Methods of administering anti-TNFα antibodies

Description of the invention

The invention relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for the treatment of a human intestinal disorder, and to kits and syringes.

Tumor necrosis factor α (TNFα) is a cytokine produced by numerous cell types including monocytes and macrophages that was originally identified for its ability to induce necrosis of certain murine tumors (see, e.g., Old, L. (1985) Science 230: 630- 632). Subsequently, the factor identified as cachectin related to cachexia was shown to be the same molecule as TNFα. TNFα is one of the shock mediators (see, e.g., 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, TNFα is involved in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infection, autoimmune diseases, graft rejection, and graft versus host reactions (see, e.g., 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 TNF? (HTNF?) In a variety of human disorders, therapeutic strategies have been developed to inhibit or counteract hTNF? Activity. In particular, antibodies that bind and neutralize hTNFa as agents for inhibiting hTNFa activity have been sought. One of the first such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas made from lymphocytes of mice immunized with hTNFa (see e.g. Hahn T; et al. (1985) Proc Natl Acad Sci USA 82: 3814-3818; Liang, CM., Et al. (1986) 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: 359370; Molier, A., et al. (1990) Cytokine 2: 162-169; U.S. Patent No. 5,231,024 to Moeller et al; European Patent Publication No. 186833 B1, Wallach, D .; European Patent Application Publication No. 218,868 A1, Old et al; European Patent Publication No. 260610 B1, Moeller, A., et al.). Although these murine anti-hTNFa antibodies often exhibited high affinity for hTNFa (e.g., Kd <10^-9 M) and have the ability to neutralize hTNFa activity, their in vivo use may be limited by problems associated with administering murine antibodies to humans, such as the short serum half-life, the inability to trigger certain effector functions in humans, and the induction of an unwanted immune response against murine antibodies in humans ( "human anti-mouse antibody" (HAMA) reaction).

In an attempt to overcome the problems associated with the use of all-murine antibodies in humans, murine anti-hTNFa antibodies were genetically engineered to be more "human-like". For example, chimeric antibodies have been generated in which the antibody chain variable regions are derived from mice and the antibody chain constant regions are derived from humans (Knight, DM, et al. (1993) Mol. Immunol. 30: 1443-1453; PCT Publication No. WO 92 (16553, Daddon, PE, et al.). In addition, humanized antibodies have also been produced in which the hypervariable domains of the antibody variable regions are derived from mice and the rest of the antibody variable regions and constant regions are derived from human (PCT Publication No. WO 92/11383, Adair, JR, et al.). However, because these chimeric and humanized antibodies still retain some murine sequences, they can still elicit an adverse immune response, a human anti-chimeric antibody (HACA) response, especially when administered over an extended period of time, e.g. for chronic indications such as rheumatoid arthritis (see e.g. Elliott, MJ, et al (1994) Lancet 344: 1125-1127; Elliot, MJ, et al (1994) Lancet 344: 1105-1110).

A more preferred hTNFa inhibitory agent as compared to murine mAbs or derivatives thereof (e.g. chimeric or humanized antibodies) would be a fully human anti-hTNFa antibody as such an agent should not induce a HAMA response, even if used for prolonged periods of time. Human monoclonal anti-hTNFα autoantibodies were generated using techniques to generate human hybridomas (Boyle, P., et al. (1993) Cell. Immunol. 152: 556-568; Boyle, P., et al. (1993) Cell. Immunol. 152: 569-581; European Patent Application Publication No. 614,984 A2, Boyle, et al.). However, these hybridoma-derived monoclonal antibodies were shown to have an affinity for hTNFa that was too low to be calculated by conventional methods, were unable to bind soluble hTNFa, and were unable to neutralize hTNFa-induced cytotoxicity (see Boyle, et al; supra; ). Moreover, the success of the technique of human hybridoma production depends on the natural presence in the peripheral blood of human lymphocytes producing autoantibodies specific for hTNFα. In some studies, plasma autoantibodies to hTPL 217 217 B1 have been detected

NFa in humans as patients (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30: 219-223; Bendtzen,

K., et al. (1990) Prog. Leukocyte Biol. 10B: 447-452), while in others they were not detected (Leusch, HG., Et al. (1991) J. Immunol. Methods 139: 145-147).

An alternative to naturally occurring human anti-hTNFa antibodies would be a recombinant hTNFa antibody. Recombinant human antibodies that bind hTNFa with a relatively low affinity (i.e. Kd ~ 10^-7 M) and fast dissociation rate (i.e. Koff ~ 10^-2 s^-1) (Griffiths, AD, et al. (1993) EMBO J. 12: 725-734). However, due to the relatively rapid dissociation kinetics, these antibodies may not be suitable for therapeutic use. In addition, a recombinant human anti-hTNFa antibody has been described that does not neutralize hTNFa activity, but rather enhances hTNFa binding to the cell surface and enhances hTNFa internalization (Lidbury, A., et al. (1994) Biotechnol. Ther. 5: 27-45; PCT Publication No. WO 92/03145, Aston, R. et al.)

Recombinant human antibodies that bind soluble hTNFa with high affinity and slow dissociation kinetics have also been described and are capable of neutralizing hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and hTNFa-induced cell activation (see patent specification). No. 6,090,382). According to conventional protocols, administration of antibodies is performed intravenously at weekly intervals. Weekly administration of antibodies and / or any drug can be expensive, inconvenient, and cause more side effects due to the frequency of administration. The downside of intravenous administration is that the injection usually requires a person with medical training.

The invention relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for the treatment of a human intestinal disorder, wherein the medicament is administered subcutaneously on a biweekly administration regimen for treating the intestinal disorder.

A preferred use is when a human antibody, or antigen binding portion thereof, that detaches from human TNF? With a Kd constant of 1 × 10 is used.^-8 M or less and constant

-3 -1 Koff speed equal to 1 x 10^-3 s^-1 or less, both determined by surface plasmon resonance, and which neutralize the cytotoxicity of human TNFα in standard in vitro L929 assay with an IC50 value of 1 x 10^-7 M or less.

The use where a human antibody or a binding portion thereof is used is preferred

-4 -1 antigen, which detaches from human TNF? At a Koff rate constant of 5 x 10^-4 s^-1 or less.

The use where a human antibody or a binding portion thereof is used is preferred

-4 -1 antigen which detaches from human TNF? At a Koff rate constant of 1 x 10^-4 s^-1 or less.

It is preferred to use a human antibody, or antigen binding portion thereof, that neutralizes the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 value of 1 × 10^-8 M or less.

It is preferred to use a human antibody, or antigen binding portion thereof, that neutralizes the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 value of 1 × 10^-9 M or less.

It is preferred to use a human antibody or antigen binding portion thereof that neutralizes the cytotoxicity of human TNF? In a standard in vitro L929 assay -10 with an IC50 value of 1 x 10^-10 M or less.

The use in which a recombinant antibody or a recombinant antigen binding portion thereof is used as the human antibody or antigen binding portion thereof is preferred.

The invention further relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for treating a human intestinal disorder, wherein the medicament is administered subcutaneously on a biweekly administration regimen for treating said intestinal disorder, and the human antibody or portion thereof. antigen binding properties have the following properties:

-3 -1

a) detach from human TNF? with a Koff rate constant of 1 x 10^-3 s^-1 or less as determined by surface plasmon resonance;

b) have a light chain CDR3 domain containing the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 3 modified 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; and

c) have a heavy chain CDR3 domain containing the amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 4 modified 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.

PL 217 217 B1

A use is preferred that uses a human antibody or antigen-binding portion thereof that detaches from human TNF? At a rate constant K_off equal to 5 x 10^- s^- or less.

The invention also relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for treating a human intestinal disorder, wherein the medicament is administered subcutaneously on a biweekly administration regimen for treating said intestinal disorder, and the human antibody or portion thereof. the antigen binding agent has a light chain variable region (LCVR) having a CDR3 domain containing the amino acid sequence of SEQ ID NO: 3, or SEQ ED NO: 3 modified by a single alanine substitution at position 1, 4, 5, 7 or 8, and have a heavy chain variable region (HCVR) having a CDR3 domain containing the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 modified by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.

It is preferred to use a human antibody or antigen binding portion thereof, the LCVR further having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and the HCVR further having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6.

It is preferred to use a human antibody or antigen binding portion thereof, the LCVR further having a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and the HCVR having a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8.

The invention also relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for the treatment of a human intestinal disorder, wherein the medicament is administered subcutaneously on a biweekly administration regimen to treat said intestinal disorder, and the human antibody or portion thereof. antigen binding agents have a light chain variable region (LCVR) containing 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.

A use is preferred when a human antibody that has an IgG1 heavy chain constant region is used.

A use is preferred when a human antibody that has an IgG4 heavy chain constant region is used.

The use where an Fab fragment is used as the human antibody is preferred.

A use in which a single chain Fv fragment is used as the human antibody is preferred.

The invention also relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for the treatment of a human intestinal disorder, wherein the medicament is administered subcutaneously on a biweekly administration regimen for treating said intestinal disorder, and the human antibody or portion thereof. the antigen binding agent has a light chain variable region (LCVR) having a CDR3 domain containing 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, SEQ ID NO: 26 or has a region heavy chain variable (HCVR) having a CDR3 domain containing 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 and SEQ ID NO: 34.

The invention also relates to the use of an isolated human anti-TNFα antibody or antigen binding portion thereof in the manufacture of a medicament for treating a human intestinal disorder, wherein the medicament is administered subcutaneously on a biweekly administration regimen for treating said intestinal disorder, and the human antibody is D2E7 antibody or the antigen-binding portion thereof.

An application where the bowel disease is an inflammatory bowel disease is preferred.

The use where the inflammatory bowel disease is Crohn's disease or ulcerative colitis is preferred.

The invention also relates to a kit which comprises a formulation comprising:

a) a pharmaceutical composition comprising an anti-TNF? antibody, or antigen binding portion thereof, and a pharmaceutically acceptable carrier; and

b) biweekly dosing instructions for a pharmaceutical composition for treating an intestinal disorder for which the anti-TNF? antibody or antigen binding portion thereof is effective.

A kit that comprises the antibody or antigen-binding portion thereof as defined above is preferred.

PL 217 217 B1

The invention also relates to a ready-made syringe, which contains a pharmaceutical composition comprising an isolated human anti-TNF? Antibody or antigen binding portion thereof for the treatment of an intestinal disorder and a pharmaceutically acceptable carrier.

A syringe that contains the antibody or antigen-binding portion thereof as defined above as a human antibody is preferred.

