Methods of manufacture of compositions for the production of anti-TNF antibodies

文档序号：440617 发布日期：2021-12-24 浏览：25次中文

阅读说明：本技术 用于产生抗tnf抗体组合物的制造方法 (Methods of manufacture of compositions for the production of anti-TNF antibodies ) 是由 K·巴尼豪斯 S·甘古利 M·格罗尼韦尔 M·A·小洛佩兹 M·尼德维德 K·D·史密斯于 2020-02-26 设计创作，主要内容包括：本发明涉及用于产生抗TNF抗体例如该抗TNF抗体是具有包含氨基酸序列SEQ ID NO：36的重链(HC)和包含氨基酸序列SEQ ID NO：37的轻链(LC)的重组抗TNF抗体的制造方法以及包含该重组抗TNF抗体的组合物。(The present invention relates to methods for producing anti-TNF antibodies, e.g., antibodies having a sequence comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (HC) comprising the amino acid sequence SEQ ID NO: 37, and a composition comprising the recombinant anti-TNF antibody.)

1. An anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells).

2. The anti-TNF antibody of claim 1, wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% of total neutral oligosaccharide species and < 1.0% of total charged oligosaccharide species.

3. The anti-TNF antibody of claim 1, wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the single neutral oligosaccharide species G0F, < 20.0% G1F, and < 5.0% G2F.

4. The anti-TNF antibody of claim 1, wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis (RMA).

5. The anti-TNF antibody of any one of claims 1 to 4, wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) compared to an anti-TNF antibody expressed in Sp2/0 cells.

6. The anti-TNF antibody of claim 5, wherein the anti-TNF antibody is a subsequent biologic.

7. A manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising:

a. culturing chinese hamster ovary cells (CHO cells) having nucleotides encoding the anti-TNF antibody;

b. expressing the anti-TNF antibody in the CHO cell; and the number of the first and second groups,

c. purifying the anti-TNF antibody.

8. The method of manufacturing according to claim 7, wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% total neutral oligosaccharide species and < 1.0% total charged oligosaccharide species, and.

9. The manufacturing process according to claim 7, wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the individual neutral oligosaccharide species G0F, < 20.0% of G1F and < 5.0% of G2F.

10. The method of manufacture of claim 7, wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis (RMA).

11. The production method according to any one of claims 7 to 10, wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to an anti-TNF antibody expressed in Sp2/0 cells.

12. The method of manufacturing of claim 11, wherein the anti-TNF antibody is a subsequent biologic.

13. A composition comprising an anti-TNF antibody, the anti-TNF antibody comprising:

(i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells).

14. The composition of claim 13, wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% of total neutral oligosaccharide species and < 1.0% of total charged oligosaccharide species.

15. The composition of claim 13, wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the single neutral oligosaccharide species G0F, < 20.0% G1F, and < 5.0% G2F.

16. The composition of claim 13, wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC).

17. The composition of any one of claims 13 to 16, wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to an anti-TNF antibody expressed in Sp2/0 cells.

18. The composition of claim 17, wherein the anti-TNF antibody is a subsequent biologic.

Technical Field

The present invention relates to methods for producing anti-TNF antibodies, e.g., antibodies having a sequence comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (HC) comprising the amino acid sequence SEQ ID NO: 37, and a composition comprising the recombinant anti-TNF antibody.

Background

TNF α is a soluble homotrimer of 17kD protein subunits. There is also a membrane bound TNF in the form of the 26kD precursor.

Cells other than monocytes or macrophages also produce TNF α. For example, human non-monocytic tumor cell lines produce TNF α as well as CD4+ and CD8+ peripheral blood T lymphocytes, and some cultured T cell lines and B cell lines also produce TNF α.

TNF α causes proinflammatory effects that lead to tissue damage such as degradation of cartilage and bone, induction of adhesion molecules, induction of procoagulant activity on vascular endothelial cells, increase adhesion of neutrophils and lymphocytes, and stimulation of macrophage, neutrophils, and vascular endothelial cells to release platelet activating factor.

