Anti-TNF antibodies, compositions and methods for treating active ankylosing spondylitis

Abstract

The present invention relates to compositions and methods utilizing anti-TNF antibodies or antigen-binding fragments thereof in the treatment of active ankylosing spondylitis (AS), for example, treatments utilizing anti-TNF antibodies having heavy chain complementarity-determining region (CDR) amino acid sequences set forth in SEQ ID NOS: 40-42 and light chain CDR amino acid sequences set forth in SEQ ID NOS: 43-45.

Description

[Technical Field]

[0001] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing having a size of 25 KB that has been submitted electronically via EFS-Web as an ASCII sequence listing with the filename "JBI6445WOPCT1Seqlist.txt" created on December 3, 2021. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to compositions and methods utilizing anti-TNF antibodies or antigen-binding fragments thereof in the treatment of active ankylosing spondylitis (AS), for example, treatments utilizing anti-TNF antibodies having heavy chain complementarity-determining region (CDR) amino acid sequences set forth in SEQ ID NOS: 40-42 and light chain CDR amino acid sequences set forth in SEQ ID NOS: 43-45.

[0003] BACKGROUND OF THE INVENTION TNFα is a soluble homotrimer of 17 kD protein subunits. A membrane-bound 26 kD precursor form of TNF also exists.

[0004] Cells other than monocytes or macrophages also produce TNFα: for example, human non-monocytic tumor cell lines produce TNFα, as do CD4+ and CD8+ peripheral blood T lymphocytes, and several cultured T and B cell lines also produce TNFα.

[0005] TNFα causes pro-inflammatory effects that result in tissue damage, such as cartilage and bone degradation, induction of adhesion molecules, induction of procoagulant activity in vascular endothelial cells, increased adhesion of neutrophils and lymphocytes, and stimulation of the release of platelet-activating factor from macrophages, neutrophils, and vascular endothelial cells.

[0006] TNFα has been implicated in infections, immune disorders, neoplastic, autoimmune, and graft-versus-host pathologies. The association of TNFα with cancer and infectious pathologies is often related to the catabolic state of the host. Cancer patients commonly suffer from weight loss associated with anorexia.

[0007] The profound wasting associated with cancer and other diseases is known as "cachexia." Cachexia involves progressive weight loss, anorexia, and a persistent decline in lean body mass in response to malignant tumor growth. The cachectic state contributes to many cancer morbidities and mortality rates. There is evidence that TNFα contributes to cachexia in cancer, infectious conditions, and other catabolic states.

[0008] TNFα is thought to play a central role in gram-negative sepsis and endotoxic shock, which include fever, fatigue, anorexia, and cachexia. Endotoxin potently activates monocyte / macrophage production and secretion of TNFα and other cytokines. TNFα and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin. Endotoxin administration to human volunteers results in acute illness with flu-like symptoms, including fever, tachycardia, increased metabolic rate, and stress hormone release. Circulating TNFα is increased in patients with gram-negative sepsis.

[0009] Therefore, TNFα has been implicated in inflammatory diseases, autoimmune diseases, viral, bacterial, and parasitic infections, malignant tumors, and / or neurodegenerative diseases, and is a useful target for certain biological treatments in diseases such as rheumatoid arthritis and Crohn's disease. Beneficial effects have been reported in open-label studies using a chimeric monoclonal antibody (cA2) against TNFα, along with suppression of inflammation and successful retreatment after relapse in rheumatoid arthritis and Crohn's disease. Beneficial results have also been reported in randomized, double-blind, placebo-controlled studies using cA2, along with suppression of inflammation in rheumatoid arthritis.

[0010] Other investigators have described mAbs specific for recombinant human TNF that had neutralizing activity in vitro. Some of these mAbs were used to map epitopes of human TNF, develop enzyme immunoassays, and aid in the purification of recombinant TNF. However, these studies do not provide a basis for generating TNF-neutralizing antibodies that can be used for in vivo diagnostic or therapeutic applications in humans due to immunogenicity, low specificity, and / or pharmaceutical unsuitability.

[0011] Neutralizing antisera or mAbs against TNF have been shown to abrogate adverse physiological changes and prevent mortality in non-human mammals following lethal challenge in experimental endotoxemia and bacteremia, for example, in rodent lethality assays and primate pathology model systems.

[0012] The putative receptor binding locus for hTNF has been disclosed, as has the receptor binding locus for TNFα, which consists of amino acids 11-13, 37-42, 49-57, and 155-157 of TNF.

[0013] Non-human mammalian, chimeric, polyclonal (e.g., antisera), and / or monoclonal antibodies (Mabs) and fragments (e.g., proteolytic digests or fusion protein products thereof) are potential therapeutics that are being investigated in some cases to attempt to treat certain diseases. However, such antibodies or fragments can elicit an immune response when administered to humans. Such an immune response can result in immune complex-mediated clearance of the antibody or fragment from the circulation, making repeated administration unsuitable for therapy, thereby reducing therapeutic benefit to the patient and limiting re-administration of the antibody or fragment. For example, repeated administration of antibodies or fragments comprising non-human portions can result in serum sickness and / or anaphylaxis. To avoid these and other problems, many approaches have been taken to reduce the immunogenicity of such antibodies and portions thereof, including chimerization and humanization, as are well known in the art. However, these and other approaches may still result in antibodies or fragments that have some immunogenicity, low affinity, low avidity, or are associated with problems in cell culture, scale-up, production, and / or low yield. Thus, such antibodies or fragments may not be ideally suited for production or use as therapeutic proteins.

[0014] Therefore, there was a need to provide an anti-TNF antibody or fragment that overcomes another of these problems, as well as an improvement over known antibodies or fragments thereof. This need led to the development of SIMPONI® (golimumab), a fully human monoclonal anti-TNF antibody.

[0015] (Summary of the Invention) In certain embodiments, the invention provides methods of treating active ankylosing spondylitis in a patient (or patients) with early stage disease for a duration of <2 years, the method comprising administering to the patient an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOs: 40-42 and light chain CDR amino acid sequences of SEQ ID NOs: 43-45, wherein after 16 weeks of treatment, the patient achieves an outcome selected from the group consisting of at least a 50% improvement from baseline in Ankylosing Spondylitis Assessment 20 (ASAS20), Ankylosing Spondylitis Assessment 40 (ASAS40), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and the outcome is maintained or improved for up to 52 weeks of treatment.

[0016] In an embodiment of the method or use, after 16 weeks of treatment, ≧70% of patients achieve an Ankylosing Spondylitis Assessment of 20 (ASAS20), ≧45% of patients achieve an Ankylosing Spondylitis Assessment of 40 (ASAS40), ≧35% of patients achieve at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and ≧25% of patients achieve Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and the percentages of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (<1.3) increase over 52 weeks of treatment.

[0017] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOs: 40-42 and light chain CDR amino acid sequences of SEQ ID NOs: 43-45, wherein after 16 weeks of treatment, ≥ 70% of patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20), ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment 40 (ASAS40), and ≥ 35% of patients achieve a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50). At least a 50% improvement from baseline is achieved, and ≧25% of patients achieve Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (<1.3) increases over 52 weeks of treatment, and the anti-TNF antibody is administered by intravenous infusion (IV) at a dose of 2 mg / kg of patient body weight at weeks 0 and 4, then every 8 weeks (q8w) thereafter, over 30±10 minutes.

[0018] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOs: 40-42 and light chain CDR amino acid sequences of SEQ ID NOs: 43-45, wherein after 16 weeks of treatment, ≥ 70% of patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20), ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment 40 (ASAS40), ≥ 35% of patients achieve at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and ≥ 25% of patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20). The percentage of patients achieving ASDAAS Inactive Disease Activity Score (<1.3), and ASAS20, ASAS40, BASDAI50, and ASDAS Inactive Disease (<1.3) increase over 52 weeks of treatment, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg by intravenous infusion (IV) over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter, and the method further comprises administering the anti-TNF antibody with or without methotrexate (MTX), sulfasalazine (SSZ), or hydroxychloroquine (HCQ).

[0019] In certain embodiments, the invention provides methods of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOs: 40-42 and light chain CDR amino acid sequences of SEQ ID NOs: 43-45, wherein after 16 weeks of treatment there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes is maintained or continues to improve over 52 weeks of treatment for ASQoL, back pain at night, and total back pain.

[0020] In certain embodiments, the invention provides methods of treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, the method comprising administering to the patient an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOS: 40-42 and light chain CDR amino acid sequences SEQ ID NOS: 43-45, wherein after 16 weeks of treatment there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, back pain at night, and total back pain, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg by intravenous infusion (IV) over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter.

[0021] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, wherein after 16 weeks of treatment there is an improvement in patient reported outcomes with respect to ankylosing spondylitis quality of life (ASQoL), night back pain, and total back pain, wherein the improvement in patient reported outcomes is The present invention provides a method for treating nighttime back pain and total back pain, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter, and the method further comprises administering the anti-TNF antibody with methotrexate (MTX), sulfasalazine (SSZ), and / or hydroxychloroquine (HCQ).

[0022] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient a composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, wherein the composition is administered by intravenous infusion (IV), and wherein after 16 weeks of treatment, ≥ 70% of the patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20) and ≥ 100% of the patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20). 45% achieve an Ankylosing Spondylitis Assessment 40 (ASAS40), ≧35% of patients achieve at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), ≧25% of patients achieve Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (<1.3) increases over 52 weeks of treatment.

[0023] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, the method comprising administering to the patient a composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, wherein the composition is administered by intravenous infusion (IV), and wherein after 16 weeks of treatment, ≥ 70% of patients achieve an Ankylosing Spondylitis Assessment Score of 20 (ASAS20), ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment Score of 40 (ASAS40), and ≥ 35% of patients achieve a Bath Ankylosing Spondylitis Assessment Score of 1 ...40). The method provides a method of achieving at least a 50% improvement from baseline in the Ankylosing Spondylitis Disease Activity Index (BASDAI50), with ≥ 25% of patients achieving Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (< 1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (< 1.3) increases over 52 weeks of treatment, wherein the composition is administered such that the anti-TNF antibody is administered at a dose of 2 mg / kg over 30 ± 10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter.

[0024] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, the method comprising administering to the patient a composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, wherein the composition is administered by intravenous infusion (IV), and wherein after 16 weeks of treatment, ≥ 70% of patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20), ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment 40 (ASAS40), and ≥ 35% of patients achieve an improvement of at least 5 from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50). and wherein ≥ 25% of patients achieve an Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (< 1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (< 1.3) increases over 52 weeks of treatment, wherein the composition is administered such that the anti-TNF antibody is administered at a dose of 2 mg / kg over 30 ± 10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter, and the method further comprises administering the composition with or without methotrexate (MTX), sulfasalazine (SSZ), or hydroxychloroquine (HCQ).

[0025] In certain embodiments, the invention provides methods of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient a composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, wherein the composition is administered by intravenous infusion (IV), and wherein after 16 weeks of treatment there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, back pain at night, and total back pain.

[0026] In certain embodiments, the invention provides methods of treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, the method comprising administering to the patient a composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOS: 40-42 and light chain CDR amino acid sequences SEQ ID NOS: 43-45, wherein the composition is administered by intravenous infusion (IV), and after 16 weeks of treatment there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, back pain at night, and total back pain, and the composition is administered such that the anti-TNF antibody is administered at a dose of 2 mg / kg over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter.

[0027] In certain embodiments, the invention provides a method of treating active ankylosing spondylitis in patients with early stage disease for a duration of <2 years, the method comprising administering to the patient a composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOS: 40-42 and light chain CDR amino acid sequences SEQ ID NOS: 43-45, wherein the composition is administered by intravenous infusion (IV), and after 16 weeks of treatment, there is improvement in patient-reported outcomes with respect to ankylosing spondylitis quality of life (ASQoL), nighttime back pain, and total back pain. and improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, nighttime back pain, and total back pain, wherein the composition is administered such that the anti-TNF antibody is administered at a dose of 2 mg / kg, administered over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter, and the method further comprises administering the composition with or without methotrexate (MTX), sulfasalazine (SSZ), or hydroxychloroquine (HCQ).

[0028] In certain embodiments, the invention provides a composition comprising an anti-TNF antibody for use in treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, wherein the anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, and wherein after 16 weeks of treatment, ≥ 70% of patients achieve an Ankylosing Spondylitis Assessment 20 (ASAS20) and ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment 40 (ASAS40). In one embodiment, ≥ 35% of patients achieve at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), ≥ 25% of patients achieve Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (< 1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (< 1.3) increases over 52 weeks of treatment.

[0029] In certain embodiments, the invention provides a composition comprising an anti-TNF antibody for use in treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, wherein the anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, the composition being administered by intravenous infusion (IV) such that the anti-TNF antibody is administered at a dose of 2 mg / kg, administered over 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter, and wherein after 16 weeks of treatment, ≥7% of the patient's disease is treated with an anti-TNF antibody. 0% of patients achieve an Ankylosing Spondylitis Assessment of 20 (ASAS20), ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment of 40 (ASAS40), ≥ 35% of patients achieve at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and ≥ 25% of patients achieve Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (< 1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (< 1.3) increases over 52 weeks of treatment.

[0030] In certain embodiments, the invention provides a composition comprising an anti-TNF antibody for use in treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, wherein the anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, the composition being administered with or without methotrexate (MTX), sulfasalazine (SSZ), or hydroxychloroquine (HCQ), and wherein after 16 weeks of treatment, ≥70% of patients achieve an ankylosing spondylitis rating of ≥1. In one embodiment, more than 45% of patients achieve an Ankylosing Spondylitis Assessment of 20 (ASAS20), ≥ 45% of patients achieve an Ankylosing Spondylitis Assessment of 40 (ASAS40), ≥ 35% of patients achieve at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and ≥ 25% of patients achieve Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (< 1.3), and the percentage of patients achieving ASAS20, ASAS40, BASDAI50, and ASDAS inactive disease (< 1.3) increases over 52 weeks of treatment.

[0031] In certain embodiments, the invention provides a composition comprising an anti-TNF antibody for use in treating active ankylosing spondylitis in patients with early stage disease of <2 years duration, wherein the anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOs: 40-42 and light chain CDR amino acid sequences of SEQ ID NOs: 43-45, and wherein after 16 weeks of treatment there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, back pain at night, and total back pain.

[0032] In certain embodiments, the invention provides a composition comprising an anti-TNF antibody for use in treating active ankylosing spondylitis in patients with early-stage disease of <2 years duration, wherein the anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg by intravenous infusion (IV) administered over 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter, and wherein after 16 weeks of treatment there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, back pain at night, and total back pain.

[0033] In certain embodiments, the invention provides a composition comprising an anti-TNF antibody for use in treating active ankylosing spondylitis in patients with early-stage disease of <2 years duration, wherein the anti-TNF antibody comprises heavy chain complementarity-determining region (CDR) amino acid sequences of SEQ ID NOS: 40-42 and light chain CDR amino acid sequences of SEQ ID NOS: 43-45, when the composition is administered with or without methotrexate (MTX), sulfasalazine (SSZ), or hydroxychloroquine (HCQ), and wherein after 16 weeks of treatment, there is improvement in patient-reported outcomes for ankylosing spondylitis quality of life (ASQoL), back pain at night, and total back pain, and the improvement in patient-reported outcomes continues over 52 weeks of treatment for ASQoL, back pain at night, and total back pain.

[0034] In certain of the above embodiments, the anti-TNF antibody comprises the variable heavy chain amino acid sequence of SEQ ID NO: 38 and the variable light chain amino acid sequence of SEQ ID NO: 39, the anti-TNF antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 36 and the light chain amino acid sequence of SEQ ID NO: 37, or the anti-TNF antibody is a golimumab antibody. [Brief explanation of the drawings]

[0035] [Figure 1]Figure 1 shows a graphical representation of an assay for the ability of TNV mAbs in hybridoma cell supernatants to inhibit TNFα binding to recombinant TNF receptors. Various amounts of hybridoma cell supernatant containing known amounts of TNV mAb were preincubated with a fixed concentration (5 ng / mL) of 125I-labeled TNFα. The mixture was transferred to a 96-well Optiplate precoated with the recombinant TNF receptor / IgG fusion protein p55-sf2. The amount of TNFα bound to the p55 receptor in the presence of mAb was determined after washing away unbound material and counting using a gamma counter. Eight TNV ​​mAb samples were tested in these experiments; however, for brevity, three of the mAbs shown by DNA sequence analysis to be identical to one of the other TNV mAbs (see section 5.2.2) are not shown here. Each sample was tested in duplicate. Results shown are representative of two independent experiments. [Figure 2A] The DNA sequence of the TNV mAb heavy chain variable region is shown. The germline gene shown is the DP-46 gene. "TNVs" indicates that the sequence shown is that of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequence define the translation initiation Met codon. A dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single-letter code) beginning with the mature mAb is shown only for the germline gene. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb heavy chain uses the J6 binding region. [Figure 2B]The DNA sequence of the TNV mAb heavy chain variable region is shown. The germline gene shown is the DP-46 gene. "TNVs" indicates that the sequence shown is that of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequence define the translation initiation Met codon. A dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single-letter code) beginning with the mature mAb is shown only for the germline gene. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb heavy chain uses the J6 binding region. [Figure 3] The DNA sequence of the TNV mAb light chain variable region is shown. The germline gene shown is a representative member of the Vg / 38K family of human kappa germline variable region genes. Dotted lines in the TNV mAb gene sequence indicate nucleotides that are identical to those in the germline sequence. The first 16 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single-letter code) of the mature mAb is shown for the germline gene only. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb light chain uses the J3 joining region. [Figure 4]The deduced amino acid sequences of the TNV mAb heavy chain variable regions are shown. The amino acid sequences (single-letter code) shown were deduced from DNA sequences determined from both uncloned and cloned PCR products. The amino acid sequences are divided into secretory signal sequence (signal), framework (FW), and complementarity-determining region (CDR) domains. The amino acid sequence of the DP-46 germline gene is shown in the line above each domain. Dotted lines indicate that the amino acid in the TNV mAb is identical to the germline gene. TNV148(B) indicates that the sequence shown relates to both TNV148 and TNV148B. "TNV" indicates that the sequence shown relates to all TNV mAbs unless a different sequence is indicated. A dashed line in the germline sequence (CDR3) indicates that the sequence is unknown or not present in the germline gene. [Figure 5] The deduced amino acid sequence of the TNV mAb light chain variable region is shown. The amino acid sequence (single-letter code) shown was deduced from DNA sequences determined from both uncloned and cloned PCR products. The amino acid sequence is divided into the secretory signal sequence (signal), framework (FW), and complementarity-determining region (CDR) domains. The amino acid sequence of the Vg / 38K-type light chain germline gene is shown in the line above each domain. Dotted lines indicate that the amino acid in the TNV mAb is identical to the germline gene. TNV148(B) indicates that the sequence shown relates to both TNV148 and TNV148B. "All" indicates that the sequence shown relates to TNV14, TNV15, TNV148, TNV148B, and TNV186. [Figure 6]Figure 1 shows a schematic diagram of the heavy and light chain expression plasmids used to generate rTNV148B-expressing C466 cells. p1783 is the heavy chain plasmid, and p1776 is the light chain plasmid. The rTNV148B variable and constant region coding domains are indicated by black boxes. The immunoglobulin enhancer in the JC intron is indicated by a gray box. Relevant restriction sites are indicated. The plasmids are shown oriented so that transcription of the Ab gene proceeds clockwise. Plasmid p1783 is 19.53 kb in length, and plasmid p1776 is 15.06 kb in length. The complete nucleotide sequences of both plasmids are known. The variable region coding sequence of p1783 can be easily replaced with alternative heavy chain variable region sequences by replacing the BsiWI / BstBI restriction fragment. The variable region coding sequence of p1776 can be replaced with alternative variable region sequences by replacing the SalI / AflII restriction fragment. [Figure 7] A graphical representation of the growth curve analysis of five rTNV148B-producing cell lines is shown. Cultures were initiated on day 0 by seeding cells in I5Q+MHX medium in T75 flasks to a viable cell density of 1.0 × 10 cells / mL in a volume of 30 mL. The cell cultures used for these studies were continuous cultures due to the transfection and subcloning procedures. Cells in the T-flasks were then thoroughly resuspended, and 0.3 mL aliquots of the culture were removed. Growth curve studies were terminated when the cell count dropped below 1.5 × 10 cells / mL. The number of viable cells in the aliquots was determined by trypan blue exclusion, and the remaining aliquot was saved for later mAb concentration determination. A human IgG ELISA was performed on all sample aliquots at the same time. [Figure 8]A graphical representation of a comparison of cell growth rates in the presence of various MHX selection concentrations is shown. Cell subclones C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional two days. Both cell cultures were then split into three cultures containing either no MHX, 0.2x MHX, or 1x MHX. One day later, new T75 flasks were seeded with the cultures at a starting density of 1x10 cells / mL, and cells were counted at 24-hour intervals for one week. The doubling time for the first five days was calculated using the formula in SOP PD32.025 and is shown above the bars. [Figure 9] A graphical representation of the stability of mAb production over time from two rTNV148B-producing cell lines is shown. After transfection and subcloning, subclones of cells from the continuous culture were used to initiate long-term continuous culture in 24-well culture dishes. Cells were cultured in I5Q medium with or without MHX selection. Cells were serially passaged by splitting the culture every 4–6 days to maintain new viable cultures while simultaneously exhausting the previous cultures. Aliquots of exhausted cell supernatants were collected immediately after the cultures were exhausted and stored until the mAb concentrations were determined. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 10] Figure 1 shows the weight change of Tg197 arthritis mouse model mice in response to an anti-TNF antibody of the present invention compared to the control in Example 4. Approximately 4-week-old Tg197 test mice were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the present invention (TNV14, TNV148, or TNV196) at 1 mg / kg or 10 mg / kg. When body weight was analyzed as the change from pre-treatment, animals treated with 10 mg / kg cA2 consistently showed higher weight gain than animals treated with D-PBS throughout the study. This weight gain was significant from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved significant weight gain by week 7 of the study. [Figure 11A]The progression of disease severity based on the arthritis index shown in Example 4 is shown. The arthritis index for the 10 mg / kg cA2 treatment group was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to demonstrate a significant decrease in AI after week 3 when compared to the D-PBS treatment group. When each was compared with others at similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there were no significant differences between the 10 mg / kg treatment groups. When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 demonstrated significantly lower AIs than 1 mg / kg cA2 at weeks 3, 4, and 7. 1 mg / kg TNV148 was also significantly lower than the 1 mg / kg TNV14 treatment group at weeks 3 and 4. While TNV196 showed a significant reduction in AI (when compared to the D-PBS-treated group) by week 6 of the study, TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 11B] The progression of disease severity based on the arthritis index shown in Example 4 is shown. The arthritis index for the 10 mg / kg cA2 treatment group was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to demonstrate a significant decrease in AI after week 3 when compared to the D-PBS treatment group. When each was compared with others at similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there were no significant differences between the 10 mg / kg treatment groups. When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 demonstrated significantly lower AIs than 1 mg / kg cA2 at weeks 3, 4, and 7. 1 mg / kg TNV148 was also significantly lower than the 1 mg / kg TNV14 treatment group at weeks 3 and 4. While TNV196 showed a significant reduction in AI (when compared to the D-PBS-treated group) by week 6 of the study, TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 11C] The progression of disease severity based on the arthritis index shown in Example 4 is shown. The arthritis index for the 10 mg / kg cA2 treatment group was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to demonstrate a significant decrease in AI after week 3 when compared to the D-PBS treatment group. When each was compared with others at similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there were no significant differences between the 10 mg / kg treatment groups. When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 demonstrated significantly lower AIs than 1 mg / kg cA2 at weeks 3, 4, and 7. 1 mg / kg TNV148 was also significantly lower than the 1 mg / kg TNV14 treatment group at weeks 3 and 4. While TNV196 showed a significant reduction in AI (when compared to the D-PBS-treated group) by week 6 of the study, TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 12] Figure 1 shows the weight change of Tg197, an arthritis mouse model mouse, in response to an anti-TNF antibody of the present invention compared to the control in Example 5. Approximately 4-week-old Tg197 test mice were assigned to one of eight treatment groups based on body weight and treated with an intraperitoneal bolus of either a control product (D-PBS) or 3 mg / kg of antibody (TNV14, TNV148) (week 0). Injections were repeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for efficacy of the test product. Serum samples obtained from animals in groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3, and 4. [Figure 13A]1 is a graph depicting the progression of disease severity based on the arthritic index in Example 5. The arthritic index for the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group, beginning at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS-treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and were also significantly lower than animals treated with TNV14 from weeks 3 to 5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13B]1 is a graph depicting the progression of disease severity based on the arthritic index in Example 5. The arthritic index for the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group, beginning at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS-treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and were also significantly lower than animals treated with TNV14 from weeks 3 to 5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13C]1 is a graph depicting the progression of disease severity based on the arthritic index in Example 5. The arthritic index for the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group, beginning at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS-treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and were also significantly lower than animals treated with TNV14 from weeks 3 to 5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 14] 1 shows the weight change of Tg197 arthritis mouse model mice in response to anti-TNF antibodies of the present invention compared to the control in Example 6. Approximately 4-week-old Tg197 test mice were assigned to one of six treatment groups based on sex and weight and treated with a single intraperitoneal bolus of either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148). The study utilized D-PBS and a 10 mg / kg cA2 control group. [Figure 15]The progression of disease severity based on the arthritis index shown in Example 6 is shown. All treatment groups showed some protection at early time points, with 5 mg / kg cA2 and 5 mg / kg TNV148 showing significant reductions in AI at weeks 1-3, and all treatment groups showing significant reductions at week 2. Later in the study, animals treated with 5 mg / kg cA2 showed some protection, with significant reductions at weeks 4, 6, and 7. Both low-dose (3 mg / kg) cA2 and TNV148 showed significant reductions at week 6, and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain significant reductions at the end of the study (week 8). There were no significant differences between any of the treatment groups (except the saline control group) at any time point. [Figure 16] Figure 1 shows the weight change of Tg197, an arthritis mouse model mouse, in response to an anti-TNF antibody of the present invention, compared to the control in Example 7. To compare the efficacy of a single intraperitoneal administration of TNV148 (derived from hybridoma cells) and rTNV148B (derived from transfected cells), approximately 4-week-old Tg197 test mice were assigned to one of nine treatment groups based on sex and weight and treated with Dulbecco's PBS (D-PBS) or a single intraperitoneal bolus of 1 mg / kg antibody (TNV148, rTNV148B). [Figure 17]Figure 1 depicts the progression of disease severity based on the arthritis index as shown in Example 7. The arthritis index for the 10 mg / kg cA2 treatment group was lower than the D-PBS control group starting at week 4 and continuing throughout the remainder of the study (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant reduction in AI at week 4. While a previous study (P-099-017) showed that TNV148 was slightly more effective in reducing the arthritis index after a single 1 mg / kg intraperitoneal bolus, this study showed slightly higher AIs from groups treated with both versions of the TNV antibody. The group treated with 1 mg / kg cA2 (except at week 6) did not have a significant increase when compared with the 10 mg / kg cA2 group, and the group treated with TNV148 was significantly higher at weeks 7 and 8, but there were no significant differences in AI between 1 mg / kg cA2, 1 mg / kg TNV148, and 1 mg / kg TNV148B at any time point in the study. [Figure 18] FIG. 1 shows a diagram of the study design for a study of intravenously administered Simponi (golimumab) in subjects with active ankylosing spondylitis (AS). [Figure 19] Figure 1 shows patient-reported outcomes at weeks 16 and 52 of treatment for a subset of ankylosing spondylitis patients with early disease (<2-year duration). The dotted line indicates patients treated with placebo who crossed over to treatment with golimumab at week 16. Data for patients treated with placebo and for patients treated with placebo and then crossed over to golimumab at week 16 are shown as gray bars. Data for patients treated with golimumab are shown as black bars. Mean (SD) change from baseline is reported, with a decrease representing an improvement in patient-reported outcome. ASQoL refers to ankylosing spondylitis quality of life.