The invention also relates to a kit which comprises a formulation comprising:

a) a pharmaceutical composition comprising an anti-TNFα antibody or antigen binding portion thereof, methotrexate and a pharmaceutically acceptable carrier; and

b) instructions for subcutaneously administering to a patient a pharmaceutical composition for treating a disorder wherein TNFα activity is undesirable, wherein said disorder is an intestinal disorder.

A kit that comprises the antibody or antigen-binding portion thereof as defined above is preferred.

The invention also relates to a finished syringe that contains a pharmaceutical composition comprising an anti-TNF? Antibody and a pharmaceutically acceptable carrier.

A syringe that comprises the antibody or antigen-binding portion thereof as defined above as the antibody is preferred.

Described herein are biweekly dosing regimens for the treatment of TNFα mediated disorders, preferably by the subcutaneous route. Biweekly dosing has many advantages over weekly dosing including, but not limited to, fewer total injections, fewer local reactions to injection (e.g. local pain and swelling), greater patient compliance (due to lower injection frequency) and lower costs for the patient as well as for the healthcare provider. Subcutaneous administration is advantageous because the patient can self administer the therapeutic substance, e.g. anti-human TNF? Antibody, which is convenient for both the patient and the health care provider.

Treatment of disorders in which TNFα activity is deleterious includes administering subcutaneous injections of antibodies to the patient every two weeks. The antibodies preferably include recombinant human antibodies that specifically bind human TNFα. In addition, treatment of disorders in which TNFα activity is deleterious involves the use of combination therapy and human antibodies are administered to the patient with another therapeutic agent, such as one or more additional antibodies that bind to other target molecules (e.g., antibodies that bind to other cytokines or which bind cell surface molecules), one or more cytokines, a soluble TNFα receptor (see e.g. PCT Publication No. WO 94/06476) and / or one or more chemicals that inhibit hTNFα production or activity (such as the cyclohexanoylidene derivatives disclosed in PCT Publication No. WO 93/19751), preferably methotrexate. The antibodies preferably include recombinant human antibodies that specifically bind human TNFα. The antibodies to be used according to the invention are characterized by high affinity binding to hTNFa and slow dissociation kinetics, and by neutralization of hTNFa activity, including hTNFa induced cytotoxicity (in vitro and in vivo) and hTNFa induced cell activation. The antibodies may be full length (e.g., an IgG1 or IgG4 antibody) or may include only the antigen binding portion (e.g., a Fab fragment, F (ab ') 2, scFv, or a single domain). The CDR3 domain of the light chain of the most preferred recombinant antibody for use according to the invention, denoted as D2E7, comprises the amino acid sequence of SEQ ID NO: 3, and the CDR3 domain of its heavy chain comprises the amino acid sequence of SEQ ID NO: 4 (shown in Appendix B / Sequence Listing). Preferably, the light chain variable region (LCVR) of the D2E7 antibody comprises the amino acid sequence of SEQ ID NO: 1 and its heavy chain variable region (HCVR) comprises the amino acid sequence of SEQ ID NO: 2. These antibodies are disclosed in US Patent No. 6,090,382.

In one embodiment, treatment of disorders in which TNFα activity is deleterious comprises inhibiting human TNFα activity by subcutaneously administering an anti-TNFα antibody every two weeks so as to treat the disorder. The disorder of the invention is an intestinal disorder, but the antibodies described herein may also be useful in the treatment of other disorders, such as an autoimmune disease (e.g. rheumatoid arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome), sepsis, infectious disease, malignancy, transplant rejection or graft versus host reaction, pulmonary disorder, bone disorder, or heart disorder.

Treatment of disorders in which TNFα activity is deleterious involves inhibiting the activity of human TNFα by subcutaneously administering an anti-TNFα antibody and methotrexate so as to

The disorder. In one aspect, methotrexate is administered together with an anti-TNF? Antibody. According to another aspect, the methotrexate is administered prior to the administration of the anti-TNF? Antibody. In yet another aspect, the methotrexate is administered following the administration of the anti-TNF? Antibody.

According to the invention, the anti-TNF? Antibody used to treat disorders in which TNF? Activity is deleterious is a human anti-TNF? Antibody. Treatment is carried out by subcutaneously administering the isolated human antibody or antigen-binding portion thereof subcutaneously every two weeks. The antibody, or antigen binding portion thereof, is preferably cleaved from human TNF? With a Kd constant of 1 x 10^-8 M or less and a Koff rate constant of 1 x 10^-3 s^-1 or less, both are determined by surface plasmon resonance, and neutralize the cytotoxicity of human TNFα in a standard in vitro L929 assay with an IC50 value of 1 x 10^-7 M or less. More preferably, the isolated human antibody, or antigen binding portion thereof, is detached

-4 -1 from human TNF? With a Koff constant of 5 x 10^-4 s^-1 or less, or even more preferably, with a Koff constant -4-1 equal to 1 x 10^-4 s^-1 or less. More preferably, the isolated human antibody, or antigen binding portion thereof, neutralizes the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 value of 1 x 10^-8 M or less, even more preferably with an IC50 value of 1 x 10^-9 M or less, and even more preferably with an IC50 value of 1 x 10^-10 M or less.

Kits can also be prepared comprising a formulation comprising a pharmaceutical composition further consisting of an anti-TNF? Antibody, methotrexate, and a pharmaceutically acceptable carrier. The kits contain instructions for subcutaneously administering the pharmaceutical composition to treat a disorder in which administration of an anti-TNF? Antibody is beneficial.

According to the invention, it is possible to treat disorders in which it is preferable to administer an anti-TNF? Antibody, comprising administering isolated human antibodies, or antigen-binding portions thereof, that bind human TNF? With high affinity, low dissociation rate, and high neutralization efficiency, so as to cure the disorder. The various aspects of the invention relate to treatment with antibodies and antibody fragments, and the pharmaceutical compositions thereof.

For an easier understanding of the invention, definitions of some terms are provided below.

As used herein, the terms "administration" and "dosing" refer to the administration of a substance (eg, anti-TNFα antibodies) to achieve a therapeutic goal (eg, treatment of a TNFα related disorder).

As used herein, the terms "biweekly dosing regimen", "biweekly dosing" and "biweekly dosing" refer to the time course of administration of a substance (e.g., anti-TNFα antibodies) to a patient so as to achieve the therapeutic goal (e.g. treatment of a TNFα related disorder). The biweekly administration regimen is not intended to be a weekly administration regimen. Preferably, the substance is administered every 9-19 days, more preferably every 11-17 days, even more preferably every 13-15 days and most preferably every 14 days.

The term "combination therapy" as used herein refers to the administration of two or more therapeutic substances, eg, an anti-TNFα antibody and a methotrexate drug. Methotrexate can be co-administered with, before, or after the administration of the anti-TNF? Antibody.

As used herein, the term "human TNF?" (Abbreviated herein as hTNF? Or simply hTNF) is intended to refer to a human cytokine that exists in a 17 kD secreted form and a 26 kD membrane-bound form that is biologically active the form consists of a trimer of 17 kD non-covalently linked molecules. The structure of TNFα is further described, for example, by Pennica, D., et al. (1984) Nature 312: 724-729; Davis, JM, et al. (1987) Biochemistry 26: 1322-1326; and Jones, EY, et al. (1989) Nature 338: 225-228. The term human TNF? Is intended to include recombinant human TNF? (RhTNF?) That can be produced by standard recombinant expression methods or purchased (R&D Systems, Catalog No. 210-TA, Minneapolis, MN).

The term "antibody" as used herein is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus 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 antibody fragments that retain the ability to specifically bind antigen (eg, hTNFa). It has been shown that the antigen-binding function of the antibody can be performed by full-length antibody fragments. Examples of binding fragments encompassed by 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 containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the 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 (Ward et al., (1989) Nature 341: 544-546) 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 encoded by separate genes, they can be joined using recombinant methods, with a synthetic linking group that allows them to be produced as a single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883 ). Such single chain antibodies are also intended to be encompassed by the term "antigen binding portion" of the antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed by this term. Dual specificity antibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linking group that is too short to allow pairing between the two domains on the same chain, thereby forcing pairing of these domains with the complementary domains of other chains and forming two antigen binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123).

In addition, the antibody, or antigen binding portion thereof, can be part of larger immunoadhesive molecules, formed by covalently or non-covalently linking the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesive molecules include the use of the streptavidin core region to generate 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 poly-histidine tag to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F (ab ') 2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. In addition, antibodies, antibody portions, and immunoadhesive molecules can be obtained using standard recombinant DNA techniques described herein.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies described herein may include amino acid residues unencoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g. in CDRs, and in particular CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into human framework sequences.

The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, produced, or isolated by recombinant techniques, such as those expressed using a recombinant expression vector that has been transfected into a host cell (described in more detail below in Section II), recombinant antibodies, combinatorial human antibody libraries (described further below in Section III), antibodies isolated from an animal (e.g., mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20: 6287 -6295), or antibodies prepared, expressed, produced, or isolated by any other means that include splicing human immunoglobulin gene sequences with other DNA sequences. In such recombinant human antibodies, the variable and constant regions are derived from human germline immunoglobulin sequences. Jed8

In some embodiments, such recombinant human antibodies are mutagenized in vitro (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences, which, while originating from and are associated with human germline VH and VL sequences, they may not naturally occur in the germline repertoire of human antibodies in vivo.

The term "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies with a different antigen specificity (e.g., 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 may have cross-reactivity to other antigens such as hTNFa molecules from other species (discussed in further detail below). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

As used herein, the term "neutralizing antibody" (or "antibody that neutralizes the activity of hTNFa") is intended to refer to an antibody whose binding to hTNFa results in inhibition of the biological activity of hTNFa. This inhibition of hTNF? Biological activity can be assessed by measuring one or more indicators of hTNF? Biological activity, such as hTNF? Induced cytotoxicity (in vitro or in vivo), hTNF? Induced cell activation and binding of hTNF? To hTNF? Receptors. These markers of hTNFa biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see Example 4). Preferably, the ability of the antibody to neutralize hTNF? Activity is assessed by inhibiting hTNF? Induced cytotoxicity of L929 cells. As an additional or alternative parameter of hTNFa activity, the ability of the antibody to inhibit hTNFa-induced ELAM-1 expression on HUVEC can be assessed as a measure of hTNFa-induced cell activation.

As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows real-time analysis of biospecific interactions by detecting changes in protein concentrations within the biological sensor matrix, e.g. using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). A more detailed description is given in 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: 268-277.

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

The term "Kd" as used herein is intended to refer to the dissociation constant for particular antibody-antigen interactions.