TNF α has long been associated with infections, immune disorders, tumor pathology, autoimmune pathology, and graft versus host pathology. The association of TNF α with cancer and infectious pathologies is generally associated with the catabolic state of the host. Weight loss in cancer patients is often associated with anorexia.

The apparent wasting associated with cancer and other diseases is called "cachexia". Cachexia includes progressive weight loss, anorexia, and persistent erosion of lean body mass by malignant growth. Cachexia results in a number of cancer morbidity and mortality. There is evidence that TNF α is associated with cachexia in cancer, infectious pathologies, and other catabolic states.

TNF α is believed to play an important role in gram-negative sepsis and endotoxic shock (including fever, malaise, anorexia, and cachexia). Endotoxin strongly activates monocyte/macrophage production and secretion of TNF α and other cytokines. TNF α and other monocyte-derived cytokines mediate metabolic and neurohormonal responses to endotoxin. Administration of endotoxin to human volunteers produces acute illness with symptoms similar to influenza, including fever, tachycardia, increased metabolic rate and stress hormone release. Circulating TNF α increases in patients with gram-negative sepsis.

Thus, TNF α is implicated in inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections, malignancies and/or neurodegenerative diseases and is a useful target for specific biotherapies of diseases such as rheumatoid arthritis and crohn's disease. The beneficial effects in open label assays using monoclonal antibodies against TNF α have been reported to be inhibition of inflammation and successful retreatment after recurrence of rheumatoid arthritis and crohn's disease. The beneficial effects of inhibiting inflammation in rheumatoid arthritis in randomized, double-blind, placebo-controlled trials have also been reported.

Antisera or mabs that have been shown to neutralize TNF in mammals other than humans can eliminate adverse physiological changes and prevent death following lethal dose challenge with experimental endotoxemia and bacteremia. This effect has been demonstrated in, for example, rodent lethality assays and primate pathology model systems.

The putative receptor binding locus for hTNF has been disclosed, and the receptor binding locus for TNF α, consisting of amino acids 11-13, 37-42, 49-57 and 155-157 of TNF, has been disclosed.

Non-human mammals, chimeric antibodies, polyclonal antibodies (e.g., antisera) and/or monoclonal antibodies (mabs) as well as fragments (e.g., proteolytic digestions or fusion protein products thereof) are potential therapeutic agents in some cases being investigated in an attempt to treat certain diseases. However, such antibodies or fragments may elicit an immune response when administered to a human. Such immune responses can lead to immune complex-mediated clearance of the antibody or fragment from circulation and render repeated administration unsuitable for treatment, thereby reducing the 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 moieties can result in seropathy and/or allergic reactions. To avoid these and other problems, various approaches have been taken to reduce the immunogenicity of such antibodies and portions thereof, including chimerization and humanization, which are well known in the art. However, these and other methods may still result in antibodies or fragments that are somewhat immunogenic, low affinity, low avidity, or problematic in cell culture, scale-up, production, and/or low yield. Thus, such antibodies or fragments may be less suitable for manufacture or use as therapeutic proteins.

Accordingly, there is a need to provide anti-TNF antibodies or fragments thereof for use as therapeutic agents in the treatment of diseases mediated by TNF α.

Disclosure of Invention

Embodiments of the invention are defined by the independent and dependent claims, respectively, appended hereto, which are incorporated by reference herein for the sake of brevity. Other embodiments, features, and advantages of various aspects of the present invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% of total neutral oligosaccharide species and > 1.0% of total charged oligosaccharide species, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the individual neutral oligosaccharide species G0F, < 20.0% of G1F, and < 5.0% of G2F, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to an anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis (RMA), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and the anti-TNF antibody is a subsequent biologic.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the individual neutral oligosaccharide species G0F, < 20.0% of G1F, and < 5.0% of G2F, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and wherein the anti-TNF antibody is a subsequent biologic.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis (RMA), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and the anti-TNF antibody is a subsequent biologic.

In certain embodiments, the invention provides an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and the anti-TNF antibody is a subsequent biologic.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% total neutral oligosaccharide species and < 1.0% total charged oligosaccharide species, and

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the single neutral oligosaccharide substance G0F, < 20.0% of G1F and < 5.0% of G2F.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the anti-TNF antibody does not have a disialylated glycan species, as determined by High Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis (RMA).