[0036] DESCRIPTION OF THE INVENTION The present invention provides isolated recombinant and / or synthetic anti-TNF human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted antibodies, and TNF anti-idiotype antibodies thereto, comprising all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3, and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5, and 6, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one anti-TNF antibody or anti-idiotype antibody. The present invention further includes, but is not limited to, methods of making and using such nucleic acids and antibodies, and anti-idiotype antibodies, including diagnostic and therapeutic compositions, methods, and devices.

[0037] As used herein, "anti-tumor necrosis factor alpha antibody," "anti-TNF antibody," "anti-TNF antibody portion," or "anti-TNF antibody fragment," and / or "anti-TNF antibody variant," etc., include any protein- or peptide-containing molecule, including molecules comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) or ligand-binding portion thereof of a heavy or light chain, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof, or at least a portion of a TNF receptor or binding protein, that can be incorporated into an antibody of the invention. Such antibodies optionally further affect a specific ligand, including, but not limited to, such antibodies modulate, decrease, increase, antagonize, agonize, reduce, mitigate, block, inhibit, abrogate, and / or interfere with at least one TNF activity or binding, or TNF receptor activity or binding, in vitro, in situ, and / or in vivo. As a non-limiting example, a suitable anti-TNF antibody, specified portion, or variant of the present invention can bind to at least one TNF or specified portion, variant, or domain thereof. A suitable anti-TNF antibody, specified portion, or variant can also optionally affect at least one TNF activity or function, such as, but not limited to, RNA, DNA, or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production, and / or synthesis. The term "antibody" is further intended to encompass antibodies, digested fragments, specified portions, and variants thereof, including antibody mimetics, or portions of antibodies or specified fragments or parts thereof that mimic antibody structure and / or function, including single-chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to mammalian TNF.For example, antibody fragments capable of binding to TNF or portions thereof, including, but not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction), and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction, and reassembly), Fv, or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the present invention (see, e.g., Colligan, Immunology, supra).

[0038] Such fragments can be produced by enzymatic cleavage, synthetically, or recombinantly, as known in the art and / or described herein. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab')2 heavy chain portion can be engineered to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. Various portions of the antibody can be chemically linked by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

[0039] As used herein, the term "human antibody" refers to a human antibody that contains substantially all parts of the protein (e.g., CDRs, framework, C L , C H Domain (e.g., C H 1. C H 2. C H 3), Hinge (V L , V H) refers to antibodies that have only minor sequence changes or mutations and are substantially non-immunogenic in humans. Similarly, antibodies designated primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals refer to antibodies specific for such species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations optionally and preferably retain or reduce immunogenicity in humans or other species compared to the unmodified antibody. Thus, a human antibody differs from a chimeric antibody or a humanized antibody. It is noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, it can include a linker peptide not found in naturally occurring human antibodies. For example, an Fv can include a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region. Such a linker peptide is considered to be of human origin.

[0040] Bispecific, heterospecific, heterobinding, or similar antibodies may also be used, which are monoclonal, preferably human or humanized, antibodies with binding specificities for at least two different antigens. In this case, one of the binding specificities is for at least one TNF protein, and the other is for any other antigen. Methods for producing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule (usually performed by affinity chromatography steps) is quite laborious, and product yields are low. Similar procedures are described, for example, in WO 93 / 08829, U.S. Pat. Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959,083 ... ,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980, WO 91 / 00360, WO 92 / 00373, EP 03089, Traunecker et al., EMBO J. 10:3655 (1991), and Suresh et al., Methods in Enzymology 121:210 (1986), each of which is incorporated herein by reference in its entirety.

[0041] Anti-TNF antibodies (also referred to as TNF antibodies) useful in the methods and compositions of the invention may optionally be characterized by high affinity binding to TNF, and optionally and preferably low toxicity. Specifically, antibodies of the invention, specific fragments, or variants thereof, in which individual components such as the variable region, constant region, and framework, individually and / or collectively, optionally and preferably, have low immunogenicity, are useful in the invention. Antibodies that can be used in the invention are optionally characterized by the ability to treat patients for extended periods of time with measurable alleviation of symptoms and low and / or acceptable toxicity. Low or acceptable immunogenicity and / or high affinity, as well as other favorable properties, can contribute to the therapeutic results achieved. "Low immunogenicity" is defined herein as a significant increase in HAHA, HACA, or HAMA responses in less than about 75%, or preferably less than about 50%, of treated patients, and / or a low increase in titers (less than about 300, preferably less than about 100, as measured by double antigen enzyme immunoassay) in treated patients (Elliott et al., Lancet 344:1125-1127 (1994), which is incorporated herein by reference in its entirety).

[0042] Utility: The isolated nucleic acids of the present invention may be used to generate at least one anti-TNF antibody or specified variant thereof, which may be used to measure or act in a cell, tissue, organ or animal (including mammals and humans) to diagnose, monitor, regulate, treat, alleviate, help prevent the occurrence of, or reduce the symptoms of at least one TNF condition selected from, but not limited to, at least one of immune disorder or disease, cardiovascular disorder or disease, infectious, malignant and / or neurological disorder or disease.

[0043] Such methods may include administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, mitigation, prevention, or reduction of symptoms, effects, or mechanisms. An effective amount may include an amount of about 0.001 to 500 mg / kg per single (e.g., bolus), multiple, or continuous administration, or an amount that achieves a serum concentration of 0.01 to 5000 μg / mL per single, multiple, or continuous administration, or any effective range or value therein, as determined using known methods described herein or known in the relevant art. References. All publications or patents cited herein are incorporated by reference in their entirety and represent the state of the art at the time of the invention and / or provide a description and enablement of the invention. A publication refers to any scientific publication or patent publication or any other information available in any media format, including all recorded electronic or printed formats. The following references are incorporated herein by reference in their entirety: Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY (1989), Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989), Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994~2001), Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997~2001).

[0044] Antibodies of the invention: At least one anti-TNF antibody of the invention comprising all of the heavy chain variable CDR regions of SEQ ID NOS: 1, 2, and 3, and / or all of the light chain variable CDR regions of SEQ ID NOS: 4, 5, and 6, can optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2002; nd Edition, Cold Spring Harbor, NY (1989), Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989), Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994~2001), Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997~2001).

[0045] Human antibodies specific for human TNF protein or fragments thereof can be raised against suitable immunogenic antigens, such as isolated and / or TNF protein, or portions thereof (including synthetic molecules such as synthetic peptides). Other specific or general mammalian antibodies can be raised as well. Preparation of immunogenic antigens and generation of monoclonal antibodies can be carried out using any suitable technique.

[0046] In one approach, hybridomas are generated using a suitable immortalized cell line (e.g., Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO) 2A, or heteromylomas, their fusion products, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art. See, for example, www.atcc.org, www.lifetech.com. Antibody-producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B-cell containing cells, or recombinant or endogenous, viral, bacterial, algae, prokaryotic, amphibian, insect, reptile, fish, mammalian, and rodent, equine, ovine, caprine, sheep, primate, eukaryotic, or any other cell that expresses heavy or light chain constant, variable, framework, or CDR sequences, either as endogenous or heterologous nucleic acid, such as genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded, or triple-stranded, hybridized, etc., or combinations thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra, Chapter 2, both of which are incorporated herein by reference in their entireties.

[0047] Antibody-producing cells can also be obtained from the peripheral blood, or preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. Any other suitable host cells can also be used to express heterologous or endogenous nucleic acid encoding an antibody of the invention, specific fragment, or variant thereof. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable, well-known methods and cloned by limiting dilution or cell sorting or other well-known methods. Cells producing antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

[0048] Other methods suitable for producing or isolating antibodies with the required specificity can be used, including, but not limited to, methods of selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, etc. display libraries available, for example, from Cambridge Antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid / Planegg, DE; Biovation, Aberdeen, Scotland, UK; BioInvent, Lund, Sweden; Dyax Corp.; Enzon; ​​Affymax / Biosite; Xoma, Berkeley, CA; Ixsys).For example, European Patent No. 368,684, International Application No. GB91 / 01134, International Application No. GB92 / 01755, International Application No. GB92 / 002240, International Application No. GB92 / 00883, International Application No. GB93 / 00605, US Patent Application No. 08 / 350260 (5 / 12 / 94), International Application No. GB94 / 01422, International Application No. GB94 / 02662, International Application No. GB97 / 01835 (CAT / MRC), International Application No. 90 / 14443, International Application No. 90 / 14424, International Application No. 90 / 14430, International Application No. US94 / 1234, International Publication No. WO92 / 18619, International Publication No. WO96 / 07754 (Scripps), European Patent No. 614 989 (MorphoSys), 95 / 16027 (BioInvent), 88 / 06630, 90 / 3809 (Dyax), U.S. Patent No. 4,704,692 (Enzon), International Application No. US91 / 02989 (Affymax), International Publication No. 89 / 06283, European Patent No. 371998, European Patent No. 550400 (Xoma), European Patent No. 229046, International Application No. US91 / 07149 (Ixsys), or stochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86 / 05803, EP 590689 (Ixsys, now Applied See, for example, "Cellular and Molecular Evolution (AME)," each of which is incorporated herein by reference in its entirety, or rely on the immunization of transgenic animals capable of producing a repertoire of human antibodies as known in the art and / or described herein (e.g., SCID mice, Nguyen et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998), each of which is incorporated herein by reference in its entirety).Such techniques include ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA, 95:14130-14135 (November 1998)), single-cell antibody production techniques (e.g., the "selected lymphocyte antibody method" (SLAM) (U.S. Patent No. 5,627,052; Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996)), gel microdroplets and flow cytometry (Powell et al., Biotechnol. 8:333-337 (1990)), One Cell Systems (Cambridge, MA), Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al., Bio / Technol. 13:787-790 (1995)), B cell selectors (Steenbakkers et al., Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers BV, Amsterdam, Netherlands (1988)).

[0049] Methods for engineering or humanizing non-human or human antibodies are similarly available and are well known in the art. Generally, humanized or engineered antibodies contain one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable, constant, or other domain of a known human sequence.

[0050] Known human Ig sequences are disclosed in numerous publications and websites, e.g., www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.html; www.public.iastate.edu / ~pedro / research_tools.html; www.mgen.uni-heidelberg.de / SD / IT / IT.html; www.whfreeman.com / immunology / CH05 / kuby05.htm; www.library.thinkquest.org / 12429 / Immune / Antibody.html; www.hhmi.org / grants / lectures / 1996 / vlab; www.path.cam.ac.uk / ~mrc7 / mikeimages.html; www.antibodyresource.com / ; www.mcb.harvard.edu / BioLinks / Immunology.html. www.immunologylink.com / ; www.pathbox.wustl.edu / ~hcenter / index.html; www.biotech.ufl.edu / ~hcl / ; www.pebio.com / pa / 340913 / 340913.html; www.nal.usda.gov / awic / pubs / antibody / ; www.m.ehime-u.ac.jp / ~yasuhito / Elisa.html; www.biodesign.com / table.asp; www.icnet.uk / axp / facs / davies / links.html; www.biotech.ufl.edu / ~fccl / protocol.html; www.isac-net.org / sites_geo.html; www.aximt1.imt.uni-marburg.de / ~rek / AEPStart.html; www.baserv.uci.kun.nl / ~jraats / links1.html; www.recab.uni-hd.de / immuno.bme.nwu.edu / ; www.mrc-cpe.cam.ac.uk / imt-doc / public / INTRO.html; www.ibt.unam.mx / vir / V_mice.html;imgt.cnusc.fr:8104 / ; www.biochem.ucl.ac.uk / ~martin / abs / index.html;antibody.bath.ac.uk / ; www.abgen.cvm.tamu.edu / lab / www.abgen.html; www.unizh.ch / ~honegger / AHOseminar / Slide01.html; www.cryst.bbk.ac.uk / ~ubcg07s / ; www.nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm; www.path.cam.ac.uk / ~mrc7 / humanisation / TAHHP.html; www.ibt.unam.mx / vir / structure / stat_aim.html; www.biosci.missouri.edu / smithgp / index.html; www.cryst.bioc.cam.ac.uk / ~fmolina / Web-pages / Pept / spottech.html; www.jerini.de / frproducts.html; www.patents.ibm.com / ibm.html.,Kabat et al. "Sequences of Proteins of Immunological Interest," US Pept. Health (1983), each of which is incorporated herein by reference in its entirety.

[0051] Such imported sequences can be used to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, binding rate constants, dissociation rate constants, avidity, specificity, half-life, or any other suitable property, as is well known in the art. Generally, some or all of the non-human or human CDR sequences are maintained while the non-human sequences in the variable and constant regions are replaced with human or other amino acids. Antibodies can also be optionally humanized while retaining high affinity for the antigen and other favorable biological properties. To this end, humanized antibodies can optionally be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the antigen-binding ability of the candidate immunoglobulin. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues directly and most substantially influence antigen binding.Humanization or engineering of the antibodies of the present invention may be accomplished using methods such as those described by Winter (Jone et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J.Mol.Biol.196:901(1987), Carter et al., Proc.Natl.Acad.Sci.USA89:4285(1992); Presta et al., J.Immunol.151:2623(1993), U.S. Patent Nos. 5723323, 5976862, 58 No. 24514, No. 5817483, No. 5814476, No. 5763192, No. 5723323, No. 5,766886, No. 571 No. 4352, No. 6204023, No. 6180370, No. 5693762, No. 5530101, No. 5585089, No. 52255 39, 4816567, International Application Nos. US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, International Application Nos. GB89 / 01334, GB91 / 01134, GB92 / 01755, International Publication Nos. WO90 / 14443, WO90 / 14424, WO90 / 14430, and EP229246 (each of which is incorporated by reference in its entirety, including the references cited therein).

[0052] Anti-TNF antibodies can also optionally be produced by immunization of transgenic animals (e.g., mice, rats, hamsters, non-human primates, etc.) capable of producing a repertoire of human antibodies, as described herein and / or known in the art. Cells producing human anti-TNF antibodies can be isolated from such animals and immortalized using suitable methods, such as those described herein.

[0053] Transgenic mice capable of producing a repertoire of human antibodies that bind to human antigens can be produced using known methods (e.g., but not limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016, and 5,789,650 issued to Lonberg et al.; WO 98 / 50433 to Jakobovits et al.; WO 98 / 24893 to Jakobovits et al.; WO 98 / 24884 to Lonberg et al.; WO 97 / 13852 to Lonberg et al.; WO 94 / 25585 to Kuc International Publication No. 96 / 34096 to Kucherlapate et al., European Patent No. 0463151(B1) to Kucherlapate et al., European Patent No. 0710719(A1) to Kucherlapate et al., European Patent No. 5,545,807 to Surani et al., International Publication No. 90 / 04036 to Bruggemann et al., European Patent No. 0438474(B1) to Bruggemann et al., European Patent No. 0814259(A2) to Lonberg et al., British Patent No. 2272440(A) to Lonberg et al., Nature 368:856-859 (1994), Int. Immunol. Int. Immunol. 6(4)579-591 (1994), Nature 2272440(A) to Lonberg et al. Genetics 7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93 (1995), and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), each of which is incorporated herein by reference in its entirety.Generally, these mice contain at least one transgene comprising DNA derived from at least one human immunoglobulin locus that has been functionally rearranged, or is capable of undergoing functional rearrangement. The endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the ability of the mice to produce antibodies encoded by endogenous genes.

[0054] Screening antibodies for specific binding to similar proteins or fragments can be successfully accomplished using peptide display libraries. This method involves screening a large sample of peptides for individual members with the desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be 3 to 5,000 or more amino acids in length, frequently 5 to 100 amino acids long, and most often about 8 to 25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have also been described. One type involves displaying peptide sequences on the surface of bacteriophage or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are described in International Patent Publication Nos. 91 / 17271, 91 / 18980, 91 / 19818, and 93 / 08278. Other systems for generating peptide libraries incorporate aspects of both in vitro chemical synthesis and recombinant methods. See International Patent Application Publication Nos. 92 / 05258, 92 / 14843, and 96 / 19256. See also U.S. Patent Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from sources such as Invitrogen (Carlsbad, CA) and Cambridge antibody Technologies (Cambridgeshire, UK).See, for example, U.S. Patent Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, and 5,856,456 assigned to Enzon; ​​U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500 assigned to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717 assigned to Affymax; and Cambridge antibody See U.S. Patent No. 5,885,793 assigned to Genentech; U.S. Patent No. 5,750,373 assigned to Genentech; U.S. Patent Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417 assigned to Xoma; Colligan, supra; Ausubel, supra; or Sambrook, supra, each of the foregoing patents and publications is incorporated herein by reference in its entirety.

[0055] The antibodies of the present invention can also be prepared using nucleic acids encoding at least one anti-TNF antibody to provide transgenic animals or mammals, such as goats, cows, horses, and sheep, that produce such antibodies in their milk. Such animals can be prepared using well-known methods. See, for example, but not limited to, U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489 (each of which is incorporated herein by reference in its entirety).

[0056] Antibodies of the present invention can further be prepared using at least one anti-TNF antibody-encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to, tobacco and corn) that produce such antibodies, specified portions, or variants in plant parts or cells cultured therefrom. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins using, for example, an inducible promoter have been successfully used to provide large quantities of recombinant proteins. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Transgenic corn has also been used to express mammalian proteins at commercial production levels with biological activity equivalent to proteins produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein. Antibodies have also been produced in large quantities from transgenic plant seeds containing antibody fragments, such as single-chain fragments (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and references cited therein. Accordingly, antibodies of the present invention can also be produced using transgenic plants by known methods. See, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October 1999); Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994) and references cited therein. See also, but not limited to, plant expression of antibodies generally. Each of the above documents is incorporated herein by reference in its entirety.

[0057] The antibodies of the present invention have a wide range of affinities (K D) can bind human TNF. In a preferred embodiment, at least one human mAb of the present invention can optionally bind human TNF with high affinity. For example, a human mAb can bind human TNF at about 10 -7 M or less, for example, 0.1 to 9.9 (or any range or value therein) x 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 or any range or value therein, including, but not limited to, K D can be combined with

[0058] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., "Antibody-Antigen Interactions," Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company: New York, NY (1992), and methods described therein.) The affinity measured for a particular antibody-antigen interaction can be different when measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen-binding parameters (e.g., K D , K. a , K. d Measurements of ) are preferably made using standardized solutions of antibody and antigen, and standardized buffers, such as those described herein.

[0059] Nucleic Acid Molecules. Using the information provided herein, such as a nucleotide sequence encoding at least 70-100% of the contiguous amino acids of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, and 8, an identified fragment, variant, or consensus sequence thereof, or a deposited vector containing at least one of these sequences, nucleic acid molecules of the invention encoding at least one anti-TNF antibody comprising all of the heavy chain variable CDR regions of SEQ ID NOS: 1, 2, and 3 and / or all of the light chain variable CDR regions of SEQ ID NOS: 4, 5, and 6 can be obtained using methods described herein or known in the art.

[0060] The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, called the antisense strand.

[0061] Isolated nucleic acid molecules of the present invention can include open reading frames (ORFs), optionally having one or more introns, including, but not limited to, nucleic acid molecules comprising at least a specified portion of at least one CDR, such as CDR1, CDR2, and / or CDR3 of at least one heavy chain (e.g., SEQ ID NOS: 1-3) or light chain (e.g., SEQ ID NOS: 4-6), nucleic acid molecules comprising coding sequences for anti-TNF antibodies or variable regions (e.g., SEQ ID NOS: 7, 8), and nucleic acid molecules that are substantially different from the above-described nucleic acid molecules but still, due to the degeneracy of the genetic code, encode at least one anti-TNF antibody described herein and / or known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one of skill in the art to generate such degenerate nucleic acid variants encoding a particular anti-TNF antibody of the present invention. See, e.g., Ausubel et al., supra. Such nucleic acid variants are encompassed by the present invention. Non-limiting examples of isolated nucleic acid molecules of the present invention include SEQ ID NOs: 10, 11, 12, 13, 14, and 15, which correspond to non-limiting examples of nucleic acids encoding the HC variable region and the LC variable region of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, respectively.

[0062] As provided herein, nucleic acid molecules of the invention, including nucleic acids encoding anti-TNF antibodies, can include those that themselves encode the amino acid sequence of an antibody fragment, sequences encoding full-length antibodies or portions of antibodies, coding sequences for antibodies, fragments, or portions, as well as additional sequences, such as at least one intron, with or without the aforementioned additional coding sequences, including, but not limited to, non-coding 5' and 3' sequences, e.g., transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., mRNA ribosome binding and stability), as well as coding sequences for at least one signal leader or fusion peptide, additional coding sequences encoding additional amino acids, e.g., amino acids that provide additional function. Thus, the antibody-encoding sequence can be fused to a marker sequence, for example, a sequence encoding a peptide that facilitates purification of the antibody, including the antibody fragment or portion to which it is fused.

[0063] Polynucleotides that selectively hybridize to the polynucleotides described herein. The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated or otherwise complementary to cDNAs in a human or mammalian nucleic acid library.

[0064] Preferably, the cDNA library contains at least 80% of the full-length sequences, preferably at least 85% or 90% of the full-length sequences, and more preferably at least 95% of the full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are typical, but not limiting, for sequences with low sequence identity to complementary sequences. Optionally, medium and high stringency conditions can be used for sequences with higher identities. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to specifically identify orthologous or paralogous sequences.

[0065] Optionally, the polynucleotides of the invention will encode at least a portion of an antibody encoded by a polynucleotide described herein. Polynucleotides of the invention encompass nucleic acid sequences available for selective hybridization to polynucleotides encoding antibodies of the invention. See, e.g., Ausubel, supra; Colligan, supra, each of which is incorporated herein by reference in its entirety.

[0066] Construction of Nucleic Acids The isolated nucleic acids of the invention can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or a combination thereof, as are well known in the art.

[0067] Nucleic acids can conveniently contain sequences in addition to the polynucleotides of the invention. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotides of the invention. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of the invention. The nucleic acids of the invention (excluding coding sequences) are optionally vectors, adapters, or linkers for cloning and / or expression of the polynucleotides of the invention.

[0068] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0069] Recombinant methods for constructing nucleic acids. The isolated nucleic acid compositions of the present invention, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning procedures known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to the polynucleotides of the present invention are used to identify desired sequences within a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0070] Nucleic acid screening and isolation methods. Probes based on the sequences of the polynucleotides of the present invention, as disclosed herein, can be used to screen cDNA or genomic libraries. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will understand that assays can be performed with varying degrees of hybridization stringency, and that either the hybridization or wash medium can be made more stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and target when duplex formation occurs. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent, such as formamide. For example, hybridization stringency can be successfully altered by changing the polarity of the reaction solution, e.g., by manipulating the formamide concentration within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization and / or wash medium. Optimally, the degree of complementarity is 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence differences in the probe and primer can be compensated for by reducing the stringency of the hybridization and / or wash medium.

[0071] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present invention without undue experimentation, based on the teachings and guidance provided herein.