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

The term "isolated nucleic acid molecule," as used herein, refers to nucleic acids encoding antibodies or antibody portions (e.g. VH, VL, CDR3) which bind to hTNFa is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion do not contain other nucleotide sequences encoding antibodies or portions of antibodies that bind antigens other than hTNFa which other sequences may naturally flank a nucleic acid in the DNA of the human genome. So, for example An isolated nucleic acid encoding the VH region of an anti-hTNFa antibody described herein does not contain any sequences encoding other VH regions that bind to antigens other than hTNFa.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of carrying a nucleic acid other than to which it has been attached. One type of vector is a "plasmid", which is a circular loop of double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors including a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell genome when introduced into the host cell and thereby replicate with the host genome. Furthermore, certain vectors are capable of directing the expression of genes with which they have been operably linked. Such vectors are described herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this specification, the terms "plasmid" and "vector" may be used interchangeably since plasmid is the most commonly used form of vector. According to the invention, however, also other forms of expression vectors, such as viral vectors (e.g. replication-defective retroviruses, adenoviruses and adeno-associated viruses) that perform 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 an individual host cell but to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell but still fall within the scope of the term "host cell" as used herein.

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

I. Human Antibodies That Bind Human TNF?

According to the invention, there are possible treatment methods in which administration of an anti-TNF? Antibody is advantageous. These methods involve subcutaneous administration every two weeks of isolated human antibodies, or antigen-binding portions thereof, that bind human TNF? With high affinity, low dissociation rate, and high neutralizing capacity. Preferably, the human antibodies described herein are recombinant, neutralizing human anti-hTNFa antibodies. The most preferred recombinant neutralizing antibody described herein is referred to herein as D2E7 (the amino acid sequence of the D2E7 VL region is shown in SEQ ID NO: 1; the amino acid sequence of the D2E7 VH region is shown in SEQ ID NO: 2). The properties of D2E7 are described in Salfeld et al. In US Patent No. 6,090,382.

In one aspect, there are methods for treating disorders where administration of an anti-TNF? Antibody is beneficial. These treatments include subcutaneous administration of D2E7 antibodies and portions of the antibodies, antibodies and antibody portions related to D2E7 subcutaneously, and other human antibodies and antibody portions with properties equivalent to D2E7, such as high affinity binding to hTNFa with poor dissociation kinetics and high capacity neutralization. In one embodiment, an isolated human antibody, or antigen-binding portion thereof, that detaches from human TNFα with a Kd constant of 1 x 10, is used for treatment.^-8 M or less and a Koff rate constant of 1 x 10^-3 s^-1 or less, both determined by surface plasmon resonance, and neutralized the cytotoxicity of human TNFα in standard in vitro L929 assay with an IC50 value of 1 x 10^-7 M or less. More preferably, the isolated human antibody or antigen binding portion thereof is cleaved from human TNFα

-4 -1 -4 -1 Koff constant equal to 5 x 10^-4 s^-1 or less, or even more preferably, with a constant Koff equal to 1 x 10^-4 s^-1 or less. More preferably, the isolated human antibody or antigen binding portion thereof neutralizes the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 value of 1 x 10^-8 M or less, even more preferably with an IC50 value of 1 x 10^-9 M or less, and even more preferably with an IC50 -10 value of 1 x 10^-10 M or less. In a preferred embodiment, the antibody is an isolated human recombinant antibody or antigen-binding portion thereof.

It is well known in the art that antibody heavy and light chain CDR3 domains play an important role in the binding specificity / affinity of an antibody for an antigen. Accordingly, according to another aspect, it is possible in accordance with the invention to treat disorders in which it is preferable to administer an anti-TNF? Antibody by subcutaneously administering human antibodies that have slow dissociation kinetics for association with hTNF? And that include the CDR3 domains of the light and heavy chain. which are structurally identical to or related to the domains of D2E7. Position 9 of the VL D2E7 CDR3 domain can be Ala or Thr without substantially affecting Koff. Accordingly, the consensus motif for the D2E7 VL CDR3 domain comprises the amino acid sequence: QRYNRAPY- (T / A) (SEQ ID NO: 3). Moreover, the 12th position of the CDR3 VH D2E7 domain may be occupied by Tyr or Asn, without substantially affecting Koff. Accordingly, the consensus motif for CDR3 VH D2E7 comprises the amino acid sequence: VSYLSTASSLD- (Y / N) (SEQ ID NO: 4). Furthermore, as shown in Example 2, the D2E7 heavy and light chain CDR3 domain can be substituted with a single alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within VH CDR3) without substantially affecting Koff. Furthermore, it will be apparent to one skilled in the art that with the susceptibility of the CDR3 VL and VH domains of D2E7 to alanine substitutions, the substitution of other amino acids within the CDR3 domain may be possible while still maintaining a low constant rate of antibody dissociation, in particular

With serous amino acids. The term "conservative amino acid substitution" as used herein means that in which one amino acid residue is replaced with another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are known in the art and include basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, methionine ), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Preferably, no more than one to five conservative amino acid substitutions are made within the CDR3 VL and / or VH domains of D2E7. More preferably no more than one to three conservative amino acid substitutions are made within the CDR3 VL and / or VH domains of D2E7. Moreover, conservative amino acid substitutions should not be made at critical amino acid positions for binding to hTNFa. Positions 2 and 5 of CDR3 VL D2E7 and positions 1 and 7 of CDR3 VH D2E7 appear to be critical for interaction with hTNFa, and therefore preferably no conservative amino acid substitutions are introduced at these positions (although an alanine substitution at position 5 of CDR3 VL D2E7 is acceptable, which described above) (see U.S. Patent No. 6,090,382).

Accordingly, in another embodiment of the invention, it is possible to treat disorders in which it is preferable to administer an anti-TNF? Antibody by subcutaneously administering an isolated human antibody or antigen binding portion thereof subcutaneously every two weeks. The antibody, or antigen binding portion thereof, preferably has the following properties:

-3 -1

a) cleaves from human TNF? at a Koff rate constant of 1 x 10^-3 s^-1 or less as determined by surface plasmon resonance;

b) has a light chain CDR3 domain containing the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 3 modified 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 containing the amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 4 modified 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 antigen binding portion thereof is cleaved from human TNF? With a Koff constant of 5 x 10^-4 s^-1 or less. Even more preferably, the antibody, or anti-4 -1 gene binding portion thereof, is detached from human TNF? With a Koff constant of 1 x 10^-4 s^-1 or less.

In yet another embodiment, the invention may treat disorders in which it is preferable to administer an anti-TNF? Antibody by subcutaneously administering an isolated human antibody or antigen binding portion thereof subcutaneously every two weeks. The antibody or antigen binding portion thereof preferably has a light chain variable region (LCVR) comprising a CDR3 domain comprising the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 3 modified by a single alanine substitution at position 1, 4, 5, 7, or 8, and heavy chain variable region (HCVR) comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 modified by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further comprises a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e. D2E7 VL CDR2) and the HCVR further comprises a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (i.e. CDR2 VH D2E7). Even more preferably, the LCVR further comprises a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e. D2E7 VL CDR1) and the HCVR comprises a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 (i.e. VH CDR1 D2E7). The framework regions for VL are preferably derived from a human germline V family_K.I, more preferably the V gene_k human germline A20 and most preferably the VL framework sequences D2E7 shown in Figures 1A and 1B of US Patent No. 6,090,382. The framework regions for VH are preferably derived from the human germline VH3 family, more preferably from the human germline VH gene DP-31 and most preferably from the sequence VH framework D2E7 shown in Figures 2A and 2B of US Patent No. 6,090,382.

In yet another embodiment, it is possible in accordance with the invention to treat disorders in which it is preferable to administer an anti-TNF? Antibody by subcutaneously administering an isolated human antibody, or antigen binding portion thereof, every two weeks. The antibody or antigen binding portion thereof preferably has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 (i.e. VL D2E7) and a heavy chain variable region (HCVR) including

The amino acid sequence of SEQ ID NO: 2 (ie VH D2E7). In some embodiments, the antibody has a heavy chain constant region such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In addition, the antibody may include a light chain constant region or a kappa light chain constant region or a lambda light chain constant region. Preferably the antibody has a kappa light chain constant region. Alternatively, the antibody portion can include, for example, a Fab fragment or a single chain Fv fragment.

An antibody or antigen binding portion thereof that comprises a D2E7 cognate CDR3 VL and VH domain may also be useful, e.g. antibodies or antigen-binding portions thereof with a light chain variable region (LCVR), which comprises a CDR3 domain containing 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) that comprises a CDR3 domain containing 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.

An antibody, or portion of an antibody, as described herein may be modified or linked to another functional molecule (e.g., another peptide or protein). Accordingly, the antibodies and antibody portions described herein are intended to include derivatized and otherwise modified forms of the human anti-hTNFa antibodies described herein, including immunoadhesive molecules. For example, an antibody or antibody portion described herein may be operably linked (by chemical conjugation, genetic fusion, non-covalent linkage, or otherwise) to one or more molecular entities, such as another antibody (e.g. bispecific antibody or dual specificity antibody), detectable agent, cytotoxic agent, pharmaceutical agent, and / or a protein or peptide that can mediate the linkage of the antibody or antibody portion to another molecule (such as the streptavidin core region or polyhistidine marker).

One type of a derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, have two differently reactive groups separated by a suitable spacer (e.g.

m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g. disuccinimidyl suberate). Such linking groups are available from Pierce Chemical Company, Rockford, IL.

Useful detectable agents to which the antibody or portion of the antibody described herein can be combined include fluorescent compounds. Exemplary detectable fluorescent agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. The antibody may also be modified by binding to detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When the antibody is modified by binding to a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when horseradish peroxidase is used as a detectable agent, the addition of hydrogen peroxide and diaminobenzidine produces a detectable colored reaction product. The antibody can also be modified by combining with biotin and detected by indirect measurement of avidin or streptavidin binding.

II. Antibody expression

The antibody or portion of the antibody for use in the present invention can be produced by recombinant expression of the immunoglobulin light and heavy chain genes in a host cell. To obtain recombinant expression of the antibody, the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the antibody light and heavy chains such that the light and heavy chains are expressed in the host cell and preferably secreted into the cell culture medium. hosts from which the antibody can be recovered. Standard recombinant DNA methodologies such as those described by Sambrook, Fritsch, and Maniatis (eds.) Are used to obtain the antibody heavy and light chain genes, to integrate the genes into recombinant expression vectors, and to introduce the vectors into host cells. 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 U.S. Patent No. 4,816,397 to Boss et al.