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% of total neutral oligosaccharide species and > 1.0% of total charged oligosaccharide species, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the single neutral oligosaccharide substance G0F, < 20.0% G1F and < 5.0% G2F, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis (RMA), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a manufacturing method for producing an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is produced by a manufacturing method comprising the steps of: a. culturing chinese hamster ovary cells (CHO cells) having a nucleotide encoding the anti-TNF antibody; b. expressing the anti-TNF antibody in CHO cells; purifying the anti-TNF antibody, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in the Sp2/0 cells, the anti-TNF antibody being a subsequent biologic.

In certain embodiments, the invention provides a composition comprising an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% of total neutral oligosaccharide species and > 1.0% of total charged oligosaccharide species, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a composition comprising an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the individual neutral oligosaccharide species G0F, < 20.0% of G1F, and < 5.0% of G2F, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to an anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a composition comprising an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a composition comprising an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells.

In certain embodiments, the invention provides a composition comprising an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the oligosaccharide profile of the anti-TNF antibody further comprises > 60.0% of the individual neutral oligosaccharide species G0F, < 20.0% of G1F, and < 5.0% of G2F, and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and wherein the anti-TNF antibody is a subsequent biologic.

In certain embodiments, the invention provides a composition comprising an anti-TNF antibody comprising: (i) comprises the amino acid sequence of SEQ ID NO: 36; and (ii) comprises SEQ ID NO: 37, wherein the anti-TNF antibody is expressed in chinese hamster ovary cells (CHO cells), and wherein the anti-TNF antibody does not have a disialylated glycan species as determined by High Performance Liquid Chromatography (HPLC), and wherein the anti-TNF antibody has a longer half-life or increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the anti-TNF antibody expressed in Sp2/0 cells, and the anti-TNF antibody is a subsequent biologic.

Drawings

Figure 1 shows a graphical representation of an assay showing the ability of TNV mAb to inhibit TNF α binding to recombinant TNF receptors in hybridoma cell supernatants. Different amounts of hybridoma cell supernatants containing known amounts of TNV mAb were used at fixed concentrations (5ng/ml)¹²⁵I-labeled TNF α pre-incubation. The mixture was transferred to a 96-well optical plate that had been previously coated with p55-sf2 (recombinant TNF receptor/IgG fusion protein). After washing away unbound material and counting using a gamma counter, the amount of TNF α bound to the p55 receptor in the presence of mAb was determined. Although eight TNV mAb samples were tested in these experiments, for simplicity, the same three mabs as one of the other TNV mabs shown by DNA sequence analysis are not shown here. Each sample was tested in duplicate. The results shown represent the results of two independent experiments.

Fig. 2A to 2B show the DNA sequences of the TNV mAb heavy chain variable regions. The germline gene shown is the DP-46 gene. "TNV'" indicates that the indicated sequences are those of TNV14, TNV15, TNV148, and TNV 196. The first three nucleotides in the TNV sequence define the translation initiation Met codon. Dots in the TNV mAb gene sequence indicate that the nucleotides are identical to those in the germline sequence. The first 19 nucleotides of the TNV sequence (underlined) correspond to the oligonucleotides used for PCR amplification of the variable region. Amino acid translation (one letter abbreviation) initiated with the mature mAb is shown only for germline genes. The three CDR domains in germline amino acid translation are marked in bold and underlined. The line labeled TNV148(B) indicates that the indicated sequence involves both TNV148 and TNV 148B. The gaps in germline DNA sequences (CDR3) were due to sequences that were not known or present in the germline gene at that time. TNV mAb heavy chain uses J6 junction regions.

Figure 3 shows the DNA sequence of the TNV mAb light chain variable region. The germline genes shown are representative members of the Vg/38K family of human kappa germline variable region genes. Dots in the TNV mAb gene sequence indicate that the nucleotides are identical to those in the germline sequence. The first 16 nucleotides of the TNV sequence (underlined) correspond to the oligonucleotides used for PCR amplification of the variable region. Amino acid translation (one letter abbreviation) of the mature mAb is shown only for germline genes. The three CDR domains in germline amino acid translation are marked in bold and underlined. The line labeled TNV148(B) indicates that the indicated sequence involves both TNV148 and TNV 148B. The gaps in germline DNA sequences (CDR3) are due to sequences that are not known or present in the germline gene. TNV mAb light chain uses J3 linker sequence.