[0072] Known methods of DNA or RNA amplification include the polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten et al.; U.S. Pat. No. 5,066,584 to Gelfan et al.; and U.S. Pat. No. 5,142,033 to Innis. No. 4,889,818 to Derrick et al., U.S. Pat. No. 4,994,370 to Silver et al., U.S. Pat. No. 4,766,067 to Biswas, and U.S. Pat. No. 4,656,134 to Ringold), and RNA-mediated amplification (U.S. Pat. No. 5,130,238 to Malek et al., trade name NASBA), which uses antisense RNA to a target sequence as a template for double-stranded DNA synthesis (the entire contents of which are incorporated herein by reference). (See, e.g., Ausubel, supra, or Sambrook, supra).

[0073] For example, polymerase chain reaction (PCR) techniques can be used to amplify the sequences of the polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, for generating nucleic acids to be used as probes for detecting the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide one of skill in the art through in vitro amplification methods can be found in Berger, Sambrook, and Ausubel, supra, as well as U.S. Pat. No. 4,683,202 to Mullis et al. (1987) and Innis et al., "PCR Protocols: A Guide to Methods and Applications," edited by Academic Press Inc., San Diego, CA (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to improve yields of long PCR products.

[0074] Synthetic methods for constructing nucleic acids. The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide that can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that chemical synthesis of DNA can be limited to sequences of about 100 or more bases, but longer sequences can be obtained by ligating shorter sequences.

[0075] Recombinant Expression Cassettes. The present invention further provides recombinant expression cassettes comprising nucleic acids of the invention. Nucleic acid sequences of the invention, e.g., cDNA or genomic sequences encoding antibodies of the invention, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. Recombinant expression cassettes typically comprise a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the invention.

[0076] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a non-heterologous form of a polynucleotide of the invention at an appropriate location (upstream, downstream, or within an intron) to up- or down-regulate expression of the polynucleotide. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0077] Vectors and Host Cells. The present invention also relates to vectors containing the isolated nucleic acid molecules of the present invention, host cells genetically engineered with recombinant vectors, and the production of at least one anti-TNF antibody by recombinant techniques well known in the art. See, e.g., Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety.

[0078] The polynucleotide can optionally be linked to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0079] The DNA insert should be operably linked to a suitable promoter. The expression construct will further contain a transcription initiation site, a transcription termination site, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will preferably contain a translation initiation site at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the translated mRNA. UAA and UAG are preferred for expression in mammalian or eukaryotic cells.

[0080] Expression vectors preferably, but are optional, include at least one selectable marker. Such markers include, for example, methotrexate (MTX) for eukaryotic cell culture, dihydrofolate reductase (DHFR; U.S. Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017), ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase (G418). Examples of suitable host cell vectors include, but are not limited to, erythrocyte synthetase, GS, U.S. Pat. Nos. 5,122,464; 5,770,359; and 5,827,739 (U.S. Pat. Nos. 5,122,464; 5,770,359; and 5,827,739) resistance genes, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria or prokaryotes (these patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are well known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other well-known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18, and Ausubel, supra, Chapters 1, 9, 13, 15, and 16.

[0081] At least one antibody of the present invention can be expressed in modified forms, such as fusion proteins, and can include not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the antibody to improve stability and persistence in host cells during purification or subsequent processing and storage. Peptide moieties can also be added to the antibody of the present invention to facilitate purification. Such regions can be removed prior to final preparation of the antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74, and Ausubel, supra, Chapters 16, 17, and 18.

[0082] Those of skill in the art are familiar with the numerous expression systems available for expression of nucleic acids encoding proteins of the invention.

[0083] Alternatively, a nucleic acid of the invention can be expressed in a host cell by being turned on (by engineering) in a host cell that contains endogenous DNA encoding an antibody of the invention. Such methods are well known in the art, such as those described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entireties.

[0084] One example of a cell culture useful for producing antibodies, specified portions, or variants thereof is mammalian cells. Mammalian cell lines are often in the form of monolayers of cells, although mammalian cell suspensions or bioreactors can also be used. Many suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Suitable host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC deposit number CRL-1580) and SP2 / 0-Ag14 cells (ATCC deposit number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0085] Expression vectors for these cells can include one or more expression control sequences such as, but not limited to, an origin of replication, a promoter (e.g., the late or early SV40 promoter, the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), the HSV tk promoter, the pgk (phosphoglycerate kinase) promoter, the EF-1α promoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulin promoter, an enhancer, and / or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., the SV40 large T Ag polyaddition site), and transcription termination sequences. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the invention are known and / or can be found, for example, in the American Type Culture Collection Catalogue of Cell Lines and Available from Hybridomas (www.atcc.org) or other known or commercial sources.

[0086] When eukaryotic host cells are used, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-781 (1983)). In addition, as is well known in the art, gene sequences for regulating replication in host cells can be incorporated into the vector.

[0087] Antibody Purification. Anti-TNF antibodies can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography (HPLC) can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0088] Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Depending on the host employed in a recombinant production procedure, the antibodies of the present invention can be glycosylated or non-glycosylated, although glycosylation is preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18, and 20; and Colligan, Protein Science, supra, chapters 12-14, all of which are incorporated herein by reference in their entireties.

[0089] Anti-TNF antibody Isolated antibodies of the invention comprising all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3 and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5, and 6 include the amino acid sequences of the antibodies disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody. Preferably, the human antibody or antigen-binding fragment binds to human TNF, thereby partially or substantially neutralizing at least one biological activity of the protein. An antibody, or specified portion or variant thereof, that partially or preferably substantially neutralizes at least one biological activity of at least one TNF protein or fragment can bind to the protein or fragment and thereby inhibit activity mediated through binding of TNF to a TNF receptor or through other TNF-dependent or -mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody capable of inhibiting TNF-dependent activity by about 20-120%, preferably at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more, depending on the assay. The ability of an anti-TNF antibody to inhibit TNF-dependent activity is preferably assessed by at least one suitable TNF protein or receptor assay described herein and / or known in the art. Human antibodies of the present invention may be of any class (e.g., IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may contain a kappa or lambda light chain. In one embodiment, the human antibody comprises an IgG heavy chain or a defined fragment, e.g., at least one isotype of IgG1, IgG2, IgG3, or IgG4. Antibodies of this type can be prepared by using transgenic mice or other transgenic non-human mammals that contain at least one human light chain (e.g., IgG, IgA) and IgM (e.g., γ1, γ2, γ3, γ4) transgene as described herein and / or known in the art. In another embodiment, the anti-human TNF human antibody comprises an IgG1 heavy chain and an IgG1 light chain.

[0090] As used herein, the term "antibody" or "antibodies" includes biosimilar antibody molecules approved under the Biologics Price Competition and Innovation Act of 2009 (BPCI Act) and similar global laws and regulations. Under the BPCI Act, an antibody may be demonstrated to be biosimilar if data demonstrate that it is "highly similar" to the reference antibody, despite minor differences in clinically inactive components, and is "expected" to produce clinical results equivalent to the reference antibody in terms of safety, purity, and potency (Endocrine Practice: February 2018, Vol. 24, No. 2, pp. 195-204). These biosimilar antibody molecules offer an abbreviated approval pathway, whereby applicants rely on clinical data from the innovator's reference antibody to secure regulatory approval. Compared to the original innovator reference antibody approved by the FDA based on successful clinical trials, biosimilar antibody molecules are referred to herein as "biosimilars." As presented herein, SIMPONI® (golimumab) is the original innovator reference anti-TNF antibody approved by the FDA based on successful clinical trials. Golimumab has been marketed in the United States since 2009.

[0091] Exemplary Sequences In various embodiments, the TNF inhibitor comprises the anti-TNF antibody SIMPONI® (golimumab), or an antigen-binding fragment thereof comprising the sequence shown below: For more information regarding the anti-TNF antibody SIMPONI® (golimumab) and other anti-TNF antibodies, see, e.g., U.S. Patent Nos. 7,250,165; 7,691,378; 7,521,206; 7,815,909; 7,820,169; 8,241,899; 8,603,778; 9,321,836; and 9,828,424.

[0092] Exemplary Anti-TNF Antibody Sequences Example - SIMPONI® (Golimumab) The heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs) are defined by Kabat.

[0093] Amino acid sequence of the golimumab heavy chain (HC) with CDRs underlined: (SEQ ID NO: 36)

[0094] TIFF2023553702000002.tif43136

[0095] Amino acid sequence of the golimumab light chain (LC) with CDRs underlined: (SEQ ID NO: 37)

[0096] TIFF2023553702000003.tif21136

[0097] Amino acid sequence of golimumab variable heavy chain (VH) with CDRs underlined: (SEQ ID NO: 38)

[0098] TIFF2023553702000004.tif19136

[0099] Amino acid sequence of the golimumab variable light chain (VL) with CDRs underlined: (SEQ ID NO: 39)

[0100] TIFF2023553702000005.tif13134

[0101] Amino acid sequence of complementarity determining region 1 (HCDR1) of golimumab heavy chain: (SEQ ID NO: 40) SYAMH

[0102] Amino acid sequence of complementarity determining region 2 (HCDR2) of golimumab antibody heavy chain: (SEQ ID NO: 41) FMSYDGSNKKYADSVKG

[0103] Amino acid sequence of complementarity determining region 3 (HCDR3) of golimumab heavy chain: (SEQ ID NO: 42) DRGIAAGGNYYYYGMDV

[0104] Amino acid sequence of complementarity determining region 1 (LCDR1) of golimumab light chain: (SEQ ID NO: 43) RASQSVYSYLA

[0105] Amino acid sequence of complementarity determining region 2 (LCDR2) of golimumab light chain: (SEQ ID NO: 44) DASNRAT

[0106] Amino acid sequence of complementarity determining region 3 (LCDRL) of golimumab light chain: (SEQ ID NO: 45) QQRSNWPPFT

[0107] At least one antibody of the present invention binds to at least one specified epitope specific to at least one TNF protein, subunit, fragment, portion, or any combination thereof. The at least one epitope can include at least one antibody binding region comprising at least a portion of the protein, preferably comprising at least one extracellular, soluble, hydrophilic, external, or cytoplasmic portion of the protein. The at least one specified epitope can include any combination of at least one amino acid sequence of at least 1-3 amino acids relative to the entire specified portion of contiguous amino acids of SEQ ID NO:9.

[0108] Generally, human antibodies or antigen-binding fragments of the invention comprise an antigen-binding region comprising at least one human complementarity-determining region (CDR1, CDR2, and CDR3) or variant of at least one heavy chain variable region, and at least one human complementarity-determining region (CDR1, CDR2, and CDR3) or variant of at least one light chain variable region. As a non-limiting example, an antibody or antigen-binding portion or variant may comprise at least one of a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 and / or a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, an antibody or antigen-binding fragment may have an antigen-binding region comprising at least a portion of at least one heavy chain CDR (i.e., CDR1, CDR2, and / or CDR3) having the corresponding CDR1, 2, and / or 3 amino acid sequence (e.g., SEQ ID NOs: 1, 2, and / or 3). In another specific embodiment, the antibody or antigen-binding portion or variant can have an antigen-binding region comprising at least a portion of at least one light chain CDR (i.e., CDR1, CDR2, and / or CDR3) having the corresponding CDR1, 2, and / or 3 amino acid sequence (e.g., SEQ ID NOs: 4, 5, and / or 6). In a preferred embodiment, the three heavy chain CDRs and three light chain CDRs of the antibody or antigen-binding fragment have the amino acid sequence of the corresponding CDRs of at least one of mAbs TNV148, TNV14, TNV15, TNV196, TNV118, TNV32, and TNV86, as described herein. Such antibodies can be prepared by preparing and expressing a nucleic acid molecule (i.e., one or more) encoding the antibody using conventional techniques for recombinant DNA technology, or by using any other suitable method, or by chemically linking various portions of the antibody (e.g., CDRs, framework) together using conventional techniques.

[0109] An anti-TNF antibody can comprise at least one heavy or light chain variable region having a defined amino acid sequence. For example, in a preferred embodiment, the anti-TNF antibody comprises at least one heavy chain variable region, optionally having the amino acid sequence of SEQ ID NO: 7, and / or at least one light chain variable region, optionally having the amino acid sequence of SEQ ID NO: 8. Antibodies that bind to human TNF and comprise defined heavy or light chain variable regions can be prepared using suitable methods, such as phage display (Katsube, Y. et al., Int J Mol. Med 1(5):863-868 (1998)) or methods employing transgenic animals, known in the art and / or described herein. For example, a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus capable of undergoing functional rearrangement can be immunized with human TNF or a fragment thereof to elicit antibody production. If desired, antibody-producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and / or as known in the art. Alternatively, antibodies, specified portions, or variants can be expressed using encoding nucleic acid or portions thereof in a suitable host cell.

[0110] The present invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in sequences substantially the same as the amino acid sequences described herein. Preferably, such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs have high affinity (e.g., K D is about 10 -9M or less). Amino acid sequences that are substantially the same as the sequences described herein include sequences containing conservative amino acid substitutions as well as amino acid deletions and / or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid with a second amino acid that has chemical and / or physical properties (e.g., charge, structure, polarity, hydrophobicity / hydrophilicity) similar to those of the first amino acid. Conservative substitutions include replacing one amino acid with another amino acid within the following group: lysine (K), arginine (R), and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine ​​(C), and glycine (G); F, W, and Y; C, S, and T.

[0111] Amino Acid Codes. The amino acids that make up the anti-TNF antibodies of the invention are often abbreviated. Amino acid designations can be provided by representing the amino acid by its single-letter code, its three-letter code, name, or three-nucleotide codon, and are well understood in the art (see Alberts, B. et al., Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc., New York (1994)).

[0112] [Table 1]

[0113] The anti-TNF antibodies of the invention may contain one or more amino acid substitutions, deletions, or additions, either due to natural mutation or human manipulation, as specified herein.

[0114] Of course, the number of amino acid substitutions a skilled artisan may make will depend on numerous factors, including those described above. Generally, the number of amino acid substitutions, insertions, or deletions for any given anti-TNF antibody, fragment, or variant will not exceed 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, e.g., 1-30, or any range or value therein, as specified herein.

[0115] Amino acids in the anti-TNF antibodies of the invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, supra, Chapter 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one TNF-neutralizing activity. Sites critical for antibody binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).

[0116] The anti-TNF antibodies of the present invention can comprise, but are not limited to, at least one portion, sequence, or combination selected from one to all of the contiguous amino acids of at least one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

[0117] The anti-TNF antibody may further optionally comprise at least one polypeptide of 70-100% of the contiguous amino acids of at least one of SEQ ID NOs: 7 and 8.

[0118] In one embodiment, the amino acid sequence of an immunoglobulin chain or portion thereof (e.g., variable region, CDR) has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) to the amino acid sequence of at least one corresponding chain of SEQ ID NO: 7, 8. For example, the amino acid sequence of the light chain variable region can be compared to the sequence of SEQ ID NO: 8, or the amino acid sequence of the heavy chain CDR3 can be compared to SEQ ID NO: 7. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) is determined using a suitable computer algorithm, as is well known in the art.

[0119] Exemplary heavy and light chain variable region sequences are set forth in SEQ ID NOs: 7 and 8. Antibodies of the invention, or specified variants thereof, can include any number of contiguous amino acid residues from an antibody of the invention, the number being selected from the group of integers consisting of 10-100% of the number of contiguous residues in an anti-TNF antibody. Optionally, this subsequence of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or more amino acids in length, or any range or value therein. Furthermore, the number of such subsequences can be any integer selected from the group consisting of 1-20, such as at least 2, 3, 4, or 5.

[0120] As those skilled in the art will recognize, the present invention includes at least one biologically active antibody of the present invention. A biologically active antibody has a specific activity that is at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 1000% of that of a natural (non-synthetic), endogenous, or related and known antibody. Methods for assaying and quantitatively measuring enzymatic activity and substrate specificity are well known to those skilled in the art.

[0121] In another aspect, the invention relates to human antibodies and antigen-binding fragments described herein that are modified by the covalent attachment of an organic moiety. Such modifications can produce antibodies or antigen-binding fragments with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain embodiments, the hydrophilic polymer group has a molecular weight of about 800 to about 120,000 daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid group or fatty acid ester group can contain about 8 to about 40 carbon atoms.

[0122] The modified antibodies and antigen-binding fragments of the present invention may contain one or more organic moieties covalently attached to the antibody, directly or indirectly. Each organic moiety attached to an antibody or antigen-binding fragment of the present invention can independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic and dicarboxylic acids. As used herein, the term "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Thus, antibodies modified by the covalent attachment of polylysine are encompassed by the present invention. Hydrophilic polymers suitable for modifying antibodies of the present invention can be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers used to modify antibodies of the present invention have, as individual molecular entities, a molecular weight of about 800 to about 150,000 daltons. For example, PEG 5000 and PEG 20,000 can be used, where the subscript is the average molecular weight of the polymer in Daltons. The hydrophilic polymer group can be substituted with 1 to about 6 alkyl groups, fatty acid groups, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by utilizing suitable methods. For example, a polymer containing an amine group can be linked to a carboxylate salt of a fatty acid or fatty acid ester, or an activated carboxylate salt on a fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be linked to a hydroxyl group on the polymer.

[0123] Fatty acids and fatty acid esters suitable for modifying antibodies of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying antibodies of the invention include, for example, n-dodecanoate (C 12 , laurate), n-tetradecanoate (C 14 , myristate), n-octadecanoate (C 18 , stearate), n-eicosanoate (C 20 , arachidate), n-docosanoate (C 22 , behenic acid), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), cis-Δ9-octadecanoate (C 18 oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C 20 , arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight-chain or branched-chain lower alkyl group. The lower alkyl group can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0124] Modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reacting with one or more modifying agents. As used herein, the term "modifying agent" refers to a suitable organic group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be linked to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activated groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Activated groups can be attached directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or to linker moieties, such as divalent C1-C 12The linker moiety can be linked via a group (wherein one or more carbon atoms can be replaced with a heteroatom such as oxygen, nitrogen, or sulfur). Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. A modifying agent containing a linker moiety can be produced by forming an amide bond between a free amine and a fatty acid carboxylate, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylic acid salt as described, or it can be reacted with maleic anhydride and the resulting product cyclized to generate an activated maleimide derivative of a fatty acid. (See, for example, WO 92 / 16221 to Thompson et al., the teachings of which are incorporated herein by reference in their entirety.)

[0125] The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, organic moieties can be attached to the antibody in a non-site-specific manner using an amine-reactive modifying agent, e.g., an NHS ester of PEG. Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of the antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibodies of the invention. Modified human antibodies and antigen-binding fragments containing organic moieties attached to specific sites on the antibodies of the invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996).

[0126] Anti-idiotypic antibodies to anti-TNF antibody compositions. In addition to monoclonal or chimeric anti-TNF antibodies, the present invention also relates to anti-idiotypic (anti-Id) antibodies specific to such antibodies of the present invention. Anti-Id antibodies are antibodies that generally recognize unique determinants associated with the antigen-binding region of another antibody. Anti-Ids can be prepared by immunizing an animal (e.g., a mouse strain) of the same species and genetic type as the Id antibody source with the antibody or its CDR-containing region. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce anti-Id antibodies. Anti-Id antibodies can also be used as "immunogens" to induce immune responses in yet another animal, generating so-called anti-anti-Id antibodies.

[0127] Anti-TNF antibody compositions. The present invention also provides at least one anti-TNF antibody composition comprising at least one, at least two, at least three, at least four, at least five, at least six, or more thereof, provided in a non-naturally occurring composition, mixture, or form, as described herein and / or known in the art. Such compositions include non-naturally occurring compositions comprising at least one or two full-length, C- and / or N-terminal deletion variants, domains, fragments, or specified variants of an anti-TNF antibody amino acid sequence selected from the group consisting of 70-100% of the contiguous amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, or a specified fragment, domain, or variant thereof. Preferred anti-TNF antibody compositions comprise at least one or two full-length, fragment, domain, or variant as at least one CDR- or LBR-containing portion of 70-100% of an anti-TNF antibody of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a specified fragment, domain, or variant thereof. More preferred compositions comprise 70-100% of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 40-99% of at least one of the specified fragments, domains, or variants thereof. Percentages of such compositions may be by weight, volume, concentration, molality, or molality as a liquid or dry solution, mixture, suspension, emulsion, or colloid, as known in the art or as described herein.

[0128] The anti-TNF antibody compositions of the present invention further comprise at least one anti-TNF antibody directed to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy, and optionally at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an anti-rheumatic drug (such as, but not limited to, methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, aurothioprine, thiazolinone ... sodium malate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterial agents (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, and other antibacterial agents), antipsoriatics, corticosteroids, Nabolic steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin (e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), vaccinations, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatics, hair The composition may comprise at least one of any suitable and effective amount of a composition or pharmaceutical composition, further comprising at least one selected from the group consisting of myomodulators, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic agents, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or similar drugs, dornase alfa (Pulmozyme), cytokines, or cytokine antagonists. Non-limiting examples of such cytokines include, but are not limited to, IL-1 through IL-23. Suitable dosages are well known in the art.For example, Wells et al., eds., Pharmacotherapy Handbook, 2. nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000 Special Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

[0129] Such anti-cancer or anti-infective agents may also include toxin molecules associated, conjugated, co-formulated, or combined with at least one antibody of the present invention. The toxin can optionally act to selectively kill pathological cells or tissues. Pathological cells may be cancer cells or other cells. Such toxins may be purified or recombinant toxins or toxin fragments containing at least one functional cytotoxic domain of a toxin selected from, but not limited to, at least one of ricin, diphtheria toxin, snake toxin, or bacterial toxin. The term toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant, or recombinant bacteria or viruses that can cause any pathology, including toxic shock, which can be fatal, in humans and other mammals. Such toxins may include, but are not limited to, enterotoxigenic Escherichia coli (E. coli) heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxin, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), streptococcal enterotoxin, and the like.Such bacteria include enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., serotype 0157 strain: H7), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus pyogenes), Shigella spp. (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Salmonella spp. (e.g., Salmonella typhi, Salmonella cholera suis, Salmonella enteritidis), Clostridium spp. (e.g., Clostridium perfringens), perfringens, Clostridium difficile, Clostridium botulinum), Campylobacter species (e.g., Campylobacter jejuni, Campylobacter fetus), Helicobacter species (e.g., Heliocbacter pylori), Aeromonas species (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides, Yersinia enterocolitica enterocolitica, Vibrio species (e.g., Vibrio cholerae, Vibrio parahemolyticus), Klebsiella species, Pseudomonas aeruginosa, and strains of Streptococci species.See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp. 1-13, Little, Brown and Co., Boston (1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiology and Control, 2nd ed., pp. 239-254, Plenum Medical Book Co., New York (1991); Mandell et al., Principles and Practice of Infectious Diseases, 3rd ed., Churchill Livingstone, New York (1990); Berkow et al., eds., The Merck Manual, 16th ed., Merck and Co., Rahway, NJ (1992); Wood et al., FEMS Microbiology Immunology, 76:121-134 (1991); Marrack et al., Science, 24:8705-711 (1990), the contents of which are incorporated herein by reference in their entireties.

[0130] The anti-TNF antibody compounds, compositions or mixtures of the present invention may further comprise at least one of any suitable auxiliary agent, including, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable auxiliary agents are preferred. Methods for preparing such sterile solutions and non-limiting examples thereof are well known in the art and are described, for example, in Gennaro, Ed., Remington's Pharmaceutical Sciences, 1894, pp. 181-184. th Edition, Mack Publishing Co. (Easton, PA) 1990. Pharmaceutically acceptable carriers suitable for the administration method, solubility, and / or stability of the anti-TNF antibody, fragment, or variant composition can be routinely selected, as known in the art or as described herein.

[0131] Pharmaceutical additives and additives useful in the present compositions include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., saccharides including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1 to 99.99% by weight or volume, alone or in combination. Exemplary protein additives include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / antibody components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0132] Suitable carbohydrate additives for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starches; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and myo-inositol. Preferred carbohydrate additives for use in the present invention are mannitol, trehalose, and raffinose.

[0133] The anti-TNF antibody composition may also contain a buffer or pH adjuster, typically a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, or phosphate buffers. A preferred buffer for use in the present composition is an organic acid salt such as citrate.

[0134] In addition, the anti-TNF antibody compositions of the invention may contain polymeric additives / additives such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0135] These and additional known pharmaceutical additives and / or excipients suitable for use in anti-TNF antibody, portion or variant compositions according to the invention are known in the art and are described, for example, in "Remington: The Science & Practice of Pharmacy," 1999; th ed., Williams&Williams, (1995) and “Physician's Desk Reference”, 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are incorporated herein by reference in their entireties. Preferred carrier or additive materials are carbohydrates (e.g., monosaccharides and alditols) and buffers (e.g., citric acid) or polymeric agents.

[0136] Formulations. As discussed above, the present invention provides stable formulations, preferably saline or phosphate buffer with selected salts, as well as preservative-containing preservative solutions and formulations, and versatile preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one anti-TNF antibody in a pharmaceutically acceptable formulation. The preserved formulations contain at least one known preservative, optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. As known in the art, concentrations of 0.001 to 5%, or 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2 , 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein may be used.Non-limiting examples include preservative-free, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01%), 0.001-2.0% phenol (e.g., 0.001-0.5%), and 0.001-2.0% phenol (e.g., 0.001-0.5%). For example, 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkyl parabens (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0137] As discussed above, the present invention provides articles of manufacture comprising packaging and at least one vial containing a solution of at least one anti-TNF antibody, optionally with a buffer and / or preservative, formulated in an aqueous diluent, the packaging comprising a label stating that the solution can be kept for 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72, or more hours. The present invention further includes articles of manufacture comprising packaging and a first vial containing lyophilized at least one anti-TNF antibody and a second vial containing an aqueous diluent of the formulated buffer or preservative, the packaging comprising a label instructing a patient to reconstitute the at least one anti-TNF antibody with the aqueous diluent to form a solution that can be kept for 24 hours or more.