In order to express D2E7 or a D2E7 related antibody, first, DNA fragments encoding the variable regions of the light and heavy chains are obtained. These DNA fragments can be obtained by amplifying and modifying the germline light and heavy chain variable sequences using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see e.g. 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 Service, NIH Publication No. 913242; Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" J. Mol. Biol. 227: 776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V78 Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24: 827-836). To obtain a DNA fragment encoding the heavy chain variable region of D2E7 or a D2E7 related antibody, a gene belonging to the VH3 family of human germline VH genes is amplified by standard PCR. Most preferably the DP-31 VH germline sequence is amplified. In order to obtain a DNA fragment encoding the variable light chain region of D2E7 or an antibody related to D2E7, a gene from the V family is amplified._K.And the human germline VL genes are standard PCR. Most preferably the germline VL A20 sequence is amplified. PCR primers suitable for use in amplifying the germline VH DP-31 and the germline VL A20 sequences can be designed based on the nucleotide sequences disclosed in the references cited above using standard methods.

After the germline VH and VL fragments have been obtained, these sequences can be mutated to encode the D2E7 or D2E7 related amino acid sequences disclosed in this specification. The amino acid sequences encoded by the germline VH and VL DNA sequences are first compared with the D2E7 or D2E7 related or D2E7 related VH and VL amino acid sequences to identify D2E7 or D2E7 related amino acid residues that differ from germline. The appropriate nucleotides of the germline DNA sequence are then mutated such that the mutant germline sequence encodes the D2E7 or related D2E7 amino acid sequence using the genetic code to determine what nucleotide changes should be made. Germline sequence mutagenesis is performed by standard methods such as PCR-based mutagenesis (in which mutated nucleotides are inserted into PCR primers such that the PCR product contains mutations) or site-directed mutagenesis.

After obtaining the DNA fragments encoding the VH and VL segments of D2E7 or related to D2E7 (by amplification and mutagenesis of the germline VH and VL genes as described above), these DNA fragments can then be manipulated using standard recombinant DNA techniques, e.g. the variable region into the genes of the full-length chain of an antibody, into the genes of a Fab fragment, or into the scFv gene. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker group. The term "operably linked" as used herein is intended in this context to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in reading frame.

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

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

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

To express the antibodies or antibody portions for use in the invention, DNAs encoding the partial or full length light and heavy chains obtained as described above are inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated to the vector such that the transcriptional and translation control sequences within the vector serve their function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the D2E7 light or heavy chain sequences or related to D2E7, the expression vector may already contain antibody constant region sequences. For example, one way to convert D2E7 or related to D2E7 VH and VL sequences into full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant regions and light chain constant regions, respectively, so that the VH segment is operably linked to the (- a) CH within the vector and the VL segment has been operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can 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 frame to the amino terminus of the antibody chain gene. The signal peptide may include an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors described herein carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" as used herein 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 e.g. Goeddel; Gene Expression Technology; Methods in EnzymoIogy 185, Academic Press, San Diego, CA (1990). It should be apparent to those skilled in the art 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 desired level of protein expression, etc. Preferred regulatory sequences for expression of a mammalian host cell include viral elements which drive high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV) promoters and / or enhancers (such as CMV promoter / enhancer), Simian 40 virus ( SV40) (such as SV4 0 promoter / enhancer), adenovirus (e.g., major adenoviral late promoter (Ad-MLP)), and polyoma virus. For a detailed description of viral regulatory elements and their sequences, see, e.g., U.S. Patent Nos. 5,168,062, Stinski, No. 4,510,245, Bell et al. and No. 4,968,615, Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors described herein can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., US Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate on a host cell into which the vector has been introduced. Preferred selectable marker genes14

These include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, expression vector (s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to include various techniques commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. While it is theoretically possible to express the antibodies for use in the present invention in prokaryotic or eukaryotic host cells, it is most preferred to express the antibodies in eukaryotic cells, and most preferably in mammalian host cells, as such eukaryotic cells, and in particular mammalian cells, are more likely to do so. and not prokaryotic cells, will assemble and secrete a properly folded and immunologically active antibody. It has been reported that prokaryotic expression of antibody genes does not effectively produce large amounts of active antibody (Boss, MA and Wood, CR (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for the expression of recombinant antibodies for use in the invention include Chinese hamster ovary (CHO cells) cells (including dhfr- CHO cells, described by UrIaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77 : 42164220, used with the DHFR selectable marker, e.g. described by RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding the antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to express the antibodies in the host cells or, more preferably, to secrete the antibodies into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to generate portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that changes to the above procedure are also possible. 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 the antibody for use in the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding the light and / or heavy chains that is not required for hTNFa binding. Antibodies for use in the invention also include molecules expressed from such truncated DNA molecules. In addition, bifunctional antibodies can be made in which one heavy chain and one light chain constitute the antibody for use in the invention and the other heavy and light chain are specific for an antigen other than hTNFa by cross-linking the antibody for use in accordance with the invention with a second antibody by standard cross-linking methods. chemical.

In a preferred recombinant expression system of an antibody or antigen binding portion thereof for use in the present invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate mediated transfection. Within a recombinant expression vector, all antibody heavy and light chain genes are operably linked to regulatory elements: CMV enhancer / AdMLP promoter to lead to high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which allows the selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. Selected transformed host cells are grown to allow expression of the antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover the antibody from the culture medium.

III. Selection of recombinant human antibodies

Recombinant human antibodies for use in accordance with the invention in addition to D2E7 or an antigen-binding portion thereof, or the antibodies related to D2E7 disclosed in this specification can be isolated by screening a recombinant combinatorial antibody library, preferably an scFv phage display library, produced using human VL and VH cDNA obtained with mRNA derived from human lymphocytes. Manufacturing and searching methodologies

Such libraries are known in the art. In addition to commercially available kits for the production of phage display libraries (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and the Stratagene Surf / ZAP phage display kit)^TM, catalog no.240612), examples of methods and reagents particularly suitable for use in the production and screening of the display of antibody libraries can be found, e.g. in U.S. Patent No. 5,223,409 to Ladner et al; PCT Publication No. WO 92/18619 to Kang et al .; PCT Publication No. WO 91/17271 to Dower et al .; PCT Publication No. WO 92/20791 to Winter et al; PCT Publication No. WO 92/15679 to Markland et al .; PCT Publication No. WO 93/01288 by Breitling et al; PCT Publication No. WO 92/01047 to McCafferty et al .; PCT Publication No. WO 92/09690 to Garrard et al .; 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) J. Mol. Biol. 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 35763580; Garrard 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 low dissociation rate constant for hTNFa, a murine anti-hTNFa antibody with high affinity and low detachment rate constant for hTNFa is first used (e.g. MAK 195, for which the hybridoma has deposit number ECACC 87 050801), to select human heavy and light chain sequences with similar binding activity for hTNFa, using epitope imprinting methods described in PCT Publication No. WO 93/06213, Hoogenboom et al. Antibody libraries used in this method preferably include scFv libraries generated and screened as described in PCT Publication No. WO 92/01047, McCafferty et al; McCafferty et al., Nature (1990) 348: 552-554; and Griffiths et al. (1993) EMBO J 12: 725-734. Libraries of scFv antibodies are preferably screened using recombinant human TNF? As the antigen.

After selecting the starting human VL and VH segments, mix and match experiments are performed in which different pairs of the initially selected VL and VH segments are screened for hTNFa binding to select preferred combinations of VL / VH pairs. Furthermore, to further increase the affinity and / or reduce the dissociation rate constant for hTNFa binding, the VL and VH segments of the preferred VL / VH pair (s) may be randomly mutated, preferably within the VH and / or VL CDR3 region, in a process analogous to the in vivo somatic mutation process responsible for the maturation of antibody affinity during the natural immune response. This in vitro affinity maturation can be achieved by amplifying the VH and VL regions using PCR primers complementary to CDR3 VH or CDR3 VL, respectively, which primers have been "bristled" with a random mixture of four nucleotide bases at certain positions so that the resulting PCR products encode VH segments and VLs into which random mutations have been introduced into the VH and / or VL region of CDR3. These randomly mutated VH and VL segments can be re-screened for binding to hTNFa, and sequences that exhibit high affinity and low dissociation rate for hTNFa binding can be selected.

After screening for and isolating an anti-hTNFa antibody for use according to the invention from a recombinant immunoglobulin display library, the nucleic acid encoding the selected antibody can be recovered from the display packaging (e.g., from the phage genome) 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 antibodies for use in the invention (e.g., linked to nucleic acid encoding additional immunoglobulin domains, such as additional constant regions). To express a recombinant human antibody recovered by screening a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into mammalian host cells as described in more detail in Section II above.

IV. Pharmaceutical Compositions and Their Administration

The antibodies and antibody portions for use in accordance with the invention can be incorporated into pharmaceutical compositions suitable for administration to a patient in the methods disclosed herein, e.g., for subcutaneous administration every two weeks. Typically, a pharmaceutical composition comprises an antibody (or antibody portion) for use in the invention and / or methotrexate and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein

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 and suitable for administration to a patient in the methods disclosed herein. Examples of pharmaceutically acceptable carriers include one or more ingredients selected from the group consisting of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, the composition more preferably comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers can further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which prolong the shelf-life or enhance the effectiveness of the antibody or antibody portion.

The compositions described herein may take a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion 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 injection or infusion solutions, such as compositions similar to those used in passively immunizing humans with other antibodies. A parenteral mode of administration is preferred (e.g., intravenously, subcutaneously, intraperitoneally, intramuscularly). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular injection. In a particularly preferred embodiment, the antibody is administered by subcutaneous injection (e.g., by subcutaneous injection every two weeks).

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, liposomes or other ordered structures suitable for high concentrations of the active ingredient. Sterile injectable solutions can be prepared by incorporating the active compound (i.e. antibody or antibody portion) 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 preparation methods are vacuum drying and freeze-drying, which produces a powder of the active ingredient with any additional desired ingredient from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, e.g. by the use of a coating, e.g. from lecithin, by maintaining the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

The antibodies and antibody portions described herein can be administered by a variety of methods known in the art, although for many therapeutic applications, the route / mode of administration according to the invention is subcutaneous injection. As is known to those skilled in the art, the route and / or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound can be combined with a carrier that will protect the compound against burst, such as in a controlled release formulation, including implants, transdermal and microencapsulated delivery systems. Biodegradable, biocompatible polymers can also be used, such as ethylene vinyl acetate, polyethylene glycol (PEG), polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such preparations are patented or known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed. Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion described herein can be administered orally, e.g., with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the compounds can be formulated with excipients and used in the form of swallow tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For non-parenteral administration of a compound described herein, it may be necessary to coat the compound or using the compound together with a material to prevent its inactivation.