Figure 4 shows the deduced amino acid sequence of the TNV mAb heavy chain variable region. The amino acid sequences shown (single letter abbreviations) were deduced from the DNA sequences determined from the unclosed PCR products and the cloned PCR products. The amino acid sequences shown are divided into secretory signal sequences (signals), Framework (FW) and Complementarity Determining Region (CDR) domains. The amino acid sequence of the DP-46 germline gene is shown on the top row of each domain. Dots indicate that the amino acids in TNV mAb are identical to germline genes. TNV148(B) indicates that the sequence shown involves both TNV148 and TNV 148B. "TNV" indicates that the indicated sequence relates to all TNV mAbs unless a different sequence is indicated. A dash in the germline sequence (CDR3) indicates that the sequence is unknown or absent in the germline gene.

Figure 5 shows the deduced amino acid sequence of the TNV mAb light chain variable region. The amino acid sequences shown (single letter abbreviations) were deduced from the DNA sequences determined from the unclosed PCR products and the cloned PCR products. The amino acid sequences shown are divided into secretory signal sequences (signals), Framework (FW) and Complementarity Determining Region (CDR) domains. The amino acid sequence of the Vg/38K type light chain germline gene is shown on the top row of each domain. Dots indicate that the amino acids in TNV mAb are identical to germline genes. TNV148(B) indicates that the sequence shown involves both TNV148 and TNV 148B. "all" indicates that the indicated sequences relate to TNV14, TNV15, TNV148B and TNV 186.

Figure 6 shows a schematic of the heavy and light chain expression plasmids used to make C466 cells expressing rTNV 148B. p1783 is a heavy chain plasmid and p1776 is a light chain plasmid. The rTNV148B variable and constant region coding domains are shown as black boxes. The immunoglobulin enhancer in the J-C intron is shown as a grey box. Relevant restriction sites are shown. The plasmid is shown oriented so that transcription of the Ab gene proceeds in a clockwise direction. The length of plasmid p1783 was 19.53kb, and the length of plasmid p1776 was 15.06 kb. The complete nucleotide sequences of both plasmids are known. The variable region coding sequence in p1783 can be readily replaced with another heavy chain variable region sequence by replacing the BsiWI/BstBI restriction fragment. The variable region coding sequence in p1776 can be replaced by another variable region sequence by replacing the SalI/AflII restriction fragment.

Figure 7 shows a graphical representation of growth curve analysis of five cell lines producing rTNV 148B. The culture was started on day 0, and the cells were seeded into I5Q + MHX medium in T75 flasks to a viable cell density of 1.0X 10 in a 30ml volume⁵Individual cells/ml. The cell cultures used for these studies have been in continuous culture since the transfection and subcloning was performed. Over the next few days, the cells in the T-flasks were thoroughly resuspended and 0.3ml aliquots of the culture were removed. When the cell count decreased to 1.5X 10 ⁵Below individual cells/ml, growth curve studies were terminated. The number of viable cells in the aliquot was determined by trypan blue exclusion and the remainder of the aliquot was stored for later mAb concentration determination. ELISA for human IgG was performed simultaneously on all sample aliquots.

Figure 8 shows a graphical representation of a comparison of cell growth rates in the presence of different concentrations of MHX selection. Cell subclones C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2mM glutamine) and cultured for an additional 2 days. The two cell cultures were then divided into three cultures that did not contain MHX, 0.2X MHX, or 1X MHX. One day later, 1X 10 of the culture was used⁵Fresh T75 flasks were inoculated at an initial density of individual cells/ml and the cells were counted at 24 hour intervals for one week. Doubling time during the first 5 days was calculated using the formula in SOP PD32.025 and shown above the bars.