[0138] The at least one anti-TNF antibody used in accordance with the present invention may be produced by recombinant means, including production from mammalian cells or transgenic preparations, or purified from other biological sources, as described herein or known in the art.

[0139] The range of at least one anti-TNF antibody included in the products of the invention is in an amount to provide a concentration in the range of about 1.0 μg / ml to about 1000 mg / ml upon reconstitution for wet / dry systems, although lower and higher concentrations are workable, depending on the intended delivery vehicle, e.g., solution formulations differ from transdermal patch, pulmonary, transmucosal, or osmotic or micropump methods.

[0140] Preferably, the aqueous diluent optionally further contains a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of the preservative used in the formulation is sufficient to produce an antibacterial effect. Such a concentration will vary depending on the preservative selected and can be easily determined by one skilled in the art.

[0141] Other additives, such as isotonicity agents, buffers, antioxidants, and preservative enhancers, can optionally and preferably be added to the diluent. Isotonicity agents, such as glycerin, are commonly used at known concentrations. Preferably, a physiologically tolerable buffer is added to provide improved pH control. The formulations can be used over a wide pH range, such as from about pH 4 to about pH 10, preferably from about pH 5 to about pH 9, and most preferably from about pH 6.0 to about pH 8.0. Preferably, the formulations of the present invention have a pH of about 6.8 to about 7.8. Suitable buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate-buffered saline (PBS).

[0142] Other additives, such as pharmaceutically acceptable solubilizers such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol), or nonionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyols, other block copolymers, and chelating agents such as EDTA and EGTA, can optionally be added to the formulation or composition to reduce aggregation. These additives are particularly useful when a pump or plastic container is used to administer the formulation. The presence of a pharmaceutically acceptable surfactant reduces the tendency of the protein to aggregate.

[0143] The formulations of the present invention can be prepared by a process comprising mixing at least one anti-TNF antibody with a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (e.g., methyl, ethyl, propyl, butyl), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. Mixing the at least one anti-TNF antibody with the preservative in the aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a quantity of at least one anti-TNF antibody in a buffered solution is combined with the desired preservative in a quantity of buffered solution sufficient to provide the desired concentrations of protein and preservative. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of components, whether or not additional additives are used, and the temperature and pH during formulation preparation are all factors that can be optimized for the dosage concentration and administration means used.

[0144] The claimed formulations can be provided to patients as clear solutions or as dual vials containing a vial of at least one lyophilized anti-TNF antibody to be reconstituted with a second vial containing water, preservatives and / or excipients, preferably phosphate buffer and / or saline, and selected salts in an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0145] The claimed products are useful for administration from immediate to over a 24 hour or longer period. Thus, the products claimed by the present invention provide significant patient benefits. The formulations of the present invention can optionally be safely stored at temperatures of about 2 to about 40° C. and retain the biological activity of the protein for extended periods; therefore, the packaging label can indicate that the solution can be stored and / or used for 6, 12, 18, 24, 36, 48, 72, or 96 hours or longer. When a preserved diluent is used, such labeling can include use for up to 1-12 months, 6 months, 1.5 years, and / or 2 years.

[0146] Solutions of at least one anti-TNF antibody of the present invention can be prepared by a process comprising mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a quantity of at least one antibody in water or a buffer is combined in an amount sufficient to provide the desired concentration of protein, and optionally, a preservative or buffer. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of components, whether or not additional additives are used, and the temperature and pH during formulation preparation are all factors that can be optimized for the dosage concentration and administration means used.

[0147] The claimed products can be provided to patients as a clear solution or as dual vials containing a vial of at least one lyophilized anti-TNF antibody that is reconstituted with a second vial containing an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0148] The claimed products can be provided indirectly to patients by providing pharmacies, clinics, or other such institutions and facilities with dual vials containing a vial of at least one lyophilized anti-TNF antibody to be reconstituted with a second vial containing a clear solution or aqueous diluent, where the clear solution may be up to 1 liter or even larger in volume, from which smaller amounts of the at least one antibody solution can be withdrawn one or more times from the larger container and transferred to smaller vials and provided by the pharmacy or clinic to customers and / or patients.

[0149] Approved devices that contain these single-vial systems include pen injector devices for delivering solutions such as BD® (pen injector device), NOvoPen® (pen injector device), AutoPen® (pen injector device), OptiPen® (pen injector device), Genotropin Pen® (pen injector device), HUMATROPEN® (pen injector device), BIojector® (pen injector device), Reco-Pen, Humaject, J-tip Needle-Free Injector, Intraject, Medi-Ject, and others, including those manufactured by, for example, Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com); Bioject, Portland, Oregon (www.bioject.com); National Medical Products, Weston These devices are manufactured or developed by Medi-Ject Corp (Minneapolis, MN, www.mediject.com), and Eppendorf Medical (Peterborough, UK, www.weston-medical.com), and Medi-Ject Corp (Minneapolis, MN, www.mediject.com). Approved devices that contain combination vial systems include pen injector systems for reconstituting lyophilized drugs in cartridges for delivery of the reconstituted solution, such as the HUMATROPEN® (pen injector device).

[0150] The presently claimed products include packaging. The packaging provides information required by regulatory agencies as well as conditions under which the product can be used. The packaging of the present invention provides instructions to the patient to reconstitute at least one anti-TNF antibody with an aqueous diluent to form a solution and use this solution for a period of 2 to 24 hours or more in the wet / dry two-vial product. In the case of a single-vial solution product, the label indicates that the solution can be used for a period of 2 to 24 hours or more. The presently claimed products are useful for human pharmaceutical product applications.

[0151] The formulations of the present invention can be prepared by a process comprising mixing at least one anti-TNF antibody and a selected buffer, preferably saline or a phosphate buffer containing a selected salt. Mixing the at least one antibody and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a quantity of at least one antibody in water or buffer is combined with a desired buffer in a quantity of water sufficient to provide the desired concentrations of protein and buffer. Variations on this process will be recognized by those skilled in the art. For example, the order of addition of components, whether or not additional additives are used, and the temperature and pH during formulation preparation are all factors that can be optimized for the administration concentration and means used.

[0152] The claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials containing a vial of at least one lyophilized anti-TNF antibody that is reconstituted with a second vial containing a preservative or buffer and additives in an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0153] At least one anti-TNF antibody in any of the stable or preserved formulations or solutions described herein can be administered to a subject in accordance with the present invention via a variety of delivery methods, as are well known in the art, such as SC or IM injection, transdermal, transpulmonary, transmucosal, implant, osmotic pump, cartridge, micropump, or other means understood by one of ordinary skill in the art.

[0154] Therapeutic Applications: The present invention also provides methods for modulating or treating at least one TNF-related disorder in a cell, tissue, organ, animal, or patient using at least one dual integrin antibody of the present invention, as known in the art or described herein.

[0155] The present invention also provides methods for modulating or treating at least one TNF-related disorder in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of obesity, an immune-related disorder, a cardiovascular disorder, an infectious disease, a malignant disorder, or a neurological disorder.

[0156] The present invention also relates to the treatment of rheumatoid arthritis, juvenile, systemic onset juvenile rheumatoid arthritis, ankylosing spondylitis, gastric ulcer, seronegative arthropathy, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis / uveitis / optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / Wegener's granulomatosis, sarcoidosis, orchitis / vasectomy repair, allergic / atopic diseases, asthma, allergic rhinitis, dermatitis, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplant, organ transplant rejection, graft versus host disease, systemic Inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningococcemia, trauma / hemorrhage, burns, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, alcoholic hepatitis, chronic inflammatory diseases, sarcoidosis, Crohn's disease, sickle cell anemia, diabetes, nephrosis, atopic disease, hypersensitivity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, any organ or tissue Transplant rejection, kidney transplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, skin allograft rejection, cartilage graft rejection, bone graft rejection, small intestine transplant rejection, fetal thymus transplant rejection, parathyroid transplant rejection, any organ or tissue xenograft rejection, allograft rejection, antireceptor hyperreaction, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity reaction, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organ Hypertrophy, endocrinopathy, monoclonal gammopathy, and skin syndromes), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin syndromes, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, post-MI open-heart syndrome, type IV hypersensitivity, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulomas due to intracellular organisms, drug hypersensitivity, metabolic / idiopathic Wilson's disease, hemacromatosis, alpha-1-antitrypsin deficiency, diabetic retinopathy,Also provided are methods for modulating or treating at least one immune-related disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of Hashimoto's thyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD), familial hemophagocytosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, toxicity, pre-eclampsia, OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy (including, but not limited to, asthenia, anemia, cachexia, etc.), chronic salicylate intoxication, and the like. See, e.g., "Merck Manual," 12th to 17th eds., Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), and "Pharmacotherapy Handbook," edited by Wells et al., 2nd ed., Appleton and Lange, Stamford, Conn. (1998, 2000), each of which is incorporated by reference in its entirety.

[0157] The present invention relates to the treatment of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic attack, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic arteriosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, cardiovascular syphilis, heart failure, cor pulmonale, primary pulmonary hypertension, arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), post-perfusion syndrome, cardiopulmonary bypass inflammatory response, chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrhythmias, ventricular fibrillation, His bundle arrhythmias, and the like. Also provided are methods for modulating or treating cardiovascular disease in a cell, tissue, organ, animal or patient, including at least one of: cardiac arrhythmias, atrioventricular block, bundle branch block, myocardial ischemic disease, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumors, aortic aneurysms and peripheral arterial aneurysms, aortic dissection, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular disorders, obliterative arterial disorders, peripheral atherosclerosis, thromboangiitis obliterans, functional peripheral arterial disorders, Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venous disease, venous thrombosis, varicose veins, arteriovenous fistula, lymphedema, lipedema, unstable angina, reperfusion injury, post-pump syndrome, ischemia-reperfusion injury, and the like. Such methods may optionally include administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.

[0158] The present invention relates to acute and chronic parasitic or infectious processes including acute or chronic bacterial infections, bacterial, viral and fungal infections, HIV infection / HIV neuropathy, meningitis, hepatitis (such as A, B or C), septic arthritis, peritonitis, pneumonia, epiglottitis, Escherichia coli 0157:h7, hemolytic uremic syndrome / embolic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, Mycobacterium tuberculosis, mycobacterium tuberculosis, and the like. Also provided are methods for modulating or treating at least one infectious disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of: Tertium avium intracellulare, Pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis / epididymitis, Legionella, Lyme disease, influenza type A, Epstein-Barr virus, viral-associated hemophagocytic syndrome, viral encephalitis / aseptic meningitis, and the like.

[0159] The present invention also provides methods for modulating or treating at least one malignant disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of leukemia, acute leukemia, acute lymphocytic leukemia (ALL), B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal carcinoma, malignant histiocytosis, malignant paraneoplastic syndrome / hypercalcemia, solid tumor, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and the like.

[0160] The present invention relates to a method for treating neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia syndrome, demyelinating diseases such as multiple sclerosis and acute transverse myelitis, extrapyramidal and cerebellar disorders such as lesions of the corticospinal system, lesions of the basal ganglia or cerebellar disorders, hyperkinetic movement disorders such as Huntington's chorea and senile chorea, drug-induced movement disorders such as those induced by drugs that block CNS dopamine receptors, hypokinetic movement disorders such as Parkinson's disease, progressive supranuclear palsy, structural lesions of the cerebellum, spinocerebellar degenerations such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degeneration, multisystem degeneration (Mencel, Dejerine-Thomas, Shi-Drager and Machado-Joseph), systemic diseases (Refsum's disease, abetalipoproteinemia, ataxia, telangiectasia and mitochondrial multisystem disorders), demyelinating core disorders. Also provided are methods for modulating or treating at least one neurological disorder in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of the following: multiple sclerosis, acute transverse myelitis, and motor unit disorders, e.g., neuromuscular atrophies (anterior horn cell degeneration, e.g., amyotrophic lateral sclerosis, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy), Alzheimer's disease, Down's syndrome in middle age, diffuse Lewy body disease, senile dementia with Lewy bodies, Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakob disease, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and dementia pugilistica. Such methods may optionally comprise administering to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant. See, e.g., The Merck Manual, 16th ed., Merck & Company, Rahway, NJ (1992).

[0161] Any of the methods of the present invention may comprise administering to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody. Such methods may optionally further comprise co-administration or combination therapy for the treatment of such immune disorders, wherein the administration of at least one anti-TNF antibody, specified portion, or variant thereof is in combination with at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an anti-rheumatic drug (such as, but not limited to, methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, or erythromycin), or a combination therapy for the treatment of such immune disorders. Antibiotics (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, and other antibacterial agents), antipsoriatics, corticosteroids, and anabolic steroids drugs, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin (e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunizing agents, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatics, The method may further comprise administering, before, simultaneously with, and / or after at least one selected from the group consisting of a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, an antimanic, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta-agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, cromolyn, epinephrine or an analogue, dornase alfa (Pulmozyme), a cytokine, or a cytokine antagonist. Suitable dosages are known in the art.For example, Wells et al., eds., Pharmacotherapy Handbook, 2. nd Edition, Appleton and Lange, Stamford, CT (2000), PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000 Special Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

[0162] TNF antagonists suitable for the compositions, combination therapies, co-administration devices and / or methods of the invention (which further include at least one antibody, specified portion, and variant thereof of the invention) include anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules that specifically bind to TNF, compounds that block and / or inhibit TNF synthesis, TNF release, or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and rolipram), A2b adenosine receptor agonists, and A2b agonists. These include, but are not limited to, adenosine receptor enhancers, compounds that block and / or inhibit TNF receptor signaling, such as mitogen-activated protein (MAP) kinase inhibitors, compounds that block and / or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors, compounds that block and / or inhibit TNF activity, such as angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril), and compounds that block and / or inhibit TNF production and / or synthesis, such as MAP kinase inhibitors.

[0163] As used herein, a "tumor necrosis factor antibody," "TNF antibody," "TNFα antibody" or "fragment," etc., reduces, blocks, inhibits, abrogates, or interferes with TNFα activity in vitro, in situ, and / or preferably in vivo. For example, suitable TNF human antibodies of the present invention are capable of binding to TNFα and include anti-TNF antibodies, antigen-binding fragments thereof, and identified mutants or domains thereof that specifically bind to TNFα. Suitable TNF antibodies or fragments may also reduce, block, abrogate, interfere with, prevent, and / or inhibit TNF RNA, DNA, or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and / or synthesis.

[0164] Chimeric antibody cA2 consists of the antigen-binding variable region of a high-affinity neutralizing murine anti-human TNFα IgG1 antibody designated A2 and the constant region of a human IgG1 kappa immunoglobulin. The human IgG1 Fc region improves the effector function of the cognate antibody, increases circulating serum half-life, and reduces the immunogenicity of the antibody. The binding activity and epitope specificity of chimeric antibody cA2 are derived from the variable region of murine antibody A2. In certain embodiments, a preferred source of nucleic acid encoding the variable region of murine antibody A2 is the A2 hybridoma cell line.

[0165] Chimeric antibody A2 (cA2) neutralizes the cytotoxic effects of both natural and recombinant human TNFα in a dose-dependent manner. Binding assays of chimeric antibody cA2 with recombinant human TNFα revealed that the affinity constant of chimeric antibody cA2 was 1.04×10 10 M -1Preferred methods for determining the specificity and affinity of monoclonal antibodies by competitive inhibition can be found in Harlow et al., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), Colligan et al., eds., "Current Protocols in Immunology," Greene Publishing Assoc. and Wiley Interscience, New York (1992-2000), Kozbor et al., "Immunol. Today," 4:72-79 (1983), Ausubel et al., eds., "Current Protocols in Molecular Biology," Wiley Interscience, New York (1987-2000), and Muller, "Meth. Enzymol." 92:589-601 (1983), which are incorporated herein by reference in their entireties.

[0166] In a specific embodiment, the murine monoclonal antibody A2 is produced by a cell line designated c134A. The chimeric antibody cA2 is produced by a cell line designated c168A.

[0167] Further examples of monoclonal anti-TNF antibodies that can be used in the present invention are described in the art (e.g., U.S. Pat. No. 5,231,024; Moller, A. et al., Cytokine 2(3):162-169 (1990); U.S. Application Serial No. 07 / 943,852 (filed September 11, 1992); Rathjen et al., WO 91 / 02078 (published February 21, 1991); Rubin et al., EPO Patent Publication No. 0 218 868 (published April 22, 1987); Yone et al., EPO Patent Publication No. 0 288 No. 088 (October 26, 1988); Liang et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (1987); Bringman et al., Hybridoma 6:489-507 (1987); and Hirai et al., J. Immunol. Meth. 96:57-62 (1987), each of which is incorporated herein by reference in its entirety.

[0168] TNF Receptor Molecules. Preferred TNF receptor molecules useful in the present invention are those that bind TNFα with high affinity (see, e.g., Feldmann et al., WO 92 / 07076 (published April 30, 1992); Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell 61:351-359 (1990); these references are incorporated herein by reference in their entireties), and optionally have low immunogenicity. In particular, the 55 kDa (p55 TNF-R) and 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the present invention. Truncated forms of these receptors containing the extracellular domain (ECD) of the receptor or a functional portion thereof (see, e.g., Corcoran et al., Eur. J. Biochem. 223:831-840 (1994)) are also useful in the present invention. Truncated forms of TNF receptors containing the ECD have been detected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)). TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, as well as derivatives and fragments or portions thereof, are further examples of TNF receptor molecules that are useful in the methods and compositions of the present invention. TNF receptor molecules that can be used in the present invention are characterized by good to excellent symptom relief and the ability to treat patients for long periods with low toxicity. Low immunogenicity and / or high affinity, as well as other yet undefined properties, may contribute to the therapeutic results obtained.

[0169] TNF receptor multimeric molecules useful in the present invention comprise all or functional portions of the ECDs of two or more TNF receptors linked via one or more polypeptide linkers or other non-peptide linkers, such as polyethylene glycol (PEG). The multimeric molecule may further comprise a signal peptide of a secretory protein to effect expression of the multimeric molecule. These multimeric molecules and methods for their production are described in U.S. Application Serial No. 08 / 437,533 (filed May 9, 1995), the contents of which are incorporated herein by reference in their entirety.

[0170] TNF immunoreceptor fusion molecules useful in the methods and compositions of the present invention comprise at least a portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These immunoreceptor fusion molecules can be assembled as monomers or hetero- or homo-multimers. Immunoreceptor fusion molecules can be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is a TNF receptor / IgG fusion protein. TNF immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al., Eur. J. Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994); Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851 and U.S. Application No. 08 / 442,133 (filed May 16, 1995), each of which is incorporated herein by reference in its entirety. Methods for producing immune receptor fusion molecules can also be found in Capon et al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538; and Capon et al., Nature 337:525-531 (1989), each of which is incorporated herein by reference in its entirety.

[0171] A functional equivalent, derivative, fragment, or region of a TNF receptor molecule refers to a portion of a TNF receptor molecule, or a portion of a TNF receptor molecule sequence encoding a TNF receptor molecule, that is of sufficient size and sequence to be functionally similar to the TNF receptor molecule that can be used in the present invention (e.g., binds TNFα with high affinity and has low immunogenicity). Functional equivalents of TNF receptor molecules also include modified TNF receptor molecules that are functionally similar to the TNF receptor molecules that can be used in the present invention (e.g., binds TNFα with high affinity and has low immunogenicity). For example, a functional equivalent of a TNF receptor molecule can contain a "SILENT" codon, or one or more amino acid substitutions, deletions, or additions (e.g., substituting one acidic amino acid for another, or substituting one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid). See Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000).

[0172] Cytokines include any known cytokine. See, for example, CopewithCytokines.com. Cytokine antagonists include, but are not limited to, any antibody, fragment, or mimetic, any soluble receptor, fragment, or mimetic, any small molecule antagonist, or any combination thereof.

[0173] Any of the methods of the present invention may include methods for treating a TNF-mediated disorder comprising administering to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy a composition or pharmaceutical composition comprising at least one anti-TNF antibody. Such methods may optionally further comprise co-administration or combination therapy for the treatment of such immune disorders, wherein the administration of at least one anti-TNF antibody, specified portion, or variant thereof is in combination with at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an anti-rheumatic drug (such as, but not limited to, methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, or the like), or a combination therapy for the treatment of such immune disorders. Antibiotics (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, and other antibacterial agents), antipsoriatics, corticosteroids, and anabolic steroids drugs, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin (e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunizing agents, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatics, The method may further comprise administering, before, simultaneously with, and / or after administration of at least one selected from a cycloplegic agent, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, an antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta-agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, cromolyn, epinephrine or an analogue, dornase alfa (Pulmozyme), a cytokine, or a cytokine antagonist.

[0174] As used herein, the term "safety," when it relates to a composition, dose, dosing regimen, treatment, or method with an anti-TNF antibody of the invention (e.g., the anti-TNF antibody golimumab), refers to a favorable risk-to-benefit ratio with an acceptable frequency and / or severity of adverse events (AEs) and serious adverse events (SAEs) compared to the standard of care or another comparator, such as another anti-TNF agent. An adverse event is an untoward medical occurrence in a patient administered a pharmaceutical product. Specifically, safety, when it relates to a composition, dose, dosing regimen, treatment, or method with an anti-TNF antibody of the invention, refers to an acceptable frequency and / or severity of adverse events, including, for example, infusion reactions, abnormal hepatic and biliary test values, infections, including TB, and malignancies.

[0175] As used herein in the context of a composition, dose, treatment regimen, therapy, or method, the terms "effective" and "effective" refer to the effectiveness of a particular composition, dose, administration, treatment, or method with an anti-TNF antibody of the invention (e.g., the anti-TNF antibody golimumab). Efficacy can be measured based on changes over the course of a disease in response to an agent of the invention. For example, an anti-TNF antibody of the invention is administered to a patient in an amount and for a time sufficient to cause an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. To determine whether the amount and duration of treatment are sufficient, various indicators reflecting the extent of the disease, disorder, or condition in question can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the disorder in question. The degree of improvement is generally determined by a physician or other appropriately trained individual, who may determine it based on signs, symptoms, biopsy, or other test results indicating improvement in clinical symptoms, or any other measure of disease activity. For example, anti-TNF antibodies of the present invention can be administered to achieve improvement in a patient's condition associated with ankylosing spondylitis (AS). Improvement in a patient's condition associated with AS can be assessed using one or more criteria, including, for example, the Ankylosing Spondylitis Disease Activity Score (ASDAS), the Bath Ankylosing Spondylitis Functional Index (BASFI), the Bath Ankylosing Spondylitis Measurement Index (BASMI), the 36-Item Short Form Health Survey Physical Health (SF-36 PCS), the 36-Item Short Form Health Survey Mental Health (SF-36 MCS), and / or the results of an Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire. The ASDAS is a disease activity score (DAS) for use in AS, developed by the International Ankylosing Spondylitis Assessment Society. The ASDAS is calculated using a formula with assessments including, for example, total back pain, duration of morning stiffness, peripheral pain / swelling, and the patient's global assessment.The BASFI is a subject's self-assessment expressed as the average of 10 questions, eight of which relate to the subject's functional anatomy and two of which relate to the subject's ability to cope with daily activities. The BASMI is a summary score calculated by converting ratings into scores for five assessments, including lateral lumbar flexion, tragus-to-parietal distance, lumbar flexion, intermalleolar distance, and cervical rotation angle. The SF-36 is a questionnaire consisting of eight scored multi-item scales, and the SF-36 PSA and SF-36 MCS are summary scores derived from the SF-36 that allow comparison of the relative burden of different diseases and the relative benefits of different treatments. The ASQoL is a self-administered patient-reported outcome instrument consisting of 18 items requiring responses to questions related to the impact of pain on sleep, mood, motivation, coping ability, activities of daily living, autonomy, relationships, and social life.

[0176] As used herein, unless otherwise indicated, the term "clinically proven" (used independently or to modify the terms "safety" and / or "effectiveness"; e.g., clinically proven safe and / or clinically proven effective) shall mean proven by a clinical trial that meets the approval standards of the U.S. Food and Drug Administration, EMEA, or a corresponding national regulatory agency. For example, a clinical trial may be a randomized, double-blind study of appropriate size used to clinically prove the efficacy of a drug.

[0177] Typically, treatment of a condition is achieved by administering a safe and effective amount or dose of at least one anti-TNF antibody composition, depending on the specific activity contained in the composition, on average, in a total range of at least about 0.01 to 500 milligrams of at least one anti-TNF antibody per kg of patient body weight per dose, preferably at least about 0.1 to 100 milligrams of antibody per kg of patient body weight per single or multiple doses. Alternatively, an effective serum concentration may comprise a serum concentration of 0.1 to 5000 μg / mL per single or multiple doses. Suitable dosages are known to medical practitioners and, of course, depend on the specific disease state, the specific activity of the administered composition, and the specific patient undergoing treatment. In some cases, achieving a desired therapeutic dose may require providing multiple doses, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.

[0178] Preferred doses are optionally 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and / or 100-500 mg / kg / dose, or any range, value, or fraction thereof, or 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, .0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 1 The concentration may comprise 9.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and / or 5000 μg / mL, or any range, value or fraction thereof.