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Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the antibody or antibody portion for use in the invention is co-formulated with and / or co-administered with one or more additional therapeutic agents. For example, an anti-hTNFa antibody or antibody portion for use in the invention can be co-formulated with and / or co-administered with methotrexate, one or more additional antibodies that bind other target molecules (e.g., antibodies that bind other cytokines or that bind surface molecules). cells), one or more cytokines, a soluble TNFα receptor (see e.g. PCT Publication No. WO 94/06476) and / or one or more chemicals that inhibit hTNF? production or activity (such as the cyclohexanoylidene derivatives disclosed in PCT Publication No. 93/19751). In addition, one or more antibodies described herein can be used in combination with two or more of the above therapeutic agents. Such combination therapies may advantageously employ lower doses of the therapeutic agents administered, thereby avoiding possible toxic effects or complications associated with the various monotherapies. The use of the antibodies or antibody portions described herein in combination with other therapeutic agents is further discussed in Section IV.

Examples of rheumatoid arthritis therapeutic agents to which an antibody or antibody portion can be combined for use in the invention include: non-steroidal anti-inflammatory drug (s) (NSAIDs); cytokine inhibitory anti-inflammatory drug (s) (CSAID); CDP-571 / BAY-10-3356 (humanized anti-TNF? Antibody; Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75kdTNFR-IgG (75kD TNF IgG receptor fusion protein; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF-IgG receptor fusion protein; Hoffmann-LaRoche); IDEC-CE9.1 / SB 210396 (anti-CD antibody with a small primate protein insert, no T cell depletion; IDEC / SmithKline; see, e.g., Arthritis & Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see e.g. Arthritis & 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 (e.g., agonist antibodies); IL-1RA (IL-1 Receptor Antagonist; Synergen / Amgen); TNFbp / s-TNFR (soluble TNF binding protein; see, e.g., Arthritis & 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, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S282); MK966 (COX-2 inhibitor; see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S81); Iloprost (see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S82); methotrexate; thalidomide (see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S282) and thalidomide related drugs (e.g. Celgen); Ieflunomide (anti-inflammatory drug and cytokine inhibitor; see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid (plasminogen activation inhibitor; see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S284); T-614 (cytokine inhibitor; see, e.g., Arthritis & amp; Rheumatism (1996) Vol. 39, No. 9 (Supplement), S282); prostaglandin E1 (see e.g. Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S282); Tenidap (a non-steroidal anti-inflammatory drug; see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S280); naproxen (a non-steroidal anti-inflammatory drug; see e.g., Neuro Report (1996) vol 7, pp. 1209-1213); meloxicam (a non-steroidal anti-inflammatory drug); ibuprofen (a non-steroidal anti-inflammatory drug); piroxicam (a non-steroidal anti-inflammatory drug); diclofenac (a non-steroidal anti-inflammatory drug); indomethacin (a non-steroidal anti-inflammatory drug); sulfasalazine (see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S281); azathioprine (see e.g. Arthritis & amp; Rheumatism (1996) Vol. 39, No. 9 (Supplement), S281); ICE inhibitor (inhibitor of the enzyme that converts the enzyme interleukin 1β); a zap-70 and / or Ick inhibitor (zap-70 tyrosine kinase inhibitor or drug); a VEGF inhibitor and / or a VEGF-R inhibitor (vascular epithelial cell growth factor inhibitors or vascular epithelial cell growth factor receptor; angiogenesis inhibitors); corticosteroid anti-inflammatory drugs (e.g. SB203580); TNF convertase inhibitors; anti-IL-12 antibodies; interleukin-11 (see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S296); interleukin-13 (see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (Supplement), S308); interleukin-17 inhibitors (see e.g. Arthritis & amp; Rheumatism (1996) Vol. 39, No. 9 (Supplement), S120); gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total radiotherapy of the lymphatic system; anti-thymocytic globulin; anti-CD4 antibodies; CD5 toxin; orally administered

Peptides and collagen; lobenzarite disodium salt; cytokine regulating agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense oligodeoxynucleotide phosphorothioates (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulfate; minocycline; anti-IL2R antibodies; lipids from marine animals and plants (fatty acids from fish and plant seeds; see e.g. DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21: 759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilosis (terafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.

Examples of inflammatory bowel disease therapeutic agents to which the antibody or antibody portion for use in the invention can be combined include the following: budesonide; epidermal growth factor; corticosteroids; cyclosporine, sulfasalazine; aminosalicylates;

6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalazine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinylimidazole compounds; CDP-571 / BAY-10-3356 (humanized anti-TNF? Antibody; Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF-IgG receptor fusion protein; Immunex; see, e.g., Arthritis & Rheumatism (1994) vol. 37, S295; J. Invest. Med. (1996) vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF-IgG receptor fusion protein; Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plow); IL-4; IL-10 and / or IL-4 agonists (e.g. agonist antibodies); interleukin-11; glucuronide or dextran conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-1 antisense oligodeoxynucleotide phosphorothioates (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.

Examples of multiple sclerosis therapeutic agents to which the antibody or antibody portion for use in the invention can be combined include the following: corticosteroids; prednisolone; methylprednisone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-ji1a (Avonex ™. Biogen); interferons (Betaseron ™; Chiron / Berlex); Copolymer 1 (Cop-1; Copaxone ™. Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; cladribine; CDP571 / BAY-10-3356 (humanized anti-TNF? Antibody; Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF-IgG receptor fusion protein; Immunex; see, e.g., Arthritis & Rheumatism (1994) vol. 37, S295; J. Invest. Med. (1996) vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF-IgG receptor fusion protein; Hoffmann-LaRoche); IL-10; IL-4; and IL-10 and / or IL-4 agonists (e.g., agonist antibodies).

Examples of sepsis therapeutic agents to which the antibody or antibody portion for use in the invention can be combined include the following: hypertonic saline solutions; antibiotics; intravenous gamma globulin; continuous hemofiltration; carbapenems (e.g. meropenem) antagonists of such cytokines as TNFα, IL-1β, IL-6 and / or IL-8 CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody Celltech / Bayer); cA2 (a Centocor chimeric anti-TNFa antibody); 75 kdTNFR-IgG (75 kD TNF-IgG receptor fusion protein; Immunex; see, e.g., Arthritis & Rheumatism (1994) vol. 37, S295; J. Invest. Med. (1996) vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF-IgG receptor fusion protein; Hoffmann-LaRoche); cytokine regulating agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (small molecule 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 low molecular weight methylxanthine; Target Therapeutics, Inc.); PGG-Glycan (aqueous soluble β1,3-glycan; Alfa-Beta Technology); apolipoprotein A1 dissolved in lipids; chiral hydroxamic acids (synthetic antibacterial agents that inhibit lipid A biosynthesis); anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPl21 (a recombinant N-terminal fragment of a human bactericidal / permeation enhancer); and synthetic anti-endotoxin peptides (SAEP; BiosYnth Research Laboratories).

Examples of adult respiratory distress syndrome (ARDS) therapeutic agents to which the antibody or antibody portion can be combined for use in accordance with

The present invention includes the following: anti-IL-8 antibodies; surfactant replacement therapy; CDP571 / BAY-10-3356 (humanized anti-TNF? Antibody; Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFRIgG (75 kD TNF-IgG receptor fusion protein; Immunex; see, e.g., Arthritis & Rheumatism (1994) vol. 37, S295; J. Invest. Med. (1996) vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNF-IgG receptor fusion protein; Hoffmann-LaRoche).

The pharmaceutical compositions described herein may contain a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion for use in the invention. The term "therapeutically effective amount" refers to an amount effective, at dosages and for required periods of time, to achieve the desired therapeutic effect. A therapeutically effective amount of the antibody or antibody portion can be varied depending upon factors such as the subject's disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit the desired response in that subject. A therapeutically effective amount also means one in which any toxic or deleterious effects of the antibody or antibody portion outweigh any of the therapeutically beneficial effects. The term "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time as required, to achieve the desired prophylactic effect. Typically, because a prophylactic dose is used in patients with pre- or earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosing regimens can be adjusted to provide the optimal response desired (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the therapeutic indications. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units used as unitary dosages for the mammals to be treated as patients; each unit containing a predetermined quantity of active compound providing the desired therapeutic effect in association with the required pharmaceutical carrier. The type of unit dosage form is dictated by and directly dependent upon (a) the unique properties of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the formulation of such active compound for treatment due to the sensitivity of the patients.

An exemplary range of a therapeutically or prophylactically effective amount of an antibody or antibody portion described herein is 10-100 mg, more preferably 20-80 mg, and most preferably about 40 mg. It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. Furthermore, it is to be understood that for any particular patient, specific modes of administration should be adjusted over time in accordance with the individual needs and professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges provided in this specification are exemplary only and are not intended to limit the practical application of the described compositions. here the composition.

V. Applications of antibodies

Knowing about their ability to bind hTNFa, anti-hTNFa antibodies or parts thereof can be used to detect hTNFa (e.g. in a biological sample such as serum or plasma) using a standard immunoassay such as an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay ( RIA) or tissue immunohistochemistry. Thus, a method of detecting hTNFa in a biological sample is possible comprising contacting the biological sample with an antibody or antibody portion described herein and detecting either the hTNFa-bound antibody (or antibody portion) or unbound antibody (or antibody portion) to thereby detect hTNF? In the biological sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive substances. 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 complexes; examples of suitable fluorescent materials include umbeliferon, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; and examples of suitable radioactive substances include¹²⁵AND, ¹³¹AND, ³⁵Wedding ³H.

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As an alternative to labeling the antibody, hTNF? Can be determined in biological fluids by a competitive immunoassay, using rhTNF? Standards labeled with a detectable substance and an unlabeled anti-hTNF? Antibody. In this assay, a biological sample, labeled rhTNFa standards, and an anti-hTNFa antibody are pooled and the amount of labeled rhTNFa standard bound to the unlabelled antibody is determined. The amount of hTNFa in the biological sample is inversely proportional to the amount of labeled hTNFa standard bound to the anti-hTNFa antibody.

Antibody D2E7 as described herein can also be used to detect TNF? From non-human species, in particular TNF? From primates (e.g., chimpanzee, baboon, marmoset, cynomolgus and rhesus macaque), porcine and mice since D2E7 can each bind to these TNF?

The antibodies and antibody portions described herein are capable of neutralizing hTNFα activity both in vitro and in vivo (see US Patent No. 6,090,382). In addition, at least some of the antibodies described herein, such as D2E7, can neutralize hTNFa activity from other species. Accordingly, the antibodies and antibody portions described herein can be used to inhibit the activity of hTNFa, e.g. in hTNF? -containing cell culture, in human subjects, or in other mammalian subjects having TNF? with which the antibody cross-reacts (e.g., chimpanzee, baboon, marmoset, cynomolgus and rhesus monkeys, pig or mouse). Thus, a method of inhibiting TNFα activity is possible which comprises contacting the TNFα with the antibody or portion of the antibody such that the TNFα activity is inhibited. Preferably, the TNFα is human TNFα. For example, in a cell culture containing or possibly containing TNF? An antibody or a portion of an antibody can be added to the culture medium to inhibit hTNF? Activity in the culture.