Figure 9 shows a graphical representation of the stability over time of mAb yield from two rTNV 148B-producing cell lines. Since transfection and subcloning were performed, cell subclones that had been in continuous culture were used to initiate long-term continuous culture in 24-well dishes. Cells were cultured in I5Q medium with and without MHX selection. Cells were serially passaged by separating the culture every 4 to 6 days to maintain a new viable culture while allowing the previous culture to deplete. Aliquots of spent cell supernatant were collected shortly after depletion of the culture and stored until mAb concentration was determined. ELISA for human IgG was performed simultaneously on all sample aliquots.

Figure 10 shows the weight change of the Tg197 in an arthritic mouse model mouse in response to an anti-TNF antibody of the invention compared to a control in example 4. At approximately 4 weeks of age, Tg197 study mice were assigned to one of 9 treatment groups based on gender and body weight and treated with a single intraperitoneal bolus dose of either 1mg/kg or 10mg/kg of either dulcoside phosphate buffer (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148 or TNV 196). When the weight was analyzed as a change compared to pre-dose, animals treated with 10mg/kg cA2 showed consistently higher weight gain throughout the study than animals treated with D-PBS. Body weight increased significantly at weeks 3 to 7. Animals treated with 10mg/kg TNV148 also achieved significant weight gain at study week 7.

Fig. 11A to 11C show progression of disease severity based on the arthritis index as described in example 4. The arthritis index was lower in the 10mg/kg cA2 treated group than in the D-PBS control group starting at week 3 and continuing for the remainder of the study (week 7). Animals treated with 1mg/kg TNV14 and animals treated with 1mg/kg cA2 showed no significant reduction in AI after week 3 when compared to the D-PBS treated group. There was no significant difference between each of the 10mg/kg treatment groups when compared to the other groups at similar doses (10mg/kg cA2 compared to 10mg/kg TNV14, 148 and 196). When comparing the 1mg/kg treatment groups, 1mg/kg TNV148 showed AI at 3, 4 and 7 weeks significantly below 1mg/kg cA 2. At 3 and 4 weeks, 1mg/kg TNV148 was also significantly lower than the 1mg/kg TNV14 treated group. Although TNV196 still showed a significant reduction in AI at study week 6 (when compared to the D-PBS treated group), TNV148 was the only 1mg/kg treatment that remained significant at the end of the study.

Figure 12 shows the weight change of the Tg197 in an arthritic mouse model mouse in response to an anti-TNF antibody of the invention, compared to a control in example 5. At approximately 4 weeks of age, Tg197 study mice were assigned to one of 8 treatment groups based on body weight and treated with either control preparation (D-PBS) or TNF antibody (TNV14, TNV148) at an intraperitoneal bolus dose of 3mg/kg (week 0). Injections were repeated for all animals at weeks 1, 2, 3 and 4. The test articles of groups 1-6 were evaluated for efficacy. Serum samples obtained from animals of groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3 and 4.

Fig. 13A to 13C are graphs showing the progression of disease severity in example 5 based on the arthritis index. The arthritis index of the 10mg/kg cA2 treated group was significantly lower than that of the D-PBS control group starting at week 2 and continuing for the remainder of the study (week 5). Animals treated with 1mg/kg or 3mg/kg cA2 and animals treated with 3mg/kg TNV14 failed to achieve any significant reduction in AI at any time throughout the study when compared to the d-PBS treated group. Animals treated with 3mg/kg TNV148 showed a significant decrease when compared to the d-PBS treated group starting at week 3 and continuing up to week 5. At study weeks 4 and 5, 10mg/kg cA2 treated animals showed a significant reduction in AI when compared to lower doses (1mg/kg and 3mg/kg) of cA2, and also significantly lower at weeks 3 to 5 than TNV14 treated animals. Although there did not appear to be a significant difference between any of the 3mg/kg treatment groups, the AI of animals treated with 3mg/kg TNV14 was significantly higher than 10mg/kg at some time points, while the AI of animals treated with TNV148 was not significantly different from animals treated with 10mg/kg cA 2.

