><entry align="center">0.5</entry><entry align="center">0.5</entry></row><row valign="middle"><entry align="center">Vascularity</entry><entry align="center">0.1</entry><entry align="center">0.1</entry><entry align="center">1 &lt;ED₅₀&lt; 5</entry></row><row valign="middle"><entry align="center">Cartilage Erosion</entry><entry align="center">0.01 &lt;ED₅₀&lt; 0.1</entry><entry align="center">0.1 &lt;ED₅₀&lt; 0.5</entry><entry align="center">0.5</entry></row><row rowsep="1" valign="middle"><entry align="center">Bone Erosion</entry><entry align="center">0.01 1&lt;ED₅₀&lt; 0.1</entry><entry align="center">0.1 &lt;ED₅₀&lt;0.5</entry><entry align="center">0.5 &lt;ED₅₀&lt; 1</entry></row></tbody></tgroup></table></tables>

0048Results from this experiment are also shown in <figref idref="f0005">Figure 5</figref>. In <figref idref="f0005">Figure 5</figref>, three slides from each limb were examined; thus, 6 slides per mouse and 12 slides per treatment group were scored for histopathology. For each treatment group, mean ± standard deviation of histopathology scores are indicated in the graph. Most of the lesions were associated with ankle joints and all appeared symmetrical (i.e. similar scores for the left and right limbs for a given mouse). Cartilage degradation resulted mostly from endosteal erosive lesions and was generally less extensive than bone erosion. Inflammation was predominantly mononuclear cells with few PMNs, but no dense PMN loci.

0049The difference among the three anti-TNF agents was most pronounced in microscopic signs of disease activity in the arthritic joints than the external manifestations measured as arthritic scores. Bone erosion in the joints was completely abolished by 0.5 mg/kg dose of D2E7. In order to achieve the same effect a much higher dose of Remicade or Enbrel, 5 mg/kg, was needed. Cartilage erosion in the joints was completely abolished by 0.1 mg/kg dose of D2E7. In order to achieve the same effect a much higher dose of Remicade, 1mg/kg, or Enbrel,5 mg/kg, was needed. Inflammation in the joints was completely abolished by 0.5 mg/kg dose of D2E7. In order to achieve the same effect higher doses of other drugs were needed: 5 mg/kg for Remicade and 10 mg/kg for Enbrel. Vascularity in the joints was completely abolished by 0.5 mg/kg dose of D2E7 or Remicade. In order to achieve the same effect a much higher dose of Enbrel, 5 mg/kg, was needed.

0050There was a clear dose-response distinction between D2E7, infliximab and etanercept in prevention of microscopic joint damage. Whereas D2E7 completely prevented bone erosion, cartilage degradation, inflammation, and vascularity at the 0.5-mg/kg dose, both infliximab and etanercept required a dose of 1 or 5 mg/kg to reach similar levels of efficacy. In sum, in the human TNF transgenic mice, Tg197, D2E7 prevented polyarthritis more potently than did etanercept or infliximab, especially at low doses.