[0179] Alternatively, the administered dose may vary depending on well-known factors such as the pharmacodynamic characteristics of the particular drug and its method and route of administration, the age, health condition, and weight of the recipient, the nature and severity of the symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. The dose of the active ingredient may typically be about 0.1 to 100 milligrams per kilogram of body weight. Typically, 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram per administration or sustained-release form is effective to obtain the desired results.

[0180] By way of non-limiting example, treatment of humans or animals may be performed using a single, infusion, or multiple doses within the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, or 40th day of administration. On at least one of the following days or in addition to the following days: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 0.1 to 100 mg / kg per day, e.g., 0.5, 0.9, 1.0, or 20 mg / kg daily, for at least one week of week 9, 50, 51, or 52, or, or additionally, for at least one year of year 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof , 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg of at least one antibody of the invention, either once or periodically.

[0181] Dosage forms (compositions) suitable for internal administration generally contain about 0.1 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.5 to 99.999% by weight based on the total weight of the composition.

[0182] For parenteral administration, antibodies can be formulated as a solution, suspension, emulsion, or lyophilized powder, either combined with a pharmaceutically acceptable parenteral vehicle or provided separately. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity and chemical stability (e.g., sodium chloride or mannitol for isotonicity, buffers and preservatives for chemical stability). The formulation is sterilized by known or suitable techniques.

[0183] Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

[0184] Alternative Administration. Many known and developed administration methods can be used in accordance with the present invention to administer a pharmaceutically effective amount of at least one anti-TNF antibody according to the present invention. Although pulmonary administration is used in the following description, other modes of administration may also be used in accordance with the present invention with suitable results.

[0185] The TNF antibodies of the invention can be delivered in a carrier, as a solution, emulsion, colloid or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or by other methods described herein or known in the art.

[0186] Parenteral Formulations and Administration. Parenteral formulations may contain common additives such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection can be prepared by using appropriate emulsifiers or wetting agents and suspending agents according to well-known methods. Injectable preparations may be non-toxic parenterally administrable diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Acceptable solvents or media include water, Ringer's solution, and isotonic saline, and sterile, fixed oils can be used as common solvents or suspending media. For these purposes, any type of fixed oil or fatty acid, including natural, synthetic, or semisynthetic fatty oils or fatty acids, natural, synthetic, or semisynthetic monoglycerides, diglycerides, or triglycerides, can be used. Parenteral administration is well known in the art and includes, but is not limited to, conventional injection means, gas-pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforator devices such as those described in U.S. Pat. No. 5,839,446, which are incorporated herein by reference in their entireties.

[0187] Alternative Delivery: The present invention further relates to administration of at least one anti-TNF antibody by parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, intravaginal, rectal, buccal, sublingual, intranasal, or transdermal means. At least one anti-TNF antibody composition may be formulated for use parenterally (subcutaneously, intramuscularly, or intravenously) or any other administration, particularly in the form of a liquid solution or suspension, particularly in semi-solid forms such as, but not limited to, creams and suppositories, for use in vaginal or rectal administration, in forms such as, but not limited to, tablets or capsules, for buccal or sublingual administration, or intranasally in forms such as, but not limited to, powders, nasal drops, or aerosols, or certain medications, or using chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or increase the drug concentration in transdermal patches (Junginger et al., In "Drug Permeation Enhancement"; Hsieh, DS (ed.), pp. 59-90 (Marcel Dekker, Inc. New York 1994, incorporated herein by reference in its entirety), or using oxidizing agents to allow application of protein and peptide containing formulations to the skin (WO 98 / 53847), or using the application of an electric field to create a transient transport pathway, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0188] Pulmonary / Intranasal Administration. For pulmonary administration, the at least one anti-TNF antibody composition is preferably delivered in a particle size effective to reach the lower airways or sinuses of the lung. According to the present invention, the at least one anti-TNF antibody can be delivered by any of a variety of inhalation or intranasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit an aerosolized formulation into the sinus cavities or alveoli of a patient, include metered-dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for pulmonary or intranasal administration of antibodies are also known in the art. All such devices can use formulations suitable for administration to dispense the antibody in an aerosol. Such aerosols can be composed of either solutions (both aqueous and non-aqueous) or solid particles. Metered-dose inhalers, such as VENTOLIN® (metered-dose inhaler), typically use a propellant gas and require actuation upon inspiration (see, e.g., WO 94 / 16970, WO 98 / 35888). Dry powder inhalers, such as Turbuhaler™ (Astra), Rotahaler® (Glaxo), DISKUS® (inhaler) (Glaxo), SPIROS® (inhaler) (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler® powder inhaler (Fisons), use breath actuation of a mixed powder (U.S. Pat. No. 4,668,218 (Astra), EP 237,507 (Astra), WO 97 / 25086 (Glaxo), WO 94 / 08552 (Dura), U.S. Pat. No. 5,458,135 (Inhale), WO 94 / 06498 (Fisons), which are incorporated herein by reference in their entireties).Nebulizers such as the AERX® (nebulizer) (Aradigm), ULTRAVENT® (nebulizer) (Mallinckrodt), and Acorn II nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871 (Aradigm); WO 97 / 22376) (the above references are incorporated herein by reference in their entireties) produce aerosols from solutions, while metered-dose inhalers, dry powder inhalers, and the like generate small particle aerosols. These specific examples of commercially available inhalation devices are intended as representative of particular devices suitable for practicing the present invention and are not intended as limiting the scope of the invention. Preferably, compositions comprising at least one anti-TNF antibody are delivered by a dry powder inhaler or nebulizer. Inhalation devices for administering at least one antibody of the present invention have several desirable characteristics. For example, delivery by inhalation devices is advantageously reliable, reproducible, and accurate. The inhalation device can optionally deliver small dry particles, eg, less than about 10 μm, preferably about 1-5 μm, for good respirability.

[0189] Administration of a TNF antibody composition as a spray. A spray containing a TNF antibody composition protein can be produced by passing a suspension or solution of at least one anti-TNF antibody through a nozzle under pressure. The nozzle size and configuration, applied pressure, and liquid feed rate can be selected to achieve a desired output and particle size. For example, an electrospray can be produced by an electric field in conjunction with a capillary or nozzle feed. Advantageously, the particles of at least one anti-TNF antibody composition protein delivered by the sprayer have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably in the range of about 2 μm to about 3 μm.

[0190] Formulations of at least one anti-TNF antibody composition protein suitable for use in a nebulizer typically comprise the antibody composition protein in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one anti-TNF antibody composition protein per ml or mg / gm of solution, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / mL or 100 mg / gm, or any range or value therein. The formulation may contain additives, buffers, isotonicity agents, preservatives, surfactants, and preferably agents such as zinc. The formulation may also contain excipients or agents for stabilizing the antibody composition protein, such as additives, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful in formulating antibody composition proteins include albumin and protamine. Typical carbohydrates useful in formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, and the like. The antibody composition protein formulation may also contain a surfactant that can reduce or prevent surface-induced aggregation of the antibody composition protein caused by atomization of the solution during aerosol formation. Various conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts generally range from 0.001 to 14% by weight of the formulation. Particularly preferred surfactants for purposes of the present invention include polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. The formulation of a protein, such as a TNF antibody, or specified portion or variant, can also include additional agents known in the art.

[0191] Administration of TNF antibody compositions by nebulizer. Antibody composition proteins can be administered by a nebulizer, such as a jet nebulizer or ultrasonic nebulizer. Jet nebulizers typically use a compressed air source to generate a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, which draws the antibody composition protein solution through a capillary tube connected to a liquid reservoir. The liquid flow from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating an aerosol. A range of configurations, flow rates, and baffle types can be used to achieve the desired performance characteristics from a given jet nebulizer. Ultrasonic nebulizers use high-frequency electrical energy, typically a piezoelectric transducer, to generate oscillatory mechanical energy. This energy is transmitted to the antibody composition protein formulation, either directly or through a coupling liquid, to create an aerosol containing the antibody composition protein. Advantageously, particles of antibody composition protein delivered by a nebulizer have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

[0192] Formulations of at least one anti-TNF antibody suitable for use in either jet or ultrasonic nebulizers typically contain a concentration of at least one anti-TNF antibody protein of about 0.1 mg to about 100 mg per ml of solution. The formulations may include additives, buffers, isotonicity agents, preservatives, surfactants, and, preferably, agents such as zinc. The formulations may also include additives or agents for stabilization of the at least one anti-TNF antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating at least one anti-TNF antibody composition protein include albumin, protamine, and the like. Typical carbohydrates useful in formulating at least one anti-TNF antibody include sucrose, mannitol, lactose, trehalose, glucose, and the like. The at least one anti-TNF antibody formulation may also include a surfactant, which may reduce or prevent surface-induced aggregation of the at least one anti-TNF antibody caused by atomization of the solution during aerosol formation. Various conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. The amount generally ranges from 0.001 to 4% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Formulations of proteins, such as antibody proteins, can also include additional agents known in the art.

[0193] Administration of TNF antibody compositions via metered dose inhalers. In metered dose inhalers (MDIs), a propellant, at least one anti-TNF antibody, and optional additives or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of a metering valve releases the mixture as an aerosol containing particles preferably less than about 10 μm in size, preferably between about 1 μm and about 5 μm, and most preferably between about 2 μm and 3 μm in size. The desired aerosol particle size can be achieved using formulations of antibody composition proteins produced by a variety of methods known to those skilled in the art, including jet milling, spray drying, critical point condensation, and the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and using hydrofluorocarbon propellants.

[0194] Formulations of at least one anti-TNF antibody for use in metered-dose inhaler devices generally comprise a fine powder containing at least one anti-TNF antibody as a suspension in a non-aqueous solvent, suspended in a propellant, for example, with the aid of a surfactant. The propellant can be any conventional substance used for this purpose, such as a chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), and the like. Preferably, the propellant is a hydrofluorocarbon. The surfactant can be selected to stabilize the at least one anti-TNF antibody as a suspension in the propellant, to protect the active agent against chemical degradation, or the like. Suitable surfactants include sorbitan trioleate, soybean lecithin, oleic acid, and the like. In some cases, a solution aerosol using a solvent such as ethanol is preferred. Formulations of the protein may also include additional agents known in the art.

[0195] Those skilled in the art will recognize that the methods of the present invention can be accomplished by pulmonary administration of at least one anti-TNF antibody composition via devices not described herein.

[0196] Oral formulations and administration. Oral formulations rely on the simultaneous administration of adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. The active ingredient compound in a solid dosage form for oral administration can be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants (magnesium stearate), parabens, preservatives (sorbic acid, ascorbic acid, α-tocopherol, etc.), antioxidants (cysteine, etc.), disintegrants, binders, thickeners, buffers, sweeteners, flavorings, perfumes, etc.

[0197] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration include emulsions, syrups, elixirs, suspensions, and solution preparations that are acceptable for medical use. These preparations can contain inert diluents commonly used in the art, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, the use of carrier compounds described in U.S. Pat. Nos. 5,879,681 and 5,5,871,753 for oral delivery of biologically active agents is known in the art.

[0198] Mucosal Formulations and Administration. Compositions and methods for administering at least one anti-TNF antibody for absorption through mucosal surfaces include emulsions containing a plurality of submicron particles, mucoadhesive macromolecules, a bioactive peptide, and an aqueous continuous phase that promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present invention include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain additives such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration may be solid and may contain additives such as lactose, or may be aqueous or oily solution nasal sprays. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (US Pat. No. 5,849,695).

[0199] Transdermal Formulations and Administration. For transdermal administration, at least one anti-TNF antibody is encapsulated in a delivery device such as a liposome or polymeric nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made from synthetic polymers such as polyhydroxy acids, such as polylactic acid, polyglycolic acid, and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers, such as collagen, polyamino acids, albumin, and other proteins, alginates, and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599).

[0200] Prolonged Administration and Formulations. It may sometimes be desirable to deliver a compound of the invention to a subject over an extended period of time, e.g., from one week to one year, via a single administration. Various sustained-release, depot, or implantable dosage forms are available. For example, dosage forms may contain pharmaceutically acceptable, non-toxic salts of compounds that have low solubility in body fluids, such as (a) acid addition salts with polybasic acids, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, and polygalacturonic acid; (b) salts with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, and cadmium, or organic cations formed, for example, from N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), e.g., zinc tannate. In addition, the compounds of the present invention, or preferably relatively insoluble salts such as those mentioned above, can be formulated into gels suitable for injection, such as aluminum monostearate gel, with, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate, and zinc pamoate. Another type of injectable sustained-release depot formulation contains the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as the polylactic acid / polyglycolic acid polymer described in U.S. Pat. No. 3,773,919. The compounds, or preferably relatively insoluble salts such as those mentioned above, can also be formulated in cholesterol-matrix silastic pellets, particularly for use in animals. Additional sustained-release depot or implant formulations, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," JR Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0201] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

[0202] Example 1: Cloning and expression of TNF antibodies in mammalian cells. A typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the antibody coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanking donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from retroviruses, e.g., RSV, HTLV-1, HIV-1, and the early promoter of cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in the practice of the present invention include, for example, pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), or pcDNA3.1 / Hygro(+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), and pBC12MI (ATCC 67109). Mammalian host cells that can be used include human Hela293, H9, and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7, and CV1, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

[0203] Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of transfected cells.

[0204] The transfected gene can also be amplified to express large quantities of the encoded antibody. The DHFR (dihydrofolate reductase) marker is useful for developing cell lines that contain hundreds or even thousands of copies of the gene of interest. Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem. J. 227:277-279 (1991); Bebbington et al., Bio / Technology 10:169-175 (1992)). Using these markers, mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for antibody production.

[0205] The expression vectors pC1 and pC4 contain a fragment of the CMV enhancer (Boshart et al., Cell 41:521-530 (1985)) in addition to the strong promoter (LTR) of the Rous sarcoma virus (Cullen et al., Molec. Cell. Biol. 5:438-447 (1985)). Multiple cloning sites with restriction enzyme cleavage sites, e.g., BamHI, XbaI, and Asp718, facilitate cloning of genes of interest. The vectors also contain the 3' intron, polyadenylation, and termination signals of the rat preproinsulin gene.

[0206] Cloning and Expression in CHO Cells. Vector pC4 is used for expression of TNF antibodies. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). This plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells transfected with these plasmids that lack dihydrofolate activity can be selected by growing the cells in selective medium (e.g., α-MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic drug methotrexate. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) has been well documented (see, e.g., F.W. Alt et al., J. Biol. Chem. 253:1357-1370 (1978); J.L. Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M.J. Page and M.A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing MTX concentrations develop resistance to the drug due to overproduction of the target enzyme, DHFR, as a result of amplification of the DHFR gene. When a second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this approach can be used to develop cell lines with more than 1,000 copies of the amplified gene. Subsequently, upon recovery of the methotrexate, cell lines are obtained which contain the amplified gene integrated into one or more chromosome(s) of the host cell.

[0207] Plasmid pC4 contains a fragment isolated from the human cytomegalovirus (CMV) immediate-early gene enhancer (Boshart et al., Cell 41:521-530 (1985)) as well as the strong promoter of the long terminal repeat (LTR) of Rous sarcoma virus (Cullen et al., Molec. Cell. Biol. 5:438-447 (1985)) to express the gene of interest. Downstream of the promoter are BamHI, XbaI, and Asp718 restriction enzyme cleavage sites, which allow for gene integration. Beyond these cloning sites, the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters, such as the human β-actin promoter, SV40 early or late promoters, or long terminal repeats from other retroviruses, such as HIV and HTLV-1, can also be used for expression. TNF can be expressed in a regulated manner in mammalian cells using Clontech's Tet-Off and Tet-On gene expression system and similar systems (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)). Other signals, such as those from the human growth hormone or globin genes, can also be used for polyadenylation of mRNA. Stable cell lines carrying the gene of interest integrated into the chromosome can also be selected upon cotransfection with a selection marker such as gpt, G418, or hygromycin. It is advantageous to initially use more than one selection marker, such as G418, plus methotrexate.

[0208] The plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art, and the vector is then isolated from a 1% agarose gel.

[0209] The isolated variable and constant region-encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase, E. coli HB101 or XL-1 Blue cells are then transformed, and bacteria containing the fragment inserted into plasmid pC4 are identified using, for example, restriction enzyme analysis.

[0210] For transfection, Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used. Five micrograms of the expression plasmid pC4 are cotransfected with 0.5 micrograms of the plasmid pSV2-neo using lipofectin. The plasmid pSV2-neo contains the dominant selectable marker, the neo gene from Tn5, which encodes an enzyme that confers resistance to a group of antibiotics, including G418. The cells are seeded in alpha minus MEM supplemented with 1 microgram / mL G418. After two days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng / mL methotrexate plus 1 microgram / mL G418. After approximately 10-14 days, single clones are trypsinized and seeded into 6-well Petri dishes or 10 mL flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest methotrexate concentration are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones growing at concentrations of 100-200 mM are obtained. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot or by reverse-phase HPLC analysis.

[0211] Example 2: Generation of high affinity human IgG monoclonal antibodies reactive with human TNF using transgenic mice. Overview. Transgenic mice containing human heavy and light chain immunoglobulin genes are used to generate high-affinity, fully human monoclonal antibodies that can be used therapeutically to inhibit TNF action for the treatment of one or more TNF-mediated diseases. (CBA / JxC57 / BL6 / J)F2 hybrid mice containing both heavy and light chain human variable and constant region antibody transgenes are immunized with human recombinant TNF (Taylor et al., Intl. Immunol. 6:579-591 (1993); Lonberg et al., Nature 368:856-859 (1994); Neuberger, M., Nature Biotech. 14:826 (1996); Fishwild et al., Nature Biotechnology 14:845-851 (1996)). Several fusions have yielded one or more panels of fully human TNF-reactive IgG monoclonal antibodies. The fully human anti-TNF antibodies are further characterized. All are IgG1κ. These antibodies are approximately 1×10 9 ~9×10 12 The unexpectedly high affinity of these fully human monoclonal antibodies makes them excellent candidates for therapeutic use in TNF-related diseases, conditions, or disorders.

[0212] Abbreviations: BSA - bovine serum albumin, CO₂ - carbon dioxide, DMSO - dimethyl sulfoxide, EIA - enzyme immunoassay, FBS - fetal bovine serum, H₂O₂ - hydrogen peroxide, HRP - horseradish peroxidase, ID - intradermal, Ig - immunoglobulin, TNF - tissue necrosis factor alpha, IP - intraperitoneal, IV - intravenous, Mab - monoclonal antibody, OD - optical density, OPD - o-phenylenediamine dihydrochloride, PEG - polyethylene glycol, PSA - penicillin, streptomycin, amphotericin, RT - room temperature, SQ - subcutaneous, v / v - volume per unit volume, w / v - weight per unit volume.

[0213] Materials and Methods Animals. Transgenic mice capable of expressing human antibodies are known in the art and are commercially available (e.g., from GenPharm International, San Jose, CA; Abgenix, Freemont, CA), which express human immunoglobulins but do not express mouse IgM or Igκ. For example, such transgenic mice contain human sequence transgenes that undergo V(D)J joining, heavy chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins (Lonberg et al., Nature 368:856-859 (1994)). Light chain transgenes can be derived, for example, in part, from yeast artificial chromosome clones containing approximately half of the germline human Vκ region. In addition, heavy chain transgenes can encode both human μ and human γ1 constant regions (Fishwild et al., Nature Biotechnology 14:845-851 (1996)) and / or γ3 constant regions. Mice from the appropriate genomic strain can be used in the immunization and fusion process to generate fully human monoclonal antibodies against TNF.

[0214] Immunization. One or more immunization schedules can be used to generate anti-TNF human hybridomas. The following exemplary immunization protocol can be followed for the first several fusions, although other similar known protocols can also be used. Several 14-20 week old female and / or surgically castrated male transgenic mice are inoculated IP or ID with 1-1000 μg of recombinant human TNF emulsified with an equal volume of TITERMAX or complete Freund's adjuvant in a final volume of 100-400 μL (e.g., 200 μL). Each mouse can also optionally receive 1-10 μg in 100 μL of physiological saline in each of 2 SQ sites. Mice can then be immunized 1-7, 5-12, 10-18, 17-25, and / or 21-34 days later with equal volumes of TNF emulsified in TITERMAX or complete Freund's adjuvant, IP (1-400 μg) and SQ (1-400 μg x 2). Mice can be bled by retroorbital puncture 12-25 and 25-40 days later without anticoagulant. Blood is then allowed to clot for 1 hour at room temperature, and serum is collected and titrated using a TNF EIA assay by known methods. If repeated injections do not result in increased titers, fusions are performed. Mice can then receive a final IV booster injection of 1-400 μg of TNF diluted in 100 μL of physiological saline. After 3 days, mice are euthanized by cervical dislocation, and spleens are aseptically removed and immersed in 10 mL of cold phosphate-buffered saline (PBS) containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B (PSA). Splenocytes are harvested by sterile perfusion of the spleen with PSA-PBS. Cells are washed once in cold PSA-PBS, counted using trypan blue dye exclusion, and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0215] Cell fusion. Fusion can be performed using a 1:1 to 1:10 ratio of mouse myeloma cells to viable spleen cells according to known, e.g., conventional, methods in the art. As a non-limiting example, spleen cells and myeloma cells can be pelleted together. The pellet can then be slowly resuspended in 1 mL of 50% (w / v) PEG / PBS solution (PEG molecular weight 1,450, Sigma) at 37°C for 30 seconds. Fusion can then be stopped by slowly adding 10.5 mL of RPMI 1640 medium (37°C) containing 25 mM Hepes over 1 minute. The fused cells are centrifuged at 500 to 1500 rpm for 5 minutes. The cells are then resuspended in HAT medium (RPMI 1640 medium containing 25 mM Hepes, 10% fetal clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 μg / mL gentamicin, 2.5% Origen culture supplement (Fisher), 10% 653-adjusted RPMI 1640 / Hepes medium, 50 μM 2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) and plated at 200 μL / well into 15 96-well flat-bottom tissue culture plates. The plates are then placed in a humidified 37 °C incubator containing 5% CO2 and 95% air for 7-10 days.

[0216] Detection of human IgG anti-TNF antibodies in mouse serum. Mouse serum can be screened for human IgG antibodies specific to human TNF using a solid-phase EIA. Briefly, plates can be coated overnight with 2 μg / mL TNF in PBS. After washing with 0.15 M saline containing 0.02% (v / v) Tween 20, wells can be blocked with 200 μL / well of 1% (w / v) BSA in PBS for 1 hour at room temperature. Plates can be used immediately or frozen at -20°C for later use. Mouse serum dilutions are incubated on the TNF-coated plates at 50 μL / well for 1 hour at room temperature. After washing, the plates are probed with 50 μL / well of Fc-specific HRP-labeled goat anti-human IgG diluted 1:30,000 in 1% BSA-PBS for 1 hour at room temperature. The plate can be washed again, and 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.01% HO and 1 mg / mL OPD) is added over 15 minutes at room temperature. Next, 25 μL / well of stop solution (4 N sulfuric acid) is added, and the OD is read at 490 nm on an automated plate spectrophotometer.

[0217] Detection of fully human immunoglobulins in hybridoma supernatants. A suitable EIA can be used to detect growth-positive hybridomas secreting fully human immunoglobulins. Briefly, 96-well pop-out plates (VWR, 610744) can be coated with 10 μg / mL goat anti-human IgG Fc in sodium carbonate buffer overnight at 4°C. The plates are washed, blocked with 1% BSA-PBS for 1 hour at 37°C, and used immediately or frozen at -20°C. Undiluted hybridoma supernatant is incubated on the plates for 1 hour at 37°C. The plates are washed and probed with HRP-labeled goat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for 1 hour at 37°C. The plates are then incubated with substrate solution as described above.

[0218] Determination of fully human anti-TNF reactivity. The above hybridomas can be simultaneously assayed for reactivity to TNF using a suitable RIA or other assay. For example, as described above, the supernatants are incubated on goat anti-human IgG Fc plates, washed, and then probed with radiolabeled TNF at an appropriate count per well for 1 hour at room temperature. The wells are washed twice with PBS, and the bound radiolabeled TNF is quantified using a suitable counter.

[0219] Human IgG1κ anti-TNF-secreting hybridomas can be expanded in cell culture and serially subcloned by limiting dilution. The resulting clonal populations are expanded, cryopreserved in freezing medium (95% FBS, 5% DMSO), and stored in liquid nitrogen.

[0220] Isotype. Determination of antibody isotype can be accomplished using an EIA format similar to that used to screen mouse immune sera for specific titrations. TNF can be coated onto 96-well plates as described above, and 2 μg / mL of purified antibody can be incubated on the plates for 1 hour at room temperature. The plates are washed and probed with HRP-labeled goat anti-human IgG1 or HRP-labeled goat anti-human IgG3 diluted 1:4000 in 1% BSA-PBS for 1 hour at room temperature. The plates are washed again and incubated with substrate solution as described above.

[0221] Binding kinetics of human anti-human TNF antibodies with human TNF. The binding characteristics of antibodies can be conveniently assessed using, for example, TNF capture EIA and BIAcore technology. Graded concentrations of purified human TNF antibodies can be assessed for binding to EIA plates coated with 2 μg / mL TNF in an assay as described above. OD can then be expressed as a semi-logarithmic plot showing relative binding efficiency.