Figure 14 shows the weight change of the Tg197 in an arthritic mouse model mouse in response to an anti-TNF antibody of the invention, compared to a control in example 6. At approximately 4 weeks of age, Tg197 study mice were assigned to one of 6 treatment groups based on gender and body weight and treated with a single intraperitoneal bolus dose of either 3mg/kg or 5mg/kg of antibody (cA2 or TNV 148). The study utilized D-PBS and a 10mg/kg cA2 control group.

Figure 15 shows progression of disease severity based on the arthritis index as described in example 6. All treatment groups showed some degree of protection at earlier time points, with 5mg/kg cA2 and 5mg/kg TNV148 showing significant reductions in AI at weeks 1 to 3, and all treatment groups showing significant reductions at week 2. At a later stage of the study, animals treated with 5mg/kg cA2 showed some degree of protection, with significant reductions at weeks 4, 6, and 7. The low dose of cA2 and TNV148 (3mg/kg) showed a significant reduction at week 6 and all treatment groups showed a significant reduction at week 7. At the end of the study (week 8), none of the treatment groups were able to maintain a significant reduction. There was no significant difference between any of the treatment groups (not including the saline control group) at any time point.

Figure 16 shows the weight change of the Tg197 of an arthritic mouse model mouse in response to an anti-TNF antibody of the invention compared to a control in example 7. The efficacy of a single intraperitoneal dose of TNV148 (from hybridoma cells) and rTNV148B (from transfected cells) was compared. At approximately 4 weeks of age, Tg197 study mice were assigned to one of 9 treatment groups based on gender and body weight and treated with a single intraperitoneal bolus dose of 1mg/kg of either du's phosphate buffer (D-PBS) or antibody (TNV148, rTNV 148B).

Figure 17 shows progression of disease severity based on the arthritis index as described in example 7. The arthritis index was lower in the 10mg/kg cA2 treated group than in the D-PBS control group starting at week 4 and continuing for the remainder of the study (week 8). Both the TNV148 treatment group and the 1mg/kg cA2 treatment group showed a significant reduction in AI at week 4. Although the previous study (P-099-017) showed that TNV148 was slightly effective in reducing the arthritis index after a single intraperitoneal bolus of 1mg/kg, the present study showed that the AI was slightly higher in both versions of the TNV antibody treatment group. Although (except for week 6) the 1mg/kg cA2 treated group did not increase significantly when compared to the 10mg/kg cA2 group and the TNV148 treated group was significantly higher at weeks 7 and 8, there was no significant difference in AI between 1mg/kg cA2, 1mg/kg TNV148, and 1mg/kg TNV148B at any time point in the study.

Figure 18 shows an overview of the 9 stages of the golimumab manufacturing process.

FIG. 19 shows a flow diagram of the stage 1 manufacturing process for the pre-incubation and amplification steps (including in-process control and process monitoring tests).

Fig. 20 shows a flow chart of the stage 2 manufacturing process steps, including in-process control and process monitoring tests.

Figure 21 shows a representative HPLC chromatogram of golimumab expressed in Sp2/0 cells using an HPLC method with fluorescence detection. Peaks associated with different species are labeled. Indicates a systemic peak not associated with golimumab.

Figure 22 shows a representative deconvolution mass spectrum of IRMA analysis of golimumab produced in Sp2/0 cells.

Figure 23 shows representative cIEF electropherograms of golimumab expressed in Sp2/0 cells, with the four major peaks labeled C, 1, 2, and 3, and the minor peaks labeled B. Internal standards of pI 7.6 and 9.5 were also labeled. Also shown are graphs representing the general relationship between the cIEF peak and reduced negative charge/sialylation degree.

Figure 24 shows a schematic overview of some of the major N-linked oligosaccharide species in golimumab IgG. The role of some enzymes in the glycosylation maturation process and some divalent cations (e.g., Mn as a cofactor) are also shown ²⁺And Cu as GalTI inhibitor²⁺) See, e.g., Biotechnol bioeng.2007, 2 months and 15 days; 96(3): 538-49; curr Drug targets.2008, 4 months; volume 9, phase 4, page 292-; j Biochem Mol biol.2002, 5 months and 31 days; vol 35, No. 3, pages 330 and 336). Note that substances with terminal sialic acid (S1 and S2) are charged substances, whereas substances lacking terminal sialic acid (G0F, G1F and G2F) are neutral substances, but the generation of charged substances depends on the presence of galactose added by the GalT1 enzyme in G1F and G2F.