In a preferred embodiment, it is possible to treat disorders in which the administration of an anti-TNF? Antibody is beneficial, comprising subcutaneously administering the antibody or antibody portion to the patient every two weeks so as to treat the disorder. In a particularly preferred embodiment, the antibody is administered subcutaneously once every two weeks. In another particularly preferred embodiment, the antibody is administered subcutaneously before, during, or after the administration of methotrexate. Preferably, the subject is a human. Alternatively, the subject may be a mammal that expresses TNF? With which an antibody cross-reacts as described herein. In addition, the subject may be a mammal into which hTNFa has been introduced (e.g., by administration of hTNFa or by expression of a transgenic hTNFa gene). An antibody described herein can be administered to a human for therapeutic purposes (discussed further below). In addition, an antibody described herein can be administered to a non-human mammal that expresses TNF? With which the antibody cross-reacts (e.g., a primate, pig, or mouse) for veterinary purposes or as part of an animal model of human disease. Considering the latter, such animal models may be useful for evaluating the therapeutic efficacy of the antibodies described herein (e.g., examining dosages and time courses of administration).

As used herein, the term "disorder in which administration of an anti-TNF? Antibody is beneficial" is intended to include diseases and other disorders in which the presence of TNF? In the subject suffering from the disorder has been shown or is suspected to be responsible for the pathophysiology of the disorder, or is a factor that contributes going to worsen the disorder, or where it has been shown that another anti-TNF? antibody or biologically active portion thereof is successfully used to treat a disease. Accordingly, a disorder in which TNF? Activity is deleterious includes a disorder in which inhibition of TNF? Activity is expected to alleviate the symptoms and / or the progress of the disorder. Such disorders can be identified, e.g., by increasing the concentration of TNF? In the biological fluid of the subject suffering from the disorder (e.g. increasing the concentration of TNF? In serum, plasma, synovial fluid, etc. of a subject) which can be detected using, e.g., an anti-TNFa antibody as described above. There are numerous examples of disorders in which TNFα activity is detrimental. According to the invention, the antibody is used to treat intestinal disorders

G. Intestinal disorders

Tumor necrosis factor is involved in the pathophysiology of inflammatory intestinal disorders (see, e.g., Tracy, KJ, et al. (1986) Science 234: 470-474; Sun, XM., Et al. (1988) J. Clin. Invest. 81: 1328-1331; MacDonald, TT, et al. (1990) Clin. Exp. Immunol. 81: 301-305). Chimeric murine anti-hTNFα antibodies have been clinically tested for the treatment of Crohn's disease (van Dullemen, HM, et al. (1995) Gastroenterology 109: 129-135). Thus, the human antibodies and antibody portions of the invention can be used to treat intestinal disorders such as idiopathic inflammatory bowel disease, which includes the two syndromes, Crohn's disease and ulcerative colitis.

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The use of the antibodies and antibody portions described herein in the treatment of other specific disorders is further discussed below.

B. Sepsis

Tumor necrosis factor has an established role in the pathophysiology of sepsis, with biological effects that include hypotension, myocardial suppression, vascular leakage, organ necrosis, stimulation of the release of toxic secondary mediators, and activation of the coagulation cascade (see, e.g., 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, the human antibodies and antibody portions described herein can be used to treat sepsis in any of its clinical settings, including septic shock, endotoxin shock, gram-negative sepsis, and toxic shock syndrome.

In addition, for the treatment of sepsis, an anti-hTNFa antibody or antibody portion described herein may be co-administered with one or more additional therapeutic agents that may further ameliorate sepsis, such as an interleukin-1 inhibitor (such as disclosed in PCT publications No. WO 92 / 16221 and WO 92/17583), the cytokine interleukin-6 (see e.g. PCT Publication No. WO 93/11793) or a platelet activating factor antagonist (see e.g. European Patent Application Publication EP 374510).

Further, in a preferred embodiment, an anti-TNF? Antibody or antibody portion as described herein is administered to a human subgroup of sepsis patients having a serum or plasma IL-6 concentration above 500 pg / ml, and more preferably 1000 pg / ml, during treatment ( see PCT Publication No. WO 95/20978, Daum, L., et al.).

C. Autoimmune diseases

Tumor necrosis factor is involved in the pathophysiology of a variety of autoimmune diseases. For example, TNFα is involved in activating tissue inflammation and causing joint damage in rheumatoid arthritis (see, e.g., 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). TNFα is also involved in promoting islet cell death and mediating the development of insulin resistance in diabetes mellitus (see, e.g., Tracey and Cerami, supra; PCT Publication No. WO 94/08609). TNFα is also involved in mediating oligodendrocyte cytotoxicity and the induction of inflammatory plaques in multiple sclerosis (see, e.g., Tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFa antibodies have been clinically tested for the treatment of rheumatoid arthritis (see, e.g., 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).

The human antibodies and antibody portions described herein can be used to treat autoimmune diseases, particularly those related to inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis. eye and nephrotic syndrome. Typically the antibody or antibody portion is administered systemically, although in certain disorders, local administration of the antibody or antibody portion to the site of inflammation may be beneficial (e.g., local administration to joints in rheumatoid arthritis or topical application to diabetic ulcers, alone or in combination with cyclohexanoylidene derivative as described in PCT Publication No. WO 93/19751).

D. Infectious diseases

Tumor necrosis factor is involved in mediating the biological effects seen in a variety of infectious diseases. For example, TNF? Is involved in mediating encephalitis and capillary thrombosis and infarction in malaria (see, e.g., Tracey and Cerami, supra). TNFα is also involved in mediating encephalitis, inducing the disruption of the blood-brain barrier, inducing septic shock syndrome, and activating venous infarction in meningitis (see e.g. Tracey and Cerami, supra). TNFα is also involved in inducing cachexia, stimulating viral proliferation, and mediating central nervous system damage in acquired immune deficiency syndrome (AIDS) (see, e.g., Tracey and Cerami, supra). Accordingly, the antibodies and antibody portions described herein can be used to treat infectious diseases, including bacterial meningitis (see, e.g. European Patent Application EP 585,705), malaria cerebral complications, AIDS and AIDS related complex (ARC) (see e.g. European Patent Application Publication No. EP 230574), as well as cytomegalovirus infection secondary to transplantation (see e.g. Fietze, E. , et al (1994) Transplantion 58: 67522

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-680). The antibodies and antibody portions described herein can also be used to alleviate symptoms associated with infectious diseases, including fever and myalgia caused by infection (such as influenza) and cachexia secondary to infection (e.g., secondary to AIDS or ARC).

E. Transplantation

Tumor necrosis factor is a key mediator of allograft rejection and graft-versus-host (GVHD) rejection and is involved in mediating the deleterious response observed when the OKT3 rat antibody directed against the CD3 T-cell receptor complex is used to inhibit rejection of kidney transplants (see e.g. ., Tracey and Cerami, supra; Eason, JD, et al (1995) Transplantion 59: 300-305; Suthanthiran, M. and Strom, TB (1994) New Engl. J. Med. 331: 365-375). Accordingly, the antibodies and antibody portions described herein can be used to inhibit graft rejection, including allograft and xenograft rejection, and to inhibit GVHD. Although the antibody or antibody portion may be used alone, it is more preferably used in combination with one or more other agents that inhibit the immune response against allograft or inhibit GVHD. For example, in one embodiment, an antibody or antibody portion described herein is used in combination with 0KT3 to inhibit responses induced by OKT3. In another embodiment, an antibody or antibody portion described herein is used in combination with one or more antibodies targeting other target molecules involved in regulating the immune response, such as CD25 (interleukin-2 receptor-α), CD11a (LFA-) 1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 (B7-1) and / or CD86 (B7-2). In yet another embodiment, an antibody or antibody portion described herein is used in combination with one or more general immunosuppressive agents such as cyclosporin A or FK506.

F. Malicious process

Tumor necrosis factor is involved in inducing cachexia, stimulating tumor proliferation, enhancing metastatic potential, and mediating cytotoxicity in malignant processes (see, e.g., Tracey and Cerami, supra). Accordingly, the antibodies and antibody portions described herein can be used to treat malignant processes, to inhibit tumor growth or metastasis, and / or to ameliorate cachexia secondary to the malignant process. The antibody or antibody portion can be administered systemically or locally to the tumor site.

G. Pulmonary disorders

Tumor necrosis factor is involved in the pathophysiology of adult respiratory distress syndrome, including stimulating endothelial leukocyte activation, targeting cytotoxicity to pneumocytes, and inducing vascular leakage (see, e.g., Tracey and Cerami, supra). Accordingly, the antibodies and antibody portions described herein can be used to treat a variety of lung disorders, including adult respiratory distress syndrome (see e.g. PCT Publication No. WO 91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The antibody or antibody portion can be administered systemically or locally to the surface of the lungs, e.g., as an aerosol.

H. Cardiac disorders

The antibodies and antibody portions described herein can also be used to treat a variety of heart disorders, including cardiac ischemia (see e.g. European Patent Application No. EP 453898) and heart failure (myocardial weakness) (see e.g. PCT Publication No. WO 94/20139).

And other

The antibodies and antibody portions described herein can also be used to treat a variety of other disorders in which TNF? Activity is deleterious. Examples of other diseases and disorders in which the pathophysiology is involved in TNF? Activity, and so that can be treated using the antibody or antibody portion, include inflammatory bone diseases and bone resorption (see, e.g., Bertolini, DR, et al. (1986) Nature 319: 516 -518; Konig, A., et al (1988) J. Bone Miner. Res. 3: 621-627; Lerner, UH and Ohlin, A. (1993) J. Bone Miner. Res. 8: 147-155; and Shankar, G. and Stern, PH (1993) Bone 14: 871-876), hepatitis, including alcoholic hepatitis (see, e.g., 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. (1994) Hepatology 20: 461-474) and viral hepatitis (Sheron, N. , et al (1991) J. Hepatol 12: 241-245; and Hussain, MJ, et al (1994) J. Clin Pathol. 47: 1112-1115), coagulation disorders (see, e.g., 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, e.g., Giroir, BP, et al. (1994) Am. J. Physiol. 267: H118-124; and Liu, XS , et al. (1994) Burns 20: 40-44), reperfusion injury (see, e.g., Scales, WE, et al. (1994) Am. J. Physiol. 267: 01122-1127; Serrick, C.,

PL 217 217 B1 et al. (1994) Transplantion 58: 1158-1162; and Yao, YM, et al. (1995) Resuscitation 29: 157-168), keloid formation (see, e.g., McCauley, RL, et al. (1992) J. Clin. Immunol. 12: 300-308), scar tissue formation and fever.