[0222] Quantitative binding constants can be obtained, for example, as follows, or by any other known suitable method: A BIAcore CM-5 (carboxymethyl) chip is placed in a BIAcore 2000 unit. HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v P20 surfactant, pH 7.4) is flowed over the flow cell of the chip at 5 μl / min until a stable baseline is obtained. A solution (100 μL) of 15 mg EDC (N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200 μL of water is added to a solution of 100 μL of 2.3 mg NHS (N-hydroxysuccinimide) in 200 μL of water. 40 μL of the resulting solution is injected onto the chip. A 6 μL solution of human TNF (15 μg / mL in 10 mM sodium acetate, pH 4.8) was injected onto the chip, resulting in an increase of approximately 500 RU. The buffer was changed to TBS / Ca / Mg / BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mM calcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 μg / mL BSA, pH 7.4) and run overnight on the chip to equilibrate it and hydrolyze or cap any unreacted succinates.

[0223] Antibodies are dissolved in running buffer at 33.33, 16.67, 8.33, and 4.17 nM. The flow rate is adjusted to 30 μL / min, and the instrument temperature is adjusted to 25°C. Two flow cells are used for the kinetic run: one with immobilized TNF (sample) and the second with underivatized flow cell (blank). 120 μL of each antibody concentration is injected over the flow cell at 30 μL / min (association phase), followed by a continuous buffer flow for 360 seconds (dissociation phase). The chip surface is regenerated (dissociation of tissue necrosis factor α / antibody complexes) by two sequential injections of 30 μL each of 2 M guanidine thiocyanate.

[0224] Data analysis is performed using BIA evaluation 3.0 or CLAMP 2.0, which are known in the art. For each antibody concentration, blank sensograms are subtracted from sample sensograms. Dissociation (k d, sec-1 ) and meetings (k a ,mol -1 sec -1 ) and perform a global fit for the dissociation constant (K D , mol) (k d / k a If the antibody affinity is high enough that the RU of the captured antibody is >100, additional dilutions of the antibody are performed.

[0225] Results and Discussion Generation of anti-human TNF monoclonal antibodies. Several fusions were performed, and each fusion produced several dozen antibodies specific to human TNF. 15 plates (1440 wells / fusion) were seeded with each fusion. Some of these were found to consist of a combination of human and mouse Ig chains. The remaining hybridomas secreted anti-TNF antibodies consisting only of human heavy and light chains. All of the human hybridomas were expected to be IgG1κ.

[0226] Binding kinetics of human anti-human TNF antibodies. ELISA analysis confirms that purified antibodies from most or all of these hybridomas bind to TNF in a concentration-dependent manner. Figures 1-2 show the results of the relative binding efficiencies of these antibodies. In this case, the avidity of the antibody for its cognate antigen (epitope) is measured. It should be noted that direct binding of TNF to an EIA plate can cause denaturation of the protein, and the apparent binding affinity may not reflect binding to the native protein. Fifty percent binding is observed across a wide range of concentrations.

[0227] Quantitative binding constants were obtained using BIAcore analysis of human antibodies, with some human monoclonal antibodies exceeding 1 × 10 -9 ~7×10 -12 K in the range D It is revealed that it has a very high affinity.

[0228] conclusion Several fusions are performed using splenocytes from hybrid mice containing human variable and constant region antibody transgenes immunized with human TNF. A set of several fully human TNF-reactive IgG monoclonal antibodies of the IgG1κ isotype is generated. The fully human anti-TNF antibodies are further characterized. Some of the antibodies generated are at 1 x 10 9 ~9×10 12 The unexpectedly high affinity of these fully human monoclonal antibodies makes them suitable for therapeutic use in TNF-dependent diseases, conditions or related states.

[0229] Example 3: Generation of human IgG monoclonal antibodies reactive with human TNFα. Summary: (CBA / JxC57BL / 6J) F2 hybrid mice (1-4) containing both heavy and light chain human variable and constant region antibody transgenes were immunized with recombinant human TNFα. One fusion, designated GenTNV, yielded eight fully human IgG1κ monoclonal antibodies that bound to immobilized recombinant human TNFα. Immediately after identification, eight cell lines were transferred to Molecular Biology for further characterization. Because these Mabs are fully human in sequence, they are expected to be less immunogenic than cA2 (Remicade) in humans.

[0230] Abbreviations: BSA - bovine serum albumin, CO2 - carbon dioxide, DMSO - dimethyl sulfoxide, EIA - enzyme immunoassay, FBS - fetal bovine serum, H2O2 - hydrogen peroxide, HC - heavy chain, HRP - horseradish peroxidase, ID - intradermal, Ig - immunoglobulin, TNF - tissue necrosis factor alpha, IP - intraperitoneal, IV - intravenous, Mab - monoclonal antibody, OD - optical density, OPD - o-phenylenediamine dihydrochloride, PEG - polyethylene glycol, PSA - penicillin, streptomycin, amphotericin, RT - room temperature, SQ - subcutaneous, TNFα - tumor necrosis factor alpha, v / v - volume per unit volume, w / v - weight per unit volume.

[0231] Introduction. Transgenic mice containing human heavy and light chain immunoglobulin genes were utilized to generate fully human monoclonal antibodies specific for recombinant human TNFα. It is anticipated that these unique antibodies can be used to therapeutically inhibit inflammatory processes involved in TNFα-mediated diseases, with the benefit of increased serum half-life and reduced immunogenic side effects, as cA2 (Remicade).

[0232] Materials and Methods Animals. Transgenic mice expressing human immunoglobulins but not mouse IgM or Igκ have been developed by GenPharm International. These mice contain functional human antibody transgenes that undergo V(D)J joining, heavy chain class switching, and somatic mutation to generate a repertoire of antigen-specific human immunoglobulins (1). The light chain transgenes are derived, in part, from yeast artificial chromosome clones containing approximately half of the germline human V loci. In addition to several VH genes, the heavy chain (HC) transgene encodes both human μ and human γ1 (2) and / or γ3 constant regions. Mice from the HCo12 / KCo5 genotype strain were used in the immunization and fusion process to generate the monoclonal antibodies described herein.

[0233] Purification of human TNFα. Human TNFα was purified from tissue culture supernatant from C237A cells by affinity chromatography using a column packed with a TNFα receptor-Fc fusion protein (p55-sf2) (5) coupled to Sepharose 4B (Pharmacia). The cell supernatant was mixed with one-ninth its volume of 10x Dulbecco's PBS (D-PBS) and passed through the column at 4 mL / min at 4°C. The column was then washed with PBS, and TNFα was eluted with 0.1 M sodium citrate, pH 3.5, and neutralized with 2 M Tris-HCl, pH 8.5. The purified TNFα was buffer exchanged into 10 mM Tris, 0.12 M sodium chloride, pH 7.5, and filtered through a 0.2 μm syringe filter.

[0234] Immunization. Female GenPharm mice, approximately 16 weeks of age, were immunized IP (200 μL) and ID (100 μL at the base of the tail) with a total of 100 μg of TNFα (lots JG102298 or JG102098) emulsified in an equal volume of Titermax adjuvant on days 0, 12, and 28. Mice were bled by retroorbital puncture on days 21 and 35 without anticoagulant. Blood was allowed to clot for 1 hour at room temperature, and serum was collected and titrated using a TNFα solid-phase EIA assay. After the day 28 injection, mice were rested for 7 weeks before undergoing the fusion designated GenTNV. Mice with specific human IgG titers of 1:160 against TNFα were then given a final IV booster injection of 50 μg of TNFα diluted in 100 μL of physiological saline. Three days later, mice were euthanized by cervical dislocation, and spleens were aseptically removed and immersed in 10 mL of cold phosphate-buffered saline (PBS) containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B (PSA). Splenocytes were harvested by sterile perfusion of the spleen with PSA-PBS. Cells were washed once in cold PSA-PBS, counted using a Coulter counter, and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0235] Cell Line. The Cell Biology Services (CBS) group received the nonsecreting mouse myeloma fusion partner 653 from Centocor's Product Development group on May 14, 1997. The cell line was expanded in RPMI medium (JRH Biosciences) supplemented with 10% (v / v) FBS (Cell Culture Labs), 1 mM sodium pyruvate, 0.1 mM NEAA, and 2 mM L-glutamine (all from JRH Biosciences), cryopreserved in 95% FBS and 5% DMSO (Sigma), and then stored in CBS's vapor-phase liquid nitrogen freezer. The cell bank was sterile (Quality Control Centocor, Malvern) and mycoplasma-free (Bionique Laboratories). Cells were maintained in logarithmic-phase culture until confluence. Prior to confluence, they were washed in PBS, counted, and viability determined by trypan blue dye exclusion (>95%).

[0236] Human TNFα was produced by a recombinant cell line, designated C237A, produced at Centocor's Molecular Biology. The cell line was expanded in IMDM medium (JRH Biosciences) supplemented with 5% (v / v) FBS (Cell Culture Labs), 2 mM L-glutamine (all from JRH Biosciences), and 0.5 μg / mL mycophenolic acid, cryopreserved in 95% FBS and 5% DMSO (Sigma), and then stored in a vapor-phase liquid nitrogen freezer at Centocor (13). The cell bank was sterile (Quality Control Centocor, Malvern) and free of mycoplasma (Bionique Laboratories).

[0237] Cell fusion. Cell fusion was performed using 653 mouse myeloma cells and live mouse spleen cells at a 1:1 ratio. Briefly, spleen cells and myeloma cells were pelleted together. The pellet was slowly resuspended in 1 mL of 50% (w / v) PEG / PBS solution (PEG molecular weight 1,450 g / mol, Sigma) at 37°C for 30 seconds. Fusion was stopped by slowly adding 10.5 mL of RPMI medium (without additives) (JRH) at 37°C for 1 minute. The fused cells were centrifuged at 750 rpm for 5 minutes. The cells were then resuspended in HAT medium (RPMI / HEPES medium containing 10% fetal bovine serum (JRH), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 μg / mL gentamicin, 2.5% Origen culture supplement (Fisher), 50 μM 2-mercaptoethanol, 1% 653-conditioned RPMI medium, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) and plated at 200 μL / well into five 96-well flat-bottom tissue culture plates. The plates were then placed in a humidified 37°C incubator containing 5% CO2 and 95% air for 7–10 days.

[0238] Detection of human IgG anti-TNFα antibodies in mouse serum. Mouse serum was screened for human IgG antibodies specific to human TNFα using a solid-phase EIA. Briefly, plates were coated overnight with 1 μg / mL TNFα in PBS. After washing with 0.15 M saline containing 0.02% (v / v) Tween 20, wells were blocked with 200 μL / well of 1% (w / v) BSA in PBS for 1 hour at room temperature. Plates were either used immediately or frozen at -20°C for later use. Mouse serum was incubated on the human TNFα-coated plates at 50 μL / well in a two-fold serial dilution for 1 hour at room temperature. After washing, the plates were probed with 50 μL / well of Fc-specific (Accurate) HRP-labeled goat anti-human IgG diluted 1:30,000 in 1% BSA-PBS for 1 hour at room temperature. The plate was washed again, and 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.01% HO and 1 mg / mL OPD) was added over 15 minutes at room temperature. Then, 25 μL / well of stop solution (4 N sulfuric acid) was added, and the OD was read at 490 nm using an automated plate spectrophotometer.

[0239] Detection of fully human immunoglobulins in hybridoma supernatants. Because GenPharm mice can produce both mouse and human immunoglobulin chains, growth-positive hybridoma clones were tested for the presence of both human light and heavy chains using two separate EIA assays. Plates were coated as described above, and undiluted hybridoma supernatants were incubated on the plates for 1 hour at 37°C. The plates were washed and probed with either an HRP-conjugated goat anti-human κ (Southern Biotech) antibody diluted 1:10,000 in 1% BSA-HBSS or an HRP-conjugated goat anti-human IgG Fc-specific antibody diluted 1:30,000 in 1% BSA-HBSS for 1 hour at 37°C. The plates were then incubated with substrate solution as described above. Hybridoma clones that did not yield positive signals in both the anti-human κ and anti-human IgG Fc EIA formats were discarded.

[0240] Isotype. Determination of antibody isotype was accomplished using an EIA format similar to that used to screen mouse immune sera for specific titers. EIA plates were coated with 10 μg / mL goat anti-human IgG (H+L) in sodium carbonate buffer overnight at 4EC and blocked as described above. Undiluted supernatants from 24-well cultures were incubated on the plates for 1 h at room temperature. Plates were washed and probed with HRP-labeled goat anti-human IgG1, IgG2, IgG3, or IgG4 (Binding Site) diluted 1:4000 in 1% BSA-PBS for 1 h at room temperature. Plates were washed again and incubated with substrate solution as described above.

[0241] Results and Discussion. Generation of fully human anti-human TNFα monoclonal antibodies. A single fusion, designated GenTNV, was performed from GenPharm mice immunized with recombinant human TNFα protein. 196 growth-positive hybrids were screened from this fusion. Eight hybridoma cell lines were identified that secreted fully human IgG antibodies reactive with human TNFα. Each of these eight cell lines secreted immunoglobulins of the human IgG1κ isotype, and all were subcloned twice by limiting dilution to obtain stable cell lines (>90% homogeneity). The cell line names and their respective C-code designations are listed in Table 1. Each of the cell lines was frozen in a 12-vial research cell bank stored in liquid nitrogen.

[0242] Parental cells harvested from wells of 24-well culture dishes for each of the eight cell lines were delivered to the Molecular Biology group on February 18, 1999 for transfection and further characterization.

[0243] [Table 2]

[0244] conclusion GenTNV fusions were performed using splenocytes from hybrid mice containing human variable and constant region antibody transgenes immunized with recombinant human TNFα prepared at Centocor. Eight fully human TNFα-reactive IgG monoclonal antibodies of the IgG1κ isotype were generated. The parent cell lines were transferred to the Molecular Biology group for further characterization and development. One of these new human antibodies may be useful for anti-inflammatory purposes, with the potential benefit of reduced immunogenicity and allergic complications compared to Remicade.

[0245] References Taylor et al., International Immunology 6:579-591 (1993). Lonberg et al., Nature 368:856-859 (1994). Neuberger, M., Nature Biotechnology 14:826 (1996). Fishwild et al., Nature Biotechnology 14:845-851 (1996). Scallon et al., Cytokine 7:759-770 (1995).

[0246] Example 4: Cloning and preparation of cell lines expressing human anti-TNFα antibodies. Summary: A panel of eight human monoclonal antibodies (mAbs), designated TNV, was found to bind to immobilized human TNFα with apparently high avidity. Seven of the eight mAbs were shown to efficiently block human TNFα binding to recombinant TNF receptors. Sequence analysis of the DNA encoding the seven mAbs confirmed that all mAbs possessed human V regions. The DNA sequences also revealed that three pairs of mAbs were identical to each other, and therefore the original panel of eight mAbs contained only four distinct mAbs, designated TNV14, TNV15, TNV148, and TNV196. Based on analysis of the deduced amino acid sequences of the mAbs and the results of in vitro TNFα neutralization data, mAbs TNV148 and TNV14 were selected for further testing.

[0247] Because the proline residue at position 75 (framework 3) of the TNV148 heavy chain was not found at that position in other human antibodies of the same subgroup during database searches, site-directed DNA mutagenesis was performed to encode a serine residue at that position to match the known germline framework e sequence. The serine-modified mAb was designated TNV148B. PCR-amplified DNA encoding the heavy and light chain variable regions of TNV148B and TNV14 was cloned into a newly prepared expression vector based on the recently cloned heavy and light chain genes of another human mAb (12B75) (U.S. Patent Application No. 60 / 236,827, filed October 7, 2000, entitled "IL-12 Antibodies, Compositions, Methods and Uses," published as WO 02 / 12500, which is incorporated herein by reference in its entirety).

[0248] P3X63Ag8.653 (653) cells or Sp2 / 0-Ag14 (Sp2 / 0) mouse myeloma cells were transfected with the respective heavy and light chain expression plasmids and screened for cell lines producing high levels of recombinant TNV148B and TNV14 (rTNV148B and rTNV14) mAbs by two rounds of subcloning. Growth curves and evaluation of the stability of mAb production over time showed that 653 transfectant clones C466D and C466C stably produced approximately 125 μg / mL of rTNV148B mAb in spent cultures, while Sp2 / 0 transfectant 1.73-12-122 (C467A) stably produced approximately 25 μg / mL of rTNV148B mAb in spent cultures. Similar analysis showed that Sp2 / 0 transfectant clone C476A produced 18 μg / mL of rTNV14 in spent cultures.

[0249] Introduction. A panel of eight mAbs derived from human TNFα-immunized GenPharm / Medarex mice (HCo12 / KCo5 genotype) were previously shown to bind to human TNFα and have a fully human IgG1κ isotype. A simple binding assay was used to determine whether exemplary mAbs of the invention might have TNFα-neutralizing activity by assessing their ability to block TNFα binding to recombinant TNF receptors. Based on these results, DNA sequence results, and some in vitro characterization of the mAb, TNV148 was selected as the mAb for further characterization.

[0250] The DNA sequence encoding the TNV148 mAb was cloned and modified to fit into a gene expression vector encoding a suitable constant region, introduced into well-characterized 653 and Sp2 / 0 mouse myeloma cells, and the resulting transfected cell lines were screened until a subclone was identified that produced 40-fold more mAb than the original hybridoma cell line.

[0251] Materials and Methods Reagents and Cells. TRIZOL reagent was purchased from Gibco BRL. Proteinase K was obtained from Sigma Chemical Company. Reverse transcriptase was obtained from Life Sciences, Inc. Taq DNA polymerase was obtained from either Perkin Elmer Cetus or Gibco BRL. Restriction enzymes were purchased from New England Biolabs. QIAquick PCR Purification Kit was obtained from Qiagen. QuikChange Site-Directed Mutagenesis Kit was purchased from Stratagene. Wizard Plasmid Miniprep Kit and RNasin were from Promega. Optiplates were obtained from Packard. 125Iodine was purchased from Amersham. Custom oligonucleotides were purchased from Keystone / Biosource International. The names, identification numbers, and sequences of the oligonucleotides used in this work are listed in Table 2.

[0252] Table 2. Oligonucleotides used to clone, manipulate, or sequence TNV mAb genes. The amino acids encoded by oligonucleotides 5'14s and HuH-J6 are shown above the sequences. The "M" amino acid residue represents the translation initiation codon. The underlined sequences in oligonucleotides 5'14s and HuH-J6 indicate the BsiWI and BstBI restriction sites, respectively. The diagonal lines in HuH-J6 correspond to the exon / intron boundary. Note that oligonucleotides whose sequences correspond to the minus strand are written in the 3'-5' orientation.

[0253] [Table 3]

[0254] One frozen vial of 653 mouse myeloma cells was obtained. The vial was thawed the same day and expanded in IMDM, 5% FBS, and 2 mM glutamine (media). These cells were maintained in continuous culture until transfection with anti-TNF DNA described herein 2–3 weeks later. Five days after thawing, some of the culture was harvested, pelleted by centrifugation, resuspended in 95% FBS, 5% DMSO, aliquoted into 30 vials, frozen, and stored for future use. Similarly, one frozen vial of Sp2 / 0 mouse myeloma cells was obtained. The vial was thawed, a new freeze-down was prepared as described above, and the frozen vials were stored in freezer boxes AA and AB at CBC. These cells were thawed and used for all Sp2 / 0 transfections described herein.

[0255] Assay for inhibition of TNF binding to the receptor. Hybridoma cell supernatant containing TNF mAb was used to determine whether the mAb binds to the recombinant TNF receptor fusion protein p55-sf2. 125 The ability to block the binding of I-labeled TNFα was assayed (Scallon et al. (1995) Cytokine 7:759-770). 50 μL of p55-sf2 at 0.5 μg / mL in PBS was added to Optiplates to coat the wells during 1 hour of incubation at 37°C. Eight serial dilutions of TNV cell supernatants were prepared in 96-well round-bottom plates using PBS / 0.1% BSA as the diluent. Cell supernatant containing anti-IL-18 mAb was included as a negative control, and the same anti-IL-18 supernatant spiked with cA2 (anti-TNF chimeric antibody, Remicade, U.S. Patent No. 5,770,198, incorporated herein by reference in its entirety) was included as a positive control. The final TNFα concentration was adjusted to 5 ng / mL. 125 I-labeled TNFα (58 Ci / g, D. Shealy) was added to 100 L of cell supernatant. The mixture was pre-incubated at room temperature for 1 hour. The coated Optiplates were washed to remove unbound p55-sf2, and 50 L of 125 The I-TNFα / cell supernatant mixture was transferred to Optiplates. After 2 hours at room temperature, the Optiplates were washed three times with PBS-Tween. 100 μL of Microscint-20 was added, and the cpm bound was determined using a TopCount gamma counter.

[0256] Amplification of V genes and DNA sequence analysis. For RNA preparation, hybridoma cells were washed once with PBS and then TRIZOL reagent was added. 7 × 10 6 ~1.7×10 7The cells were resuspended in 1 mL of TRIZOL. After adding 200 μL of chloroform, the tube was vigorously shaken. The sample was centrifuged at 4°C for 10 minutes. The aqueous phase was transferred to a new microfuge tube, and an equal volume of isopropanol was added. The tube was vigorously shaken and incubated at room temperature for 10 minutes. The sample was then centrifuged at 4°C for 10 minutes. The pellet was washed once with 1 mL of 70% ethanol and briefly dried in a vacuum oven. The RNA pellet was resuspended in 40 μL of DEPC-treated water. The quality of the RNA preparation was determined by fractionating 0.5 μL in a 1% agarose gel. The RNA was stored in a -80°C freezer until use.

[0257] To prepare heavy and light chain cDNA, a mixture containing 3 μL of RNA and 1 μg of either oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) (see Table 1) was prepared in an 11.5 μL volume. The mixture was incubated at 70°C in a water bath for 10 minutes and then cooled on ice for 10 minutes. A separate mixture was prepared consisting of 2.5 μL of 10x reverse transcriptase buffer, 10 μL of 2.5 mM dNTPs, 1 μL of reverse transcriptase (20 units), and 0.4 μL of the ribonuclease inhibitor RNasin (1 unit). 13.5 μL of this mixture was added to 11.5 μL of the cold RNA / oligonucleotide mixture, and the reaction was incubated at 42°C for 40 minutes. The cDNA synthesis reaction was then stored in a -20°C freezer until use.

[0258] Unpurified heavy and light chain cDNAs were used as templates for PCR amplification of the variable region coding sequences. Five oligonucleotide pairs (366 / 354, 367 / 354, 368 / 354, 369 / 354, and 370 / 354, Table 1) were simultaneously tested for their ability to prime amplification of heavy chain DNA. Two oligonucleotide pairs (362 / 208 and 363 / 208) were simultaneously tested for their ability to prime amplification of light chain DNA. PCR reactions were performed using 2 units of PLATINUM™ High-Fidelity (HIFI) Taq DNA Polymerase in a total volume of 50 μL. Each reaction contained 2 μL of cDNA reaction mixture, 10 pmoles of each oligonucleotide, 0.2 mM dNTPs, 5 μL of 10× HIFI buffer, and 2 mM magnesium sulfate. The thermal cycler program was 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 62°C for 30 s, and 68°C for 1.5 min, followed by a final incubation at 68°C for 10 min.

[0259] To prepare PCR products for direct DNA sequencing, they were purified using a QIAquick™ PCR Purification Kit according to the manufacturer's protocol. DNA was eluted from the spin column using 50 μL of sterile water and then dried to a volume of 10 μL using a vacuum dryer. Next, DNA sequencing reactions were set up in a total volume of 20 μL, containing 1 μL of purified PCR product, 10 μM oligonucleotide primers, 4 μL of BigDye Terminator™ ready reaction mix, and 14 μL of sterile water. The heavy chain PCR product, generated with the oligonucleotide pair 367 / 354, was sequenced using oligonucleotide primers 159 and 360. The light chain PCR product, generated with the oligonucleotide pair 363 / 208, was sequenced using oligonucleotides 34 and 163. The thermal cycler program for sequencing was 25 cycles (96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 4 minutes), followed by overnight at 4°C. Reaction products were fractionated through polyacrylamide gels and detected using an ABI 377 DNA sequencer.

[0260] Site-directed mutagenesis to change the amino acid Pro in TNV148 mAb 75A single nucleotide change was made in the TNV148 heavy chain variable region DNA sequence to replace with a serine residue. Complementary oligonucleotides 399 and 400 (Table 1) were designed, and this change was made using QuikChange™ site-directed mutagenesis, as described by the manufacturer. The two oligonucleotides were first fractionated on a 15% polyacrylamide gel, and the major band was purified. Mutagenesis reactions were prepared using either 10 ng or 50 ng of TNV148 heavy chain plasmid template (p1753), 5 μL of 10× reaction buffer, 1 μL of dNTP mix, 125 ng of primer 399, 125 ng of primer 400, and 1 μL of Pfu DNA polymerase. Sterile water was added to bring the total volume to 50 μL. The reaction mixture was then incubated in a thermal cycler programmed to incubate at 95°C for 30 seconds, followed by 14 cycles of incubation at 95°C for 30 seconds, 55°C for 1 minute, 64°C for 1 minute, 68°C for 7 minutes, followed by 30°C for 2 minutes (1 cycle). These reactions were designed to incorporate the mutagenic oligonucleotide into an otherwise identical newly synthesized plasmid. To remove the original TNV148 plasmid, 1 μL of DpnI endonuclease, which cuts only the original methylated plasmid, was added, and the sample was then incubated at 37°C for 1 hour. 1 μL of the reaction was then used to transform Epicurian Coli XL1-Blue supercompetent Escherichia coli (E. coli) using the standard heat shock method, and transformed bacteria were identified after plating on LB-ampicillin agar plates. Plasmid minipreps were prepared using the Wizard™ kit as described by the manufacturer. After eluting the sample from the Wizard™ column, the plasmid DNA was further purified by precipitating it with ethanol and then resuspending it in 20 μL of sterile water. DNA sequence analysis was then performed to identify plasmid clones with the desired base changes and to confirm that no other base changes had been inadvertently introduced into the TNV148 coding sequence.Using the same parameters described in section 4.3, 1 μL of the plasmid was subjected to a cycle sequencing reaction prepared with 3 μL of BigDye mix, 1 μL of pUC19 forward primer, and 10 μL of sterile water.

[0261] Construction of Expression Vectors from 12B75 Genes. Several recombinant DNA steps were performed to prepare new human IgG1 and human Kappa expression vectors from previously cloned genomic copies of the 12B75-encoding heavy and light chain genes, respectively (as disclosed in U.S. patent application Ser. No. 60 / 236,827, filed Oct. 7, 2000, entitled "IL-12 Antibodies, Compositions, Methods and Uses," published as WO 02 / 12500, and incorporated herein by reference in its entirety). The final vectors were designed to allow for simple, one-step replacement of existing variable region sequences with any appropriately designed, PCR-amplified variable region.

[0262] To modify the 12B75 heavy chain gene in plasmid p1560, a 6.85 kb BamHI / HindIII fragment containing the promoter and variable region was transferred from p1560 to pUC19 to generate p1743. The smaller size of this plasmid compared to p1560 allowed the use of QuikChange™ mutagenesis (using oligonucleotides BsiWI-1 and BsiWI-2) to introduce a unique BsiWI cloning site immediately upstream of the translation start site, according to the manufacturer's protocol. The resulting plasmid was designated p1747. To introduce a BstBI site at the 3' end of the variable region, a 5' oligonucleotide primer was designed with SalI and BstBI sites. This primer, along with the pUC reverse primer, was used to amplify a 2.75 kb fragment from p1747. This fragment was then cloned back into the naturally occurring SalI and HindIII sites in the 12B75 variable region, thereby introducing a unique BstBI site. The resulting intermediate vector, designated p1750, was capable of accepting variable region fragments with BsiWI and BstBI ends. To prepare versions of the heavy chain vector in which the constant region was also derived from the 12B75 gene, the BamHI-HindIII insert of p1750 was transferred to pBR322, which contains an EcoRI site downstream of the HindIII site. The resulting plasmid, p1768, was then digested with HindIII and EcoRI and ligated to a 5.7 kb HindIII-EcoRI fragment from p1744, a subclone obtained by cloning the large BamHI-BamHI fragment from p1560 into pBC. The resulting plasmid, p1784, was then used as a vector for TNV Ab cDNA fragments with BsiWI and BstBI ends. Additional work was performed to prepare expression vectors p1788 and p1798, which contain the IgG1 constant region from the 12B75 gene and differ from each other by the extent to which they contain the 12B75 heavy chain JC intron.

[0263] To modify the 12B75 light chain gene in plasmid p1558, a 5.7 kb SalI / AflII fragment containing the 12B75 promoter and variable region was transferred from p1558 into the XhoI / AflII sites of plasmid L28. This new plasmid, p1745, provided a smaller template for the mutagenesis step. A unique SalI restriction site was introduced at the 5' end of the variable region by QuikChange™ mutagenesis using oligonucleotides (C340salI and C340sal2). The resulting intermediate vector, p1746, contained unique SalI and AflII restriction sites into which variable region fragments could be cloned. Any variable region fragments cloned into p1746 would preferably be joined to the 3' half of the light chain gene. To prepare a restriction fragment from the 3' half of the 12B75 light chain gene that could be used for this purpose, oligonucleotides BAHN-1 and BAHN-2 were annealed to each other to form a double-stranded linker containing the restriction sites BsiW1, AflII, HindII, and NotI, and containing ends that could be ligated into the KpnI and SacI sites. This linker was cloned between the KpnI and SacI sites of pBC to yield plasmid p1757. A 7.1-kb fragment containing the 12B75 light chain constant region, generated by digesting p1558 with AflII followed by partial digestion with HindIII, was cloned between the AflII and HindII sites of p1757 to yield p1762. This new plasmid contained unique BsiWI and AflII sites where a BsiWI / AflII fragment containing the promoter and variable region could join and transfer the two halves of the gene.

[0264] cDNA cloning and assembly of expression plasmids. All RT-PCR reactions (see above) were treated with Klenow enzyme to further fill in the DNA ends. The heavy chain PCR fragment was digested with the restriction enzymes BsiWI and BstBI and then cloned between the BsiWI and BstBI sites of plasmid L28 (L28 was used because the 12B75-based intermediate vector p1750 had not yet been prepared). DNA sequence analysis of the cloned insert indicated that the resulting construct was correct and that no errors had been introduced during PCR amplification. The identification numbers assigned to these L28 plasmid constructs (TNV14, TNV15, TNV148, TNV148B, and TNV196) are shown in Table 3.

[0265] The BsiWI / BstBI inserts of the heavy chains of TNV14, TNV148, and TNV148B were transferred from the L28 vector into the newly prepared intermediate vector p1750. The identification numbers assigned to these intermediate plasmids are shown in Table 2. This cloning step and subsequent steps were not performed for TNV15 and TNV196. Next, the variable regions were transferred into two different human IgG1 expression vectors. Using the restriction enzymes EcoRI and HindIII, the variable regions were transferred into Centocor's previously used IgG1 vector p104. The resulting expression plasmids encoding IgG1 of the Gm(f+) allotype were designated p1781 (TNV14), p1782 (TNV148), and p1783 (TNV148B) (see Table 2). The variable regions were also cloned upstream of the IgG1 constant region derived from the 12B75 (GenPharm) gene. These expression plasmids encoding IgG1 of the G1m(z) allotype are also listed in Table 3.

[0266] Table 3. Plasmid identification numbers for the various heavy and light chain plasmids. The L28 vector or pBC vector represents the initial Ab cDNA clone. The inserts of these plasmids were transferred into incomplete 12B75-based vectors to generate intermediate plasmids. One additional transfer step resulted in the final expression plasmids that were either linearized and then introduced into cells or used to purify the mAb gene insert before transfection of cells. ND = not performed.

[0267] [Table 4]

[0268] The light chain PCR products were digested with restriction enzymes SalI and SacII and then cloned between the SalI and SacII sites of the plasmid pBC. Two different light chain versions, differing by one amino acid, were designated p1748 and p1749 (Table 2). DNA sequence analysis confirmed that these constructs had the correct sequence. The SalI / AflII fragments of p1748 and p1749 were then cloned between the SalI and AflII sites of the intermediate vector p1746 to generate p1755 and p1756, respectively. These 5' halves of the light chain genes were then joined to the 3' halves of the genes by transferring the BsiWI / AflII fragments from p1755 and p1756 to the newly prepared construct p1762, generating the final expression plasmids p1775 and p1776, respectively (Table 2).

[0269] Cell Transfection, Screening, and Subcloning. A total of 15 mouse myeloma cell lines were transfected with various TNV expression plasmids (see Table 3 in the Results and Discussion section). These transfections were differentiated by whether (1) the host cells were Sp2 / 0 or 653; (2) the heavy chain constant region was encoded by Centocor's previous IgG1 vector or the 12B75 heavy chain constant region; (3) the mAb was TNV148B, TNV148, TNV14, or the new HC / LC combination; (4) the DNA was a linearized plasmid or purified Ab gene insert; and (5) the presence or absence of a complete JC intron sequence in the heavy chain gene. In addition, some transfections were repeated to increase the likelihood of screening a large number of clones.

[0270] Sp2 / 0 and 653 cells were each transfected with a mixture of heavy and light chain DNA (8–12 μg each) by electroporation under standard conditions previously described (Knight DM et al. (1993) Molecular Immunology 30:1443–1453). For transfections 1, 2, 3, and 16, the appropriate expression plasmids were linearized by restriction enzyme digestion prior to transfection. For example, SalI and NotI restriction enzymes were used to linearize the TNV148B heavy chain plasmid p1783 and light chain plasmid p1776, respectively. For the remaining transfections, DNA inserts containing only the mAb gene were isolated from the plasmid vector by digesting the heavy chain plasmid with BamHI and the light chain plasmid with BsiWI and NotI. The mAb gene inserts were then purified by agarose gel electrophoresis and Qiex purification resin. Cells transfected with the purified gene insert were co-transfected with 3–5 μg of PstI-linearized pSV2gpt plasmid (p13) as a source of selection marker. After electroporation, cells were seeded into 96-well tissue culture dishes in IMDM, 15% FBS, and 2 mM glutamine and incubated at 37°C in a 5% CO2 incubator. After 2 days, an equal volume of IMDM, 5% FBS, 2 mM glutamine, and 2×MHX selection medium (1×MHX = 0.5 μg / mL mycophenolic acid, 2.5 μg / mL hypoxanthine, and 50 μg / mL xanthine) was added, and the plates were incubated for an additional 2–3 weeks to allow colonies to form.

[0271] Cell supernatants collected from wells containing colonies were assayed for human IgG by ELISA as described. Briefly, various dilutions of cell supernatants were incubated in 96-well EIA plates coated with polyclonal goat anti-human IgG Fc fragment, followed by detection of bound human IgG using alkaline phosphatase-conjugated goat anti-human IgG (H+L) and an appropriate color substrate. A standard curve using the same purified mAb measured in the cell supernatants as a standard was included on each EIA plate to allow quantification of human IgG in the supernatants. Cells in those colonies that appeared to produce the most human IgG were passaged into 24-well plates for further production evaluation in spent cultures to identify the highest-producing parental clones.

[0272] The parent clone with the highest production was subcloned to identify subclones with higher production potential and prepare more homogeneous cell lines. 96-well tissue culture plates were seeded with one cell per well or four cells per well in IMDM, 5% FBS, 2 mM glutamine, and 1x MHX and incubated at 37°C in a 5% CO2 incubator for 12–20 days until colonies appeared. Cell supernatants were collected from wells containing one colony per well and analyzed by ELISA as described above. Selected colonies were passaged into 24-well plates, and after culture exhaustion, the highest-producing subclones were identified by quantifying human IgG levels in their supernatants. This process was repeated when the selected first subclones were subjected to a second round of subcloning. The best subclones from the second round were selected as cell lines for further development.

[0273] Characterization of cell subclones. The best subclones from the second round were selected and growth curves were performed to assess mAb production levels and cell growth characteristics. A T75 flask was cultured with 1 x 10 cells in 30 mL of IMDM, 5% FBS, 2 mM glutamine, and 1 x MHX (or serum-free medium). 5 Cells were seeded at 1 × 10 cells / mL. 300 μL aliquots were removed at 24-hour intervals to measure viable cell density. Viable cell counts were 1 × 10 cells / mL.5 Analysis continued until the antibody concentration was below 100 cells / mL. Aliquots of the harvested cell supernatants were assayed for the concentration of antibody present. ELISA assays were performed using rTNV148B or rTNV14 JG92399 as standards. Samples were incubated for 1 hour on ELISA plates coated with polyclonal goat anti-human IgG Fc, and bound mAb was detected with alkaline phosphatase-conjugated goat anti-human IgG (H+L) at a 1:1000 dilution.

[0274] A separate growth curve analysis was also performed on the two cell lines to compare their growth rates in the presence of various amounts of MHX selection agent. Cell lines C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional two days. Both cell cultures were then split into three cultures containing either no MHX, 0.2x MHX, or 1x MHX (1x MHX = 0.5 μg / mL mycophenolic acid, 2.5 μg / mL hypoxanthine, 50 μg / mL xanthine). After one day, 1x10 cells were placed in a new T75 flask and cultured. 5 Cultures were seeded at a starting density of 1000 cells / mL and cells were counted at 24-hour intervals for 1 week. Aliquots for mAb production were not collected. Doubling times for these samples were calculated using the formula provided in SOP PD32.025.

[0275] Additional studies were performed to assess the stability of mAb production over time. Cultures were grown in IMDM, 5% FBS, and 2 mM glutamine in 24-well plates, either with or without MHX selection. When the cultures became confluent, they were split into new cultures, and the old cultures were then depleted. At this time, aliquots of the supernatant were taken and stored at 4°C. Aliquots were removed over a period of 55 to 78 days. At the end of this period, the supernatants were tested for the amount of antibody present by anti-human IgG Fc ELISA, as outlined above.

[0276] Results and Discussion Inhibition of TNF binding to recombinant receptors A simple binding assay was performed to determine whether the eight TNV ​​mAbs contained in the hybridoma cell supernatants could inhibit TNFα binding to the receptor. The concentration of TNV mAb in each cell supernatant was first determined by standard ELISA analysis of human IgG. Next, the recombinant p55 TNF receptor / IgG fusion protein p55-sf2 was coated onto an EIA plate and assayed in the presence of various amounts of TNV mAb. 125 I-labeled TNFα was allowed to bind to the p55 receptor. As shown in Figure 1, all but one of the eight TNV ​​mAbs (TNV122) efficiently blocked TNFα binding to the p55 receptor. In fact, the TNV mAbs appeared more effective at inhibiting TNFα binding than the cA2 positive control mAb spiked into the negative control hybridoma supernatant. These results were interpreted as indicating that the TNV mAbs likely block the biological activity of TNFα in cell-based assays and in vivo, and therefore, further analysis is warranted.

[0277] DNA sequence analysis Confirmation that the RNA encodes a human mAb As a first step in characterizing seven TNV mAbs (TNV14, TNV15, TNV32, TNV86, TNV118, TNV148, and TNV196) that exhibited TNFα blocking activity in receptor binding assays, we isolated total RNA from the seven hybridoma cell lines producing these mAbs. Each RNA sample was then used to prepare human antibody heavy or light chain cDNAs containing the complete signal sequence, complete variable region sequence, and partial constant region sequence of each mAb. These cDNA products were then amplified in PCR reactions, and the PCR-amplified DNA was directly sequenced without first cloning the fragment. The sequenced heavy chain cDNA was >90% identical to DP-46, one of five human germline genes present in mouse (Figure 2). Similarly, the sequenced light chain cDNA was either 100% or 98% identical to one of the human germline genes present in mouse (Figure 3). These sequence results confirmed that the RNA molecules transcribed into cDNA and sequenced encoded human antibody heavy and light chains. It should be noted that because the variable regions were PCR amplified using oligonucleotides that map to the 5' end of the signal sequence coding sequence, the first few amino acids of the signal sequence may not represent the actual sequence of the original TNV translation product, but represent the actual sequence of the recombinant TNV ​​mAb.

[0278] Specific neutralizing mAb Analysis of the cDNA sequences of the entire variable regions of both the heavy and light chains of each mAb revealed that TNV32 was identical to TNV15, TNV118 was identical to TNV14, and TNV86 was identical to TNV148. The results of receptor binding assays were consistent with the DNA sequence analysis; both TNV86 and TNV148 were approximately four-fold better than both TNV118 and TNV14 at blocking TNF binding. Therefore, subsequent work focused only on four unique TNV mAbs: TNV14, TNV15, TNV148, and TNV196.

[0279] Relationships between the four mAbs DNA sequence analysis revealed that the heavy chain-encoding genes of the four TNV mAbs were all highly homologous to each other and appeared to all originate from the same germline gene, DP-46 (Figure 2). Additionally, because each of the heavy chain CDR3 sequences was highly similar and of the same length, and because they all used the J6 exon, they likely arose from a single VDJ gene rearrangement event followed by somatic changes that made each mAb unique. DNA sequence analysis revealed that there were only two distinct light chain genes in the four mAbs (Figure 3). The light chain variable region coding sequences in TNV14 and TNV15 are identical to each other and to representative germline sequences of the Vg / 38K family of human kappa chains. The light chain coding sequences of TNV148 and TNV196 are identical to each other but differ from the germline sequences at two nucleotide positions (Figure 3).

[0280] The deduced amino acid sequences of the four mAbs revealed the relatedness of the actual mAbs. The four mAbs contain four distinct heavy chains (Figure 4) but only two distinct light chains (Figure 5). The differences between the TNV mAb sequences and the germline sequences were mostly limited to the CDR domains, but three of the mAb heavy chains also differed from the germline sequence in the framework regions (Figure 4). Compared to the DP-46 germline-encoded Ab framework regions, TNV14 was identical, TNV15 differed by one amino acid, TNV148 differed by two amino acids, and TNV196 differed by three amino acids.

[0281] cDNA Cloning, Site-Directed Mutagenesis, and Assembly of Final Expression Plasmids. cDNA Cloning. Based on the DNA sequences of the PCR-amplified variable regions, new oligonucleotides were designed for another PCR amplification to accommodate the cloned coding sequences within the expression vectors. For the heavy chain, the product of this second PCR was digested with restriction enzymes BsiWI and BstBI and cloned into the plasmid vector L28 (plasmid identification number shown in Table 2). For the light chain, the product of the second PCR was digested with SalI and AflII and cloned into the plasmid vector pBC. Individual clones were then sequenced to confirm that their sequences were identical to previous sequences obtained from direct sequencing of the PCR products, revealing the most abundant nucleotide at each position in a potentially heterogeneous population of molecules.

[0282] Site-directed mutagenesis to alter TNV148. mAbs TNV148 and TNV196 were consistently observed to be four-fold more potent than the next best mAb (TNV14) in neutralizing TNFα bioactivity. However, as noted above, the TNV148 and TNV196 heavy chain framework sequences differ from the germline framework sequences. Comparison of the TNV148 heavy chain sequence with other human antibodies showed that many other human mAbs contain an Ile residue at position 28 of framework 1 (counting only the mature sequence), while the Pro residue at position 75 of framework 3 is a rare amino acid at that position.

[0283] A similar comparison of the TNV196 heavy chain suggested that three amino acids in framework 3 that differ from the germline sequence may be rare in human mAbs. These differences could potentially render TNV148 and TNV196 immunogenic when administered to humans. Because TNV148 has only one amino acid residue of interest, and this residue is not thought to be important for TNFα binding, we used site-directed mutagenesis techniques to change a single nucleotide in the TNV148 heavy chain coding sequence (of plasmid p1753) so that the germline Ser residue was encoded instead of the Pro residue at position 75. The resulting plasmid was called p1760 (see Table 2). The resulting gene and mAb were called TNV148B to distinguish it from the original TNV148 gene and mAb (see Figure 5).

[0284] Assembly of Final Expression Plasmids. New antibody expression vectors were prepared based on the 12B75 heavy and light chain genes previously cloned as genomic fragments. Different TNV ​​expression plasmids were prepared (see Table 2); in each case, the 5' flanking sequence, promoter, and intron enhancer were derived from the respective 12B75 gene. For the light chain expression plasmid, the complete JC intron, constant region coding sequence, and 3' flanking sequence were also derived from the 12B75 light chain gene. For the heavy chain expression plasmid that resulted in the final product cell lines (p1781 and p1783, see below), the human IgG1 constant region coding sequence was derived from Centocor's previously used expression vector (p104). Importantly, the final product cell line reported here expresses a TNV mAb of a different allotype (Gm(f+)) than the original hybridoma-derived TNV mAb (G1m(z)). This is because the 12B75 heavy chain gene from GenPharm mice encodes an Arg residue at the C-terminal end of the CH1 domain, whereas Centocor's IgG1 expression vector p104 encodes a Lys residue at that position. Other heavy chain expression plasmids (e.g., p1786 and p1788) in which the JC intron, complete constant region coding sequence, and 3' flanking sequence are derived from the 12B75 heavy chain gene have been prepared, but cell lines transfected with these genes were not selected as production cell lines. The vectors were carefully designed to allow for one-step cloning of subsequent PCR-amplified V regions to yield the final expression plasmids.

[0285] The PCR-amplified variable region cDNAs were transferred from the L28 or pBC vectors into intermediate 12B75-based vectors that provided the promoter region and part of the JC intron (see Table 2 for plasmid identification numbers). Restriction fragments containing the 5' halves of the antibody genes were then transferred from these intermediate vectors to the final expression vectors that provided the 3' halves of the respective genes to form the final expression plasmids (see Table 2 for plasmid identification numbers).

[0286] Cell transfection and subcloning. Expression plasmids were either linearized by restriction digestion, or the antibody gene inserts in each plasmid were purified from the plasmid backbone. Sp2 / 0 and 653 mouse myeloma cells were transfected with heavy and light chain DNA by electroporation. Fifteen different transfections were performed, most of which were unique as defined by Ab, specific characteristics of the Ab gene, whether the gene was on the linearized whole plasmid or a purified gene insert, and the host cell line (summarized in Table 4). Cell supernatants from clones resistant to mycophenolic acid were assayed for the presence of human IgG by ELISA and quantified using purified rTNV148B as a reference standard curve.

[0287] The highest producing rTNV148B cell line Ten of the 653 highest-producing parental lines from rTNV148B transfection 2 (producing 5–10 μg / mL in spent 24-well cultures) were subcloned to screen for higher-producing cell lines and prepare a more homogeneous cell population. Two of the subclones of parental lines 2.320, 2.320-17, and 2.320-20 produced approximately 50 μg / mL in spent 24-well cultures, a five-fold increase over their parental lines. A second subcloning of subcloned lines 2.320-17 and 2.320-20 resulted in the production of approximately 50 μg / mL of erythrocyte leukemia virus (ECV) in spent 24-well cultures.

[0288] The identification numbers of the heavy and light chain plasmids encoding each mAb are shown. For transfections performed with purified mAb gene inserts, plasmid p13 (pSV2gpt) was included as a source of the gpt selection marker. The heavy chain constant region was encoded either by the same human IgG1 expression vector used to encode Remicade ("old") or by the constant region contained within the 12B75 (GenPharm / Medarex) heavy chain gene ("new"). H1 / L2 refers to the "new" mAb composed of the TNV14 heavy chain and the TNV148 light chain. Plasmids p1783 and p1801 differ only by the extent to which their heavy chain genes contain a JC intron. The transfection number, which defines the first digit in the gene name of the cell clone, is shown on the right. The rTNV148B-producing cell lines C466 (A, B, C, D) and C467A described herein were derived from transfections no. 2 and 1, respectively. The rTNV14-producing cell line C476A was derived from transfection no. 3.

[0289] [Table 5]

[0290] ELISA assays on spent 24-well culture supernatants showed that these second-round subclones all produced 98–124 μg / mL, an increase of at least twofold over the first-round subclones. These 653 cell lines were assigned C-code designations, as shown in Table 5.

[0291] Three of the highest-producing Sp2 / 0 parental lines from rTNV148B transfection 1 were subcloned. Two rounds of subcloning of parental line 1.73 led to the identification of a clone that produced 25 μg / mL in spent 24-well cultures. This Sp2 / 0 cell line was designated C467A (Table 5).

[0292] Highest producing rTNV14 cell line Three of the highest-producing Sp2 / 0 parental lines from rTNV14 transfection 3 were subcloned once. Subclone 3.27-1 was found to be the highest producer in spent 24-well cultures, with a production of 19 μg / mL. This cell line was designated C476A (Table 5).

[0293] Table 5. Summary of selected generating cell lines and their C codes. The first digit of the original clone name indicates which transfection the cell line was derived from. All of the C-encoded cell lines reported here were derived from transfections with restriction enzyme-linearized heavy and light chain whole plasmids.

[0294] [Table 6]

[0295] Characterization of subcloned cell lines To more carefully characterize the growth characteristics of the cell lines and determine mAb production levels on a large scale, growth curve analyses were performed using T75 cultures. Results showed that each of the four C466 series of cell lines produced mAb at 1.0 × 10 6 ~1.25×10 6 The results showed that the cells reached a peak cell density of 1.0 × 10 cells / mL and a maximum mAb accumulation level of 110–140 μg / mL (Figure 7). In contrast, the highest producing Sp2 / 0 subclone, C467A, reached 2.0 × 10 cells / mL. 6 A peak cell density of 100 cells / mL and a maximum mAb accumulation level of 25 μg / mL were reached (Figure 7). Growth curve analysis was not performed on the rTNV14-producing cell line C476A.

[0296] Further growth curve analysis was performed to compare growth rates at different concentrations of MHX selection. This comparison was prompted by recent observations that C466 cells cultured in the absence of MHX appeared to grow faster than the same cells cultured in normal amounts of MHX (1x). Because cytotoxic concentrations of compounds such as mycophenolic acid tend to be measured over several orders of magnitude, it was thought possible to significantly speed up cell doubling times without sacrificing the stability of mAb production by using lower concentrations of MHX. Cell lines C466A and C466B were cultured either without MHX, with 0.2x MHX, or with 1x MHX. Viable cell counts were performed at 24-h intervals for 7 days. Results revealed MHX concentration-dependent cell growth rates (Figure 8). Cell line C466A exhibited a doubling time of 25.0 h in 1x MHX but only 20.7 h without MHX. Similarly, cell line C466B exhibited a doubling time of 32.4 h in 1x MHX but only 22.9 h without MHX. Importantly, the doubling times of both cell lines in 0.2x MHX were more similar to those observed without MHX than in 1x MHX (Figure 8). This observation suggests that enhanced cell performance may be achieved by using less MHX in bioreactors where doubling time is a critical parameter. However, while stability study results (see below) suggest that cell line C466D is capable of stably producing rTNV148B for at least 60 days in the absence of MHX, the stability study also demonstrated higher mAb production levels when cells were cultured in the presence of MHX compared to the absence of MHX.

[0297] Stability studies were performed on cultures with or without MHX selection to assess mAb production from various cell lines over a period of approximately 60 days. Not all cell lines maintained high mAb production. After just 2 weeks of culture, production from clone C466A was approximately 45% lower than at the start of the study. Production from clone C466B also appeared to be significantly reduced. However, clones C466C and C466D maintained fairly stable production, with C466D exhibiting the highest absolute production level (Figure 9).