The present invention is described with reference to the following figures.

Figures 1A and 1B show the American College of Rheumatology 20 (ACR20) and ACR50 responses in rheumatoid arthritis (RA) patients following subcutaneous administration of D2E7 antibody every week for a total of twelve weeks (1A), or subcutaneous administration of D2E7 antibody and methotrexate at the second week (1B) for a total of twenty-four weeks. These data indicate that the every other week dosing is as effective as the weekly dosing.

Figure 2 shows the ACR20, ACR50, and ACR70 responses in RA patients following subcutaneous administration of the D2E7 antibody and methotrexate every other week for twenty-four weeks.

Figures 3A and 3B depict changes over time in tender joint number (3A) and swollen joint number (3B) over twenty-four weeks in RA patients following subcutaneous administration of D2E7 and methotrexate every other week for twenty-four weeks.

Figure 4 shows the results from a Brief Health Survey (SF-36) of RA patients after subcutaneous administration of the D2E7 antibody and methotrexate every other week for twenty-four weeks. RP, physical meaning; PF, physical function; BP, body pain; GH, general health; V, vitality; SE, social functioning; RE, emotional state; and MH, mental health.

Figure 5 shows the percentage of subjects giving an ACR response after a single intravenous injection of the D2E7 antibody and methotrexate in patients suffering from RA.

The present invention is further illustrated by the following examples.

Example 1

Treatment with anti-TNF? Antibody

Efficacy of D2E7 after subcutaneous (sc) administration

In this study, twenty-four patients with active RA were treated with weekly doses of 0.5 mg / kg D2E7 (n = 18) or placebo (n = 6) by sc injection for three months. Patients in this study had a mean disease duration of 10.1 years, a Disease Activity Score (DAS) of 4.87, and received a mean of 3.4 DMARDs (Disease-Modifying Antirheumatic Drugs) prior to study enrollment; re-reflecting significant disease activity. Patients who responded to treatment continued treatment with the open-label D2E7 antibody, while patients who failed to respond to treatment with 0.5 mg / kg or who lost DAS response at 0.5 mg / kg were increased to 1 mg / kg at a dose of 0.5 mg / kg. sc injection after the twelfth week of the study.

The first patients enrolled received up to sixty injections and thus were treated with the study drug for sixty weeks. The efficacy of sc dosing was similar to that of intravenous injections. Up to 78% of patients achieved a DAS and ACR20 response within the first weeks of treatment. Subcutaneous administration of D2E7 at 0.5 mg / kg / week reduced swollen joint count (SWJ) by 54%, tender joint count (TJC) by 61% and CRP by 39% over twelve weeks compared to baseline, while in the placebo group all parameters increased. After completion of this placebo-controlled study period, patients continued treatment for up to fourteen months with continued efficacy. These results therefore indicate that subcutaneous administration of D2E7 at a dose of 0.5 mg / kg / week can be safely used by patients themselves with good local tolerance.

Administration of D2E7 and methotrexate

In this study, patients received either sc or intravenous (IV) placebo or D2E7 at a dose of 1 mg / kg in addition to their treatment (methotrexate) MTX. Fifty-four patients were enrolled and eighteen patients received iv D2E7 and sc placebo, eighteen patients received iv placebo and sc D2E7, and eighteen patients received placebo iv and sc Patients received the second dose just after they lost their blinded response status, no earlier than four weeks after the first dose. Subsequently, all patients received open-label sc injections of D2E7 every two weeks.

The demographic characteristics of the study population in this study included a mean RA duration of 11.1 years, prior exposure to a mean of 3.6 DMARDs (non-MTX), and mean DAS at study entry of 4.81. On day twenty-ninth, 72% of IV D2E7-treated patients and 44% of D2E7 sc-treated patients achieved a DAS response by 24

This was equated to only 28% of placebo-treated patients (shown in Fig. 5). Of the responders in this study, 28% of placebo-treated patients maintained an ACR20 response until Day 29, compared with 72% of IV D2E7 and 67% of D2E7 sc treated patients who maintained responses for one to three months.

Example 2

Dose for total body weight of subcutaneously administered anti-TNF? Antibody

Subcutaneous administration of D2E7 every week

Two hundred and eighty-four RA patients were enrolled in this study and were designed to determine the optimal dose for total body weight of D2E7 administered subcutaneously. Patients were randomized to receive 20, 40, or 80 mg of D2E7 or placebo weekly for twelve weeks, at which time placebo-treated patients were switched to a blinded trial of 40 mg D2E7 / week.

Approximately 49% of patients achieved ACR20 at 20 mg, 55% of patients achieved ACR20 at 40 mg, and 54% of patients achieved ACR20 at 80 mg, while only 10% of placebo patients achieved ACR20 (shown in Figure 1A) . Approximately 23% of patients achieved ACR50 with 20 mg, 27% of patients achieved ACR50 with 40 mg and 20% of patients achieved ACR50 with 80 mg, and only 2% of patients on placebo achieved ACR50. These data illustrate that subcutaneous administration of D2E7, especially at a dose of 40 mg / week, produces a good response.

Example 3

Subcutaneous administration every two weeks with anti-TNFa antibody, subcutaneous administration every two weeks D2E7

The clinical effects, safety, immunogenicity and tolerability of RA patients with partial responses to MTX following subcutaneous (sc) injections of placebo or D2E7 at several dose levels every other week for up to twenty-four weeks in combination with continued MTX treatment were studied.

Study design

A placebo-controlled, double-blind, randomized, multicentre study was conducted in patients with RA who had insufficient efficacy or tolerance to MTX. During the course of the trial, patients continued treatment at a constant dose of MTX within the dose range specified in the inclusion criteria described below.

This study consisted of two parts:

1) "washout period of previous drug prior to administration of another drug" of four weeks prior to the first dose of drug during which DMARD withdrawal (except MTX); and

2) "placebo-controlled period" during which patients were randomized to one of four cohorts of sixty-seven to receive placebo, 20, 40 or 80 mg of D2E7 (as a dose for total body weight) administered every other week sc for up to 24 weeks. Each dose of study drug was administered as two sc injections of 1.6 ml each. The patient received the first dose from healthcare professionals as part of patient training. Subsequent doses were administered by the patients themselves at the study site under direct observation of trained personnel for the first four weeks. Thereafter, the doses were administered outside the study site by the patient, a trained patient designated by the patient, or by medical personnel. The drug was dispensed for four or five weeks after each clinical evaluation. Patients were tested serially during weeks one, two, three, four, six, eight, twelve, sixteenth, twenty-four and twenty-four, with joint examinations being performed by an ungrouped assessor, independent of the treating physician.

Two hundred and seventy-one RA patients were enrolled in this study. The study population was representative of the North American moderate to severe RA population: approximately 70% of female patients, and predominantly over the age of forty. The population was selected using predefined inclusion and exclusion criteria known to those of skill in the art, e.g. the patient must be diagnosed with RA according to the criteria outlined by the American College of Rheumatology (ACR) revised 1987 (set out in Appendix A).

Results

Figures 1B and 2-4 show that subcutaneous treatment with D2E7 every two weeks combined with methotrexate was significantly better than placebo in reducing the signs and symptoms of RA over twenty-four weeks. All three doses of D2E7 were statistically significantly more effective than weekly placebo. Moreover, D2E7 at the dose of 40 mg and 80 mg had better efficacy than the dose of 20 mg.

Appendix A

ACR definition of RA

1987 Criteria and Functions of the 1987 Classification Scheme for Rheumatoid Arthritis (RA)

<td>Criterion</td><td>Definition</td>

<td>1. Inflammation of 3 or more areas of the joints</td><td>At least 3 joint areas simultaneously showed soft tissue swelling or fluid (not just bone hypertrophy) as observed by a physician. The 14 possible joint areas are right or left PIP, MDP, wrist, elbow, knee, ankle and MTP joints.</td>

<td>2. Arthritis of the hand Wrist MCP MCP or MCP wrist and wrist</td><td>Soft tissue swelling or fluid (not just bone hypertrophy) of the specified area as monitored by your doctor. Where 2 areas were specified, they had to be affected simultaneously.</td>

<td>3. Symmetrical swelling (arthritis)</td><td>Simultaneous involvement of the same joint areas (as defined in 1 on both sides of the body (bilateral PIP, MCP or MTP disease is acceptable despite the lack of absolute symmetry)</td>

<td>4. Serum rheumatoid factor</td><td>Demonstration of abnormal serum rheumatoid factor levels by any method that is positive in &lt;5% of healthy controls</td>

<td>5. Radiographic changes in rheumatoid arthritis</td><td>Radiographic changes typical of rheumatoid arthritis on the anterior anterior radiographs of the hand and wrist, which must include erosions or obvious bone decalcification located in or most clearly adjacent to the affected joints (osteoarthritis alone does not qualify)</td>

A patient is defined as having RA if he falls in 1 of the 5 RA subsets listed in Table 7 and has a clinical diagnosis of RA by his physician. Criteria 1, 2 and 3 must be present for at least 6 weeks. Arthritis and Rheumatism, vol. 31, no. 3 (March 1988)

PL 217 217 B1

Sequence Listing &lt;110&gt; Abbott Laboratories (Bertnuda) Ltd.iin.