[0298] conclusion From an initial panel of eight human mAbs against human TNFα, TNV148B and TNV14 were selected as preferred based on several criteria, including protein sequence and TNF-neutralizing potency. Cell lines producing >100 μg / mL rTNV148B and >19 μg / mL rTNV14 were prepared.

[0299] Example 5: Testing of arthritic mice with anti-TNF antibodies and controls using a single bolus injection Approximately 4-week-old Tg197 test mice were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148, or TNV196) at either 1 mg / kg or 10 mg / kg.

[0300] Results: When body weight was analyzed as a change from pre-treatment, animals treated with 10 mg / kg cA2 consistently gained more weight throughout the study than animals treated with D-PBS. This weight gain was significant from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved significant weight gain by week 7 of the study. (See Figure 10.)

[0301] Figures 11A-11C show the progression of disease severity based on the arthritic index. The arthritic index for the 10 mg / kg cA2 treatment group was lower than that of the D-PBS control group, starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to demonstrate a significant decrease in AI after week 3 when compared to the D-PBS treatment group. There were no significant differences between the 10 mg / kg treatment groups when compared to similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196). When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 demonstrated significantly lower AIs than 1 mg / kg cA2 at weeks 3, 4, and 7. TNV148 at 1 mg / kg was also significantly lower than the 1 mg / kg TNV14-treated group at weeks 3 and 4. TNV196 showed a significant reduction in AI (when compared with the D-PBS-treated group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study.

[0302] Example 6: Study of arthritic mice using anti-TNF antibodies and controls as multiple bolus doses Approximately 4-week-old Tg197 test mice were assigned to one of eight treatment groups based on body weight and treated with an intraperitoneal bolus of control article (D-PBS) or 3 mg / kg of antibody (TNV14, TNV148) (week 0). Injections were repeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for test article efficacy. Serum samples obtained from animals in groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3, and 4.

[0303] Results: No significant differences were observed when body weight was analyzed as a change from pre-treatment. Animals treated with 10 mg / kg cA2 consistently gained more weight than animals treated with D-PBS throughout the study (see Figure 12).

[0304] Figures 13A-13C show the progression of disease severity based on the arthritis index. The arthritis index for the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group, starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS-treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and were also significantly lower than animals treated with TNV14 from weeks 3 to 5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2.

[0305] Example 7: Study in arthritic mice using anti-TNF antibodies and controls as a single intraperitoneal bolus Approximately 4-week-old Tg197 test mice were assigned to one of six treatment groups based on sex and weight and treated with a single intraperitoneal bolus of either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148). The study utilized D-PBS and 10 mg / kg cA2 control groups.

[0306] When body weight was analyzed as a change from pre-treatment, all treatments achieved similar weight gain. Animals treated with either 3 or 5 mg / kg TNV148 or 5 mg / kg cA2 significantly increased their body weight early in the study (weeks 2 and 3). Only animals treated with TNV148 maintained significant weight gain at later time points. Animals treated with both 3 and 5 mg / kg TNV148 showed significant gains at week 7, and animals treated with 3 mg / kg TNV148 were still significantly elevated 8 weeks after injection (see Figure 14).

[0307] Figure 15 depicts the progression of disease severity based on the arthritis index. All treatment groups showed some protection at early time points, with 5 mg / kg cA2 and 5 mg / kg TNV148 showing significant reductions in AI at weeks 1-3, and all treatment groups showing significant reductions at week 2. Later in the study, animals treated with 5 mg / kg cA2 showed some protection, with significant reductions at weeks 4, 6, and 7. Both low-dose (3 mg / kg) cA2 and TNV148 showed significant reductions at week 6, and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain significant reductions at the end of the study (week 8). There were no significant differences between any of the treatment groups (except the saline control group) at any time point.

[0308] Example 8: Study in arthritic mice using anti-TNF antibodies and controls as a single intraperitoneal bolus between anti-TNF antibodies and modified anti-TNF antibodies To compare the efficacy of a single intraperitoneal injection of TNV148 (derived from hybridoma cells) and rTNV148B (derived from transfected cells), approximately 4-week-old Tg197 test mice were assigned to one of nine treatment groups based on sex and weight and treated with Dulbecco's S PBS (D-PBS) or a single intraperitoneal bolus of 1 mg / kg antibody (TNV148, rTNV148B).

[0309] When body weight was analyzed as a change from pre-treatment, animals treated with 10 mg / kg cA2 consistently gained more weight throughout the study than animals treated with D-PBS. This weight gain was significant at week 1 and weeks 3-8. Animals treated with 1 mg / kg TNV148 also achieved significant weight gain at weeks 5, 6, and 8 of the study. (See Figure 16.)

[0310] Figure 17 depicts the progression of disease severity based on the arthritic index. The arthritic index for the 10 mg / kg cA2 treatment group was lower than the D-PBS control group starting at week 4 and continuing throughout the remainder of the study (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant reduction in AI at week 4. While a previous study (P-099-017) showed that TNV148 was slightly more effective in reducing the arthritic index after a single 1 mg / kg intraperitoneal bolus, this study showed slightly higher AIs from groups treated with both versions of the TNV antibody. The group treated with 1 mg / kg cA2 (except at week 6) did not have a significant increase when compared with the 10 mg / kg cA2 group, and the group treated with TNV148 was significantly higher at weeks 7 and 8, but there were no significant differences in AI between 1 mg / kg cA2, 1 mg / kg TNV148, and 1 mg / kg TNV148B at any time point in the study.

[0311] Example 9: Anti-TNF antibodies for the treatment or prevention of ankylosing spondylitis overview A multicenter, randomized, double-blind, placebo-controlled study of the anti-TNFα monoclonal antibody golimumab administered intravenously in subjects with active ankylosing spondylitis.

[0312] SIMPONI® (golimumab) is a fully human monoclonal antibody with the immunoglobulin G1 (IgG1) heavy chain isotype (G1m[z] allotype) and the kappa light chain isotype. Golimumab has a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37. The molecular weight of golimumab ranges from 149,802 to 151,064 daltons. Golimumab binds to human tumor necrosis factor alpha (TNFα) with high affinity and specificity and neutralizes TNFα biological activity.

[0313] Objectives and Hypothesis Main purpose The primary objective of this study is to evaluate the efficacy of IV golimumab 2 mg / kg in subjects with active ankylosing spondylitis (AS) by assessing the reduction of signs and symptoms of AS.

[0314] secondary purpose Secondary objectives of golimumab are to evaluate: Efficacy related to improvements in physical function, range of motion, health-related quality of life, and other health outcomes ·Safety Pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity

[0315] hypothesis To address the primary objective of the study, the statistical hypothesis (alternative hypothesis) is that IV golimumab 2 mg / kg is statistically superior to placebo in reducing signs and symptoms in subjects with active AS based on the primary efficacy endpoint.

[0316] The primary endpoint of this study is the proportion of subjects achieving a 20% improvement from baseline on the Ankylosing Spondylitis Assessment (ASAS) International Working Group scale (referred to as ASAS20) at week 16. This endpoint was chosen because it is well accepted by regulatory authorities and the clinical AS community.

[0317] Study design overview This is a Phase 3, multicenter, randomized, double-blind, placebo-controlled study comparing the efficacy and safety of IV golimumab with placebo in subjects with active AS who have an inadequate response to or are intolerant to NSAIDs. Approximately 200 subjects will be randomized at approximately 40 investigational sites. All subjects will be randomly assigned to receive either golimumab 2 mg / kg or placebo via IV infusion at Week 0, Week 4, and every 8 weeks (q8w) thereafter through Week 52. At Week 16, all subjects receiving placebo infusions will cross over and begin receiving IV golimumab infusions.

[0318] Subjects in the golimumab IV treatment group will continue to receive golimumab IV infusion. Database lock will be scheduled at Weeks 28 and 60. Subjects will be followed for adverse events (AEs) and serious adverse events (SAEs) for at least 8 weeks after the last study treatment administration. The end of the study will be defined as when the last subject completes the 60-week visit.

[0319] Target population Eligible subjects were men or women aged 18 years or older with a diagnosis of AS for at least 3 months, defined as "definite" by the revised New York criteria, and symptoms of active disease, as evidenced by a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) of ≥ 4 and a Visual Analogue Scale (VAS) of ≥ 4 for total back pain on a 0-10 cm scale, respectively. Subjects were required to have a C-reactive protein (CRP) level of ≥ 0.3 mg / dL.

[0320] Other key characteristics of the study population were: Current users of methotrexate (MTX), sulfasalazine (SSZ), and hydroxychloroquine (HCQ) and low-dose oral corticosteroids are tolerated and should begin the study on stable doses of these medications. Subjects with prior exposure to no more than one biologic anti-TNFα agent (other than golimumab) will be accepted for enrollment in this study, but will be limited to a maximum of 20% of the study population. Subjects with complete spinal ankylosis, defined as the presence of bridging syndesmotic spur formation at all intervertebral levels of the cervical and lumbar spine visualized on lateral spinal radiographs, will be accepted for inclusion in this study but will be limited to a maximum of 10% of the study population.

[0321] Screening for eligible subjects will occur within 6 weeks prior to administration of study drug.

[0322] Subjects must also meet the inclusion and exclusion criteria.

[0323] Dosage and Administration At the initial screening visit, all subjects deemed potentially eligible for the study will provide informed consent according to the protocol-specified inclusion and exclusion criteria for enrollment in the study. At the randomization visit, subjects will be reassessed, and if all specified inclusion and exclusion criteria are met, they will be randomized to receive either golimumab IV infusion or placebo IV infusion. Randomization will be stratified by geographic region and prior use of anti-TNFα therapy.

[0324] Prior to the first test infusion, subjects will be randomly assigned in a 1:1 ratio to one of the following two treatment groups: Group 1 (n=100): Subjects receive IV placebo infusions at weeks 0, 4, and 12. Subjects cross over to IV golimumab 2 mg / kg at week 16 and receive doses at weeks 16, 20, and q8w thereafter. Group 2 (n=100): Subjects receive 2 mg / kg IV golimumab at weeks 0, 4, and q8w thereafter. Subjects receive an IV placebo infusion at week 16 to maintain blinding.

[0325] All infusions are completed over 30±10 minutes.

[0326] Efficacy assessment / endpoint The efficacy assessments selected for this study were established in previous trials of therapeutic biologic agents for the treatment of AS. The patient-reported outcomes (PROs) selected for this study are consistent with clinically relevant measurements accepted in the medical literature for other trials of AS and applicable US / EU regulatory guidance documents.

[0327] Ankylosing spondylitis response assessments include: ·Bath Ankylosing Spondylitis Functional Index (BASFI) Patient's overall rating ·All back pain ·Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) Short-Form 36-Item Health Survey (SF-36) ·Bath Ankylosing Spondylitis Measurement Index (BASMI) Ankylosing Spondylitis Quality of Life (ASQoL) Questionnaire Chest expansion Back pain at night Enthesitis index Medical Outcomes Study Sleep Scale Work Limitations Questionnaire (WLQ) Productivity Visual Analogue Scale EuroQol-5D (EQ-5D) questionnaire

[0328] Primary endpoint The primary endpoint of this study is the proportion of ASAS20 responders at week 16. A study will be considered positive if it demonstrates that the proportion of subjects with ASAS20 at week 16 is statistically significantly greater in the golimumab group compared to the placebo group.

[0329] Key secondary endpoints The following major secondary analyses will be performed: Endpoints are listed in order of importance as specified below. 1. The proportion of subjects achieving ASAS40 at week 16. 2. The proportion of subjects achieving at least a 50% improvement from baseline in BASDAI at Week 16. 3. Change from baseline in BASFI at week 16.

[0330] Pharmacokinetic evaluation Blood samples will be collected at selected visits to evaluate the PK of IV golimumab in adult subjects with AS. If study drug is administered at that visit, the pharmacokinetic sample should be drawn from a different arm than the IV infusion line. Specifically, at visits 0, 4, 12, 20, 36, and 52, two samples for serum golimumab concentration will be collected: one sample will be collected immediately before the infusion and the other will be collected 1 hour after the end of the infusion. For each of the remaining visits, only one sample for serum golimumab concentration will be collected. This sample should be collected before the infusion if a study drug infusion is administered at that visit. Random PK samples will also be drawn for population PK analysis at visits between Weeks 12 and 20, except at Weeks 12, 16, or 20. This sample should be collected at least 24 hours before or after the study drug infusion. Where applicable, serum for measurements of both golimumab concentration and antibodies to golimumab will be derived from the same blood draw.

[0331] Immunogenicity assessment To assess the immunogenicity of golimumab in adult subjects with AS, serum samples for detection of antibodies to golimumab will be collected according to a time and event schedule.

[0332] Biomarker evaluation Biomarker samples are collected to gain a molecular understanding of inter-individual variability in clinical outcomes, which may help identify population subgroups that respond differently to drugs. Biomarker samples may be used to help address emerging questions and enable the development of safer, more effective, and ultimately personalized therapies in the future.

[0333] Pharmacogenetic (DNA) evaluation Genomic testing will be conducted to study the link between specific genes and disease or drug response. DNA studies related only to golimumab or to the disease for which this drug was developed will be conducted. Genome-wide pharmacogenetic and / or epigenetic testing will be conducted in consenting subjects in this study. Subjects participating in this portion of the study will be required to sign a separate informed consent form. Furthermore, subjects may withdraw such consent at any time without affecting their participation in other aspects of the study or their future participation in the study.

[0334] Pharmacogenetic blood samples will be collected to enable pharmacogenetic studies as needed (local regulations permitting). Subject participation in pharmacogenetic studies is voluntary.

[0335] Safety evaluation Based on the safety profiles of other anti-TNFα agents and the golimumab safety data to date, several AEs of interest have been identified and will be monitored and evaluated in this study, including infusion reactions, hepatobiliary test abnormalities, infections including TB, and malignancies.

[0336] statistical methods Most data will be summarized using simple descriptive summary statistics such as n, mean, SD, median, IQ range, minimum, and maximum for continuous variables, and counts and percentages for discrete variables.

[0337] Categorical variables, such as the proportion of subjects responding to treatment, are compared using the Cochran-Mantel-Haenszel (CMH) test stratified by prior use of anti-TNFα therapy. Generally, prior ANOVA, using anti-TNFα therapy as a factor, is used to analyze continuous variables unless otherwise specified. All statistical tests are performed with an α of 0.05 (two-tailed). In addition to statistical analysis, graphical data displays (eggs, line graphs) and subject lists can also be used to summarize / present data.

[0338] Group Set The population set is the intention-to-treat population (i.e., all randomized subjects). Subjects included in the efficacy analysis will be summarized according to their assigned treatment group, regardless of whether they received their assigned treatment.

[0339] Safety and PK analyses will include all subjects who received at least one dose of study treatment.

[0340] Endpoint Analysis Primary endpoint analysis To address the primary objective, the proportion of subjects with an ASAS20 response at Week 16 (primary endpoint) will be compared between the placebo and golimumab groups using a stratified CMH test (Yes / No) prior to the use of anti-TNFα therapy at a significance level of 0.05 (two-sided test). In this primary efficacy analysis, data from all randomized subjects will be analyzed according to their assigned treatment group, regardless of whether they received their actual treatment. If a subject has data for at least one ASAS component at Week 16, a last observation carried forward (LOCF) procedure will be used to allocate missing ASAS components. If a subject does not have data for all ASAS components at Week 16, the subject will be considered a non-responder.

[0341] Primary secondary endpoint analysis The following primary secondary analyses are conducted in order of importance as specified below. 1. Compare the proportion of subjects achieving ASAS40 at week 16 between treatment groups. 2. The proportion of subjects achieving at least a 50% improvement from baseline in the BASDAI at Week 16 will be compared between treatment groups. 3. Compare the change from baseline in BASFI at week 16 between treatment groups.

[0342] To control for Type I error rates for multiplicity, the first major secondary endpoint will be tested only if the primary endpoint achieves statistical significance at the 0.05 significance level (two-sided). Subsequent major secondary endpoints will be tested only if the primary endpoint and the preceding major secondary endpoints are statistically significant at the 0.05 significance level (two-sided).

[0343] Safety analysis overview Routine safety assessments will be performed. The occurrence and type of AEs, SAEs, and reasonably related AEs, including infusion reactions and infections, including TB, will be summarized by treatment group. The number of subjects with abnormal laboratory parameters (hematology and chemistry) based on NCI CTCAE toxicity grades will be summarized. Additionally, the number of subjects with ANA and anti-dsDNA antibodies will be summarized, as well as the relationship between antibodies to golimumab and infusion reactions.

[0344] All safety analyses will be performed using the population of all subjects who received at least one dose of study medication. Analyses will be performed using the treatments that subjects actually received.

[0345] Additionally, graphical data displays (eggs, line graphs) and object lists can also be used to summarize / present the data.

[0346] Abbreviation: AE Adverse Event AS ankylosing spondylitis ASAS20 Ankylosing Spondylitis Assessment 20 ASQoL Ankylosing Spondylitis Quality of Life ASSERT Ankylosing Spondylitis Trial for the Evaluation of Recombinant Infliximab Therapy BASDAI Bath Ankylosing Spondylitis Disease Activity Index BASFI Bath Ankylosing Spondylitis Functional Index BASMI Bath Ankylosing Spondylitis Measurement Index BCG Bacillus Calmette-Guerin CHF Congestive Heart Failure CMH Cochran-Mantel-Haenszel CRP C-reactive protein DAS Disease Activity Score DBL Database Lock DMARDs Disease-modifying antirheumatic drugs DMC Data Monitoring Committee DNA deoxyribonucleic acid ECG electrocardiogram eCRF Electronic Case Reporting Form eDC Electronic Data Capture EQ-5D EuroQol-5D EQ VAS EQ Visual Analogue Scale EU European Union GCP Good Clinical Practice HBV Hepatitis B virus HCQ Hydroxychloroquine HCV Hepatitis C virus HIV human immunodeficiency virus HRQOL Health-related quality of life IB Investigational Drug Brochure ICF Informed Consent Form ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IEC Independent Ethics Committee IgG1 immunoglobulin G1 IMA Independent Musculoskeletal Assessment Factor IRB Institutiona...

Claims

1. 1. A pharmaceutical composition for use in a method of treating active ankylosing spondylitis in a patient with early stage disease for a period of <2 years, said pharmaceutical composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs:40-42 and light chain CDR amino acid sequences SEQ ID NOs:43-45, said method comprising the step of administering said anti-TNF antibody to said patient, wherein after 16 weeks of treatment, said patient achieves an outcome selected from the group consisting of Ankylosing Spondylitis Assessment 20 (ASAS20), Ankylosing Spondylitis Assessment 40 (ASAS40), at least a 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and said outcome is maintained or improved up to 52 weeks of treatment.

2. 2. The pharmaceutical composition of claim 1, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg of the patient's body weight at weeks 0 and 4, then every 8 weeks (q8w) thereafter, such that the anti-TNF antibody is administered by intravenous infusion (IV) over 30±10 minutes.

3. 2. The pharmaceutical composition of claim 1, wherein the anti-TNF antibody comprises a variable heavy chain amino acid sequence of SEQ ID NO:38 and a variable light chain amino acid sequence of SEQ ID NO:

39.

4. The pharmaceutical composition of claim 1 , wherein the anti-TNF antibody comprises a heavy chain amino acid sequence of SEQ ID NO:36 and a light chain amino acid sequence of SEQ ID NO:

37.

5. The pharmaceutical composition of claim 1 , wherein the anti-TNF antibody is a golimumab antibody.

6. The pharmaceutical composition of claim 5, wherein the method further comprises administering the anti-TNF antibody together with methotrexate (MTX), sulfasalazine (SSZ) and / or hydroxychloroquine (HCQ).

7. A pharmaceutical composition for use in a method of treating active ankylosing spondylitis in a patient with early stage disease for a period of <2 years, said pharmaceutical composition comprising an anti-TNF antibody comprising heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 40-42 and light chain CDR amino acid sequences SEQ ID NOs: 43-45, said method comprising the step of administering said anti-TNF antibody to said patient, wherein after 16 weeks of treatment there is an improvement in patient reported outcomes in terms of ankylosing spondylitis quality of life (ASQoL), back pain at night and total back pain, and said improvement in patient reported outcomes is maintained or continues to improve over 52 weeks of treatment in ASQoL, back pain at night and total back pain.

8. 8. The pharmaceutical composition of claim 7, wherein the anti-TNF antibody is administered at a dose of 2 mg per kg of the patient's body weight at weeks 0 and 4, then every 8 weeks (q8w) thereafter, such that the anti-TNF antibody is administered by intravenous infusion (IV) over 30±10 minutes.

9. 8. The pharmaceutical composition of claim 7, wherein the anti-TNF antibody comprises a variable heavy chain amino acid sequence of SEQ ID NO:38 and a variable light chain amino acid sequence of SEQ ID NO:

39.

10. The pharmaceutical composition of claim 7 , wherein the anti-TNF antibody comprises a heavy chain amino acid sequence of SEQ ID NO:36 and a light chain amino acid sequence of SEQ ID NO:

37.

11. The pharmaceutical composition of claim 7 , wherein the anti-TNF antibody is a golimumab antibody.

12. The pharmaceutical composition of claim 11, wherein the method further comprises administering the anti-TNF antibody together with methotrexate (MTX), sulfasalazine (SSZ) and / or hydroxychloroquine (HCQ).

13. 1. A pharmaceutical composition for use in a method of treating active ankylosing spondylitis in a patient with early stage disease for a period of <2 years, said pharmaceutical composition comprising an anti-TNF antibody comprising a variable heavy chain amino acid sequence of SEQ ID NO:38 and a variable light chain amino acid sequence of SEQ ID NO:39, said method comprising administering to said patient said anti-TNF antibody composition by intravenous infusion (IV), whereby said anti-TNF antibody is administered at a dose of 2 mg / kg of said patient's body weight at weeks 0 and 4, then every 8 weeks (q8w) thereafter. over 30±10 minutes, wherein after 16 weeks of treatment, the patient achieves an outcome selected from the group consisting of Ankylosing Spondylitis Assessment 20 (ASAS20), Ankylosing Spondylitis Assessment 40 (ASAS40), at least 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and said outcome is maintained or improved up to 52 weeks of treatment.

14. The pharmaceutical composition of claim 13 , wherein the anti-TNF antibody comprises a heavy chain amino acid sequence of SEQ ID NO:36 and a light chain amino acid sequence of SEQ ID NO:

37.

15. The pharmaceutical composition of claim 13, wherein the anti-TNF antibody is a golimumab antibody.

16. The pharmaceutical composition of claim 13, wherein the method further comprises administering the composition together with methotrexate (MTX), sulfasalazine (SSZ) and / or hydroxychloroquine (HCQ).

17. 1. A composition comprising an anti-TNF antibody for use in the treatment of active ankylosing spondylitis in patients with early stage disease for a period of <2 years, wherein the anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs:40-42 and light chain CDR amino acid sequences SEQ ID NOs:43-45, and wherein after 16 weeks of treatment the patient achieves an outcome selected from the group consisting of Ankylosing Spondylitis Assessment 20 (ASAS20), Ankylosing Spondylitis Assessment 40 (ASAS40), at least 50% improvement from baseline in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and Ankylosing Spondylitis Disease Activity Score (ASDAS) inactive disease (<1.3), and wherein the outcome is maintained or improved up to 52 weeks of treatment.

18. The composition of claim 17 , wherein the anti-TNF antibody comprises a variable heavy chain amino acid sequence of SEQ ID NO:38 and a variable light chain amino acid sequence of SEQ ID NO:

39.

19. The composition of claim 17 , wherein the anti-TNF antibody comprises a heavy chain amino acid sequence of SEQ ID NO:36 and a light chain amino acid sequence of SEQ ID NO:

37.

20. The composition of claim 17, wherein the anti-TNF antibody is a golimumab antibody.

21. 18. The composition of claim 17, wherein the anti-TNF antibody is administered at a dose of 2 mg per kg of the patient's body weight by intravenous infusion (IV) administered over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter.

22. 18. The composition of claim 17, further comprising methotrexate (MTX), sulfasalazine (SSZ) and / or hydroxychloroquine (HCQ).

23. 1. A composition comprising an anti-TNF antibody for use in the treatment of active ankylosing spondylitis in patients with early stage disease for a period of <2 years, wherein said anti-TNF antibody comprises heavy chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 40-42 and light chain CDR amino acid sequences SEQ ID NOs: 43-45, and after 16 weeks of treatment there is an improvement in patient reported outcomes in terms of ankylosing spondylitis quality of life (ASQoL), back pain at night and total back pain, and said improvement in patient reported outcomes is maintained or continues to improve over 52 weeks of treatment in ASQoL, back pain at night and total back pain.

24. The composition of claim 23 , wherein the anti-TNF antibody comprises a variable heavy chain amino acid sequence of SEQ ID NO:38 and a variable light chain amino acid sequence of SEQ ID NO:

39.

25. The composition of claim 23 , wherein the anti-TNF antibody comprises the heavy chain amino acid sequence of SEQ ID NO:36 and the light chain amino acid sequence of SEQ ID NO:

37.

26. 24. The composition of claim 23, wherein the anti-TNF antibody is a golimumab antibody.

27. 24. The composition of claim 23, wherein the anti-TNF antibody is administered at a dose of 2 mg per kg of the patient's body weight by intravenous infusion (IV) administered over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter.

28. 28. The composition of claim 27, further comprising methotrexate (MTX), sulfasalazine (SSZ) and / or hydroxychloroquine (HCQ).

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