&lt;120&gt; Methods of administering anti-TNFa antibodies &lt;130 BBI-168PC &lt;140&gt; PCT / US02 / 17790 &lt;141&gt; 2002-06-05 &lt;15Q&gt; 60/296961 &lt;151&gt; 2001-06-08 &lt;160&gt; 37 &lt;170 FastSEO for Windows Version 4.0 &lt;210&gt; 1 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; trick &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;220 &lt;223&gt; mutant human antibody &lt;400&gt; 1

<td colspan="6">Asp Ile Gin Met Thr Gin</td><td rowspan="2">Cheese</td><td colspan="5" rowspan="2">Pro Cheese Cheese Leu Ser 10</td><td rowspan="2">Ala</td><td rowspan="2">Cheese</td><td rowspan="2">Val 15</td><td rowspan="2">Gly</td>

<td colspan="4"> 1</td><td colspan="2">s</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr</td><td>How much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>Ala</td><td>Cheese</td><td>Gin</td><td>Gly</td><td>How much</td><td>Arg</td><td>Asn</td><td>Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Pro</td><td>Lys</td><td>Leu</td><td>Leu</td><td>how much</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Phe</td><td>Cheese</td><td rowspan="2">Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Glu</td><td>Asp</td><td>Val</td><td>Ala</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>cys</td><td>Gin</td><td>Arg</td><td>Tyr</td><td>Asn</td><td>Arg</td><td>Ala</td><td>Pro</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Phe</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>How much</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210 2 &lt;2ll&gt; 121 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400&gt; 2

<td>Glu</td><td>Val</td><td>Gin</td><td>Leu</td><td>Val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Leu</td><td>Val</td><td>Gin</td><td>Pro</td><td>Gly</td><td rowspan="2">Arg</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Asp</td><td rowspan="2">Asp</td><td rowspan="2">Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>Ala</td><td>Underworld</td><td>His</td><td>Trp</td><td>Val</td><td>Arg</td><td>Gin</td><td>Ala</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Trp</td><td>Val</td>

PL 217 217 B1

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>Cheese</td><td>Ala</td><td>How much</td><td>Thr</td><td rowspan="2">Trp</td><td>Asn</td><td>Cheese</td><td>Gly</td><td>His</td><td>How much</td><td>Asp</td><td>Tyr</td><td>Ala</td><td>Asp Ser</td><td>Val</td>

<td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Glu</td><td colspan="2">Gly Arg</td><td>Phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Asn</td><td>Leu cheese</td><td>Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td>so</td>

<td>Leu</td><td>Gin</td><td>Underworld</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Ala</td><td>Val</td><td>Tire Tyr</td><td>Cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ala</td><td>Bald</td><td>val</td><td>Cheese</td><td>Tyr</td><td>Leu</td><td>Cheese</td><td>Thr</td><td>Ala</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>ASp</td><td>Tyr Trp</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td>

<td>Gin</td><td rowspan="2">Gly</td><td>Thr</td><td>Leu</td><td>Val</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 3 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;22O&gt;

&lt;223&gt; mutant human antibody &lt;22O&gt;

&lt;221&gt; VARIETY &lt;222&gt; &lt;91 &lt;223&gt; Xaa Thr or Ala &lt;400&gt; 3

Gin Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 S &lt;210&gt; 4 &lt;2łl&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;220&gt;

&lt;22l&gt; VARIETY &lt;222&gt; (12) &lt;223&gt; Xaa &lt;= Tyr or Asn &lt;400&gt; 4

Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 15 10 &lt;210 5 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 5

Ala Ala Ser Thr Leu Gin Ser 1 5

PL 217 217 B1 &lt;210&gt; 6 &lt;211&gt; 1?

&lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; $

Ala Ile Thr Trp Asn Sec Gly His Ile Asp Tyr Ala Asp Ser Val Glu 15 10 15

Gly &lt;210&gt; 7 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;22 3&gt; mutant human antibody &lt;4O0&gt; 7

Arg Ala Ser Gin Gly Ile Arg Asn Tyr Leu Ala

10 &lt;2l0&gt; 8 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;22 3&gt; mutant human antibody &lt;400&gt; e.

Asp Tyr Ala Met His

5 &lt;210&gt; 9 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213; » Artificial sequence &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 9

<td>Asp</td><td>How much</td><td>Gin</td><td>Underworld</td><td>The</td><td>Gin</td><td>Sey</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Wow</td><td>Cheese</td><td>Ala</td><td>Cheese</td><td>How much</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td colspan="2">Asp Arg</td><td>Val</td><td>Thr</td><td>How much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>Ala</td><td>Cheese</td><td>Gin</td><td>Gly</td><td>How much</td><td>Arg</td><td>Asn</td><td>Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin'</td><td>Bald</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Pro</td><td>Bald</td><td>Leo</td><td>Leu</td><td>How much</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Phe</td><td>Cheese</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

PL 217 217 B1

Gin Asp Val Ala The Tyr Tyr Cys Gin Łys Tyr Asn Ser Ala Pro Tyr 85 90 95

Ala Phe Gly Gin Gly Thr Lys Val Glu Ile Łys 100 105 &lt;210&gt; 10 &lt;21ł&gt; 121 &lt;212&gt; PAT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 10

<td>Gin</td><td>Val</td><td>Gin</td><td>Wow</td><td>tfai</td><td>Glu</td><td>Cheese</td><td>Gly</td><td colspan="2">Gly Gly</td><td>Wow</td><td>Val</td><td>Gin</td><td>Pro Gly Arg</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Wow</td><td>Cheese</td><td>Cys</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Asp Asp Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>Ala</td><td>Underworld</td><td>His</td><td>Trp</td><td>Val</td><td>Arg</td><td>Gin</td><td>Ala</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Wow</td><td>Asp Trp Val</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>Cheese</td><td>Ala</td><td>How much</td><td>Thr</td><td>Trp</td><td>Asn</td><td>Cheese</td><td>Gly</td><td>His</td><td>How much</td><td colspan="2">Asp Tyr</td><td>Ala</td><td>Asp Ser Val</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td>

<td>Gly</td><td>Gly</td><td>Arg</td><td>Phe</td><td>Ala</td><td>Val</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Asn</td><td>Ala</td><td>Bald</td><td>Asn</td><td>Ala Leu Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Wow</td><td>Gin</td><td>Underworld</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Ala</td><td>Val</td><td>Tyr Tyr Cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td>

<td>Thr</td><td>Bald</td><td>Ala</td><td>Cheese</td><td>Tyr</td><td>Leu</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Wow</td><td>Asp</td><td>Asn Trp Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td>

<td>Gin</td><td>Gly</td><td>Thr</td><td>Łea</td><td>Val</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;2l0&gt; 11 &lt;2ll&gt; 9 &lt;212&gt; PRT &lt;2l3&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 11

Gin Łys Tyr Asn Ser Ala Pro Tyr Ala 1 5 &lt;210 »12 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220» &lt;223 »mutated human antibody &lt;400» 12

Gin Lys Tyr Asn Arg Ala Pro Tyr Ala 1 5 &lt;210&gt; 13 &lt;211&gt; 9

PL 217 217 B1 &lt;212&gt; PAT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 13

Gin Lys Tyr Gin Arg Ala Pro Tyr Thr 1 5 &lt;210 14 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213? Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400 14

Gin Lys Tyr Ser Ser Ala Pro Tyr Thr 1 5 &lt;210&gt; 15 &lt;211&gt; 9 &lt;2l2&gt; PRT &lt;213? Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400&gt; 15

Gin Lys Tyr Asn Ser Ala Pro Tyr Thr 1 5 &lt;210 16 &lt;211&gt; 9 &lt;212&gt; PAT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400 16

Gin Lys Tyr Asn Arg Ala Pro Tyr Thr 1 5 &lt;210 1?

&lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400 13

Gin Lys Tyr Asn Ser Ala Pro Tyr Tyr 1 5

PL 217 217 B1

<td>&lt;210&gt; 16 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Dummy sequence</td>

<td> &lt;220&gt;</td>

<td>&lt;223&gt; mutant human antibody</td>

<td> &lt;400&gt; 16</td>

Gin Lys Tyr Asn Ser Ala Pro Tyr Asn 1 5 &lt;210&gt; n &lt;211&gt; 9 &lt;212&gt; PAT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 19

Gin Lys Tyr Thr Ser Ala Pro Tyr Thr 1 S &lt;210&gt; 20 &lt;211&gt; 9 &lt;212&gt; PAT &lt;213&gt; artificial sequence &lt;220&gt;

&lt;22 3&gt; mutant human antibody &lt;400&gt; 20

Gin lys Tyr Asn Arg Ala Pro Tyr Asn 1 5 '&lt;210&gt; 21 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 21

Gin Lys Tyr Asn Ser Ala Ala Tyr Ser 1 5 &lt;210&gt; 22 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody

PL 217 217 B1 &lt;400&gt; 22

Gin Gin Tyr Asn Ser Ala Pro Asp Thr l 5 &lt;210 23 &lt;211&gt; 9 &lt;212&gt; PAT &lt;213&gt; Dummy &lt;22O&gt;

&lt;223&gt; mutant human antibody &lt;40G&gt; 23

Gin Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5 &lt;210 24 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400&gt; 24

Gin Lys Tyr Ile Ser Ala Pro Tyr Thr 1 5 &lt;210 25 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;40G&gt; 25

Gin Lys Tyr Asn Arg Pro Pro Tyr Thr 1 5 &lt;210&gt; 26 &lt;211&gt; 9 &lt;212&gt; PR?

&lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;40O 26

Gin Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5 &lt;210 2? &lt;210 12 &lt;212&gt; PR?

Artificial sequence &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400 27

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 15 10 &lt;210&gt; 28 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 28

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys 15 10 &lt;210 29 &lt;211&gt; 12 &lt;212&gt; PAT &lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400 29

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr 15 10 &lt;21O&gt; 30 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; mutant human antibody &lt;400&gt; 30

Ala ser Tyr Leu Ser Thr Ser Ser Sar Leu Asp Asp 15 10 &lt;210&gt; 31 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400&gt; 31

Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr 15 10

PL 217 217 B1 &lt;210&gt; 32 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220 &lt;223 mutant human antibody &lt;400&gt; 32

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr 1 $ 10 &lt;210 33 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220 &lt;223? mutant human antibody &lt;400 33

Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr 15 10 &lt;2ł0&gt; 34 &lt;211? 12 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220?

&lt;223&gt; mutant human antibody &lt;400 34

Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr 15 10 &lt;210 35 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213? Artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400 35

Val Ser Tyr Łeu Ser Thr Ala Ser Ser Leu ftsp Asn 15 10 &lt;210&gt; 36 &lt;211&gt; 321 &lt;212&gt; DNA &lt;213? Artificial sequence &lt;220?

&lt;223&gt; mutant human antibody

GB 217 217 B1 &lt;400 36 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtagggga cagagtcacc 60 atcacttgtc gggcaagtca gggcątcaga aattacttag cctggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgęt gcatccactt tgcaatcagg ggtcccatct 180 cggttcagtg gcagtggatc tgggacsgat ttcactctca ccaccagcag cctacagcct 240 gaagatgttg caacttatta ctgtcaaagg tataaccgtg caccgtatac ttttggccag 300 gggaccaagg tggaaatcaa a 321 &lt;210&gt; 37 &lt;211&gt; 363 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220 &lt;223&gt; mutant human antibody &lt;400 37 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ccggcaggtc cctgagactc 60 tcctgtgcgg cctctggatt cacctttgat gattatgcca tgeactgggt ccggcaagct 120 ccagggaagg gcctggaatg ggtetcagct atcacttgga atagtggtca catagactat 180 gcggactctg tggagggccg attcaccatc tccagagaca acgceaagąa ctccctgtat 240 ctgcaaatga acagtctgag agctgaggat acggccgtat attactgtgc gaaagtcteg 300 taccttagca ccgcgtcctc ccttgactat tggggccaag gtaccctggt caccgtctcg 360 AGT 363