Figure 25 shows representative HPLC chromatograms of the oligosaccharide profiles of golimumab expressed in Sp2/0 cells and golimumab expressed in CHO cells. The peak of eluted anthranilic acid-labeled N-glycans was identified with a hash of all peaks above baseline. Brackets and/or labels are used to indicate the set of peaks corresponding to total neutral, total charged, mono-sialylated and di-sialylated oligosaccharide species.

Detailed Description

The present invention provides compositions comprising anti-TNF antibodies having a heavy chain comprising SEQ ID NO: 36 and a light chain comprising SEQ ID NO: 37 (LC).

As used herein, "anti-tumor necrosis factor alpha antibody", "anti-TNF antibody portion" or "anti-TNF antibody fragment" and/or "anti-TNF antibody variant" and the like include any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one Complementarity Determining Region (CDR) of a heavy or light chain or a ligand binding portion thereof, 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 also affect specific ligands, such as but not limited to such antibodies modulate, decrease, increase, antagonize, agonize, moderate, alleviate, block, inhibit, abrogate, and/or interfere with at least one TNF activity or binding, or with a 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 at least one TNF or a specified portion, variant, or domain thereof. Suitable anti-TNF antibodies, specified portions or variants 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 also intended to encompass antibodies, digested fragments, specified portions and variants thereof, including antibody mimetics or antibody portions that comprise structures and/or functions that mimic an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind mammalian TNF. For example, the invention encompasses antibody fragments capable of binding TNF or a portion thereof, including, but not limited to, Fab fragments (e.g., obtained by papain digestion), Fab 'fragments (e.g., obtained by pepsin digestion and partial reduction), and F (ab') ₂Fragments (e.g. by pepsin)Enzymatic digestion), a facb fragment (e.g., by plasmin digestion), a pfc' fragment (e.g., by pepsin or plasmin digestion), an Fd fragment (e.g., by pepsin digestion, partial reduction, and reaggregation), an Fv or scFv fragment (e.g., by molecular biology techniques) (see, e.g., Colligan, Immunology, supra).

Such fragments may be produced by enzymatic cleavage, synthesis, or recombinant techniques as are known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, the code F (ab')₂The combined genes for the heavy chain portion were designed to include the CH encoding the heavy chain₁DNA sequence of a domain and/or hinge region. The various portions of the antibody can be chemically linked together by conventional techniques or can be prepared as a continuous protein using genetic engineering techniques.

The term "human antibody" as used herein refers to substantially every part of a protein therein (e.g., CDR, framework, C)_LDomain, C_HDomains (e.g., C)_H1、C_H2 and CH3), hinge (V)_L、V_H) Are substantially non-immunogenic in humans, with only minor sequence changes or variations. Similarly, antibodies that specify genera primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals, refer to specific antibodies of such species, sub-genera, sub-families, families. Furthermore, chimeric antibodies include any combination of the above. Such alterations or variations optionally and preferably maintain or reduce immunogenicity in humans or other species relative to the unmodified antibody. Thus, human antibodies are distinct from chimeric or humanized antibodies. It should be 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. In addition, when the human antibody is a single chain antibody, it may comprise a linking peptide not present in natural human antibodies. For example, the Fv can comprise a linker peptide, such as two to about eight, that links the heavy chain variable region and the light chain variable region Glycine or other amino acid residues. Such linker peptides are considered to be of human origin.

Bispecific antibodies (e.g.) A xenospecific antibody, a xenoconjugated antibody or similar antibody, which are monoclonal, preferably human or humanized antibodies having binding specificity for at least two different antigens. In the present case, one of the binding specificities is directed against at least one TNF protein and the other binding specificity is directed against any other antigen. Methods of making bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression 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 assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually by an affinity chromatography step, can be inefficient due to low product yields, and different strategies have been developed to facilitate bispecific antibody production.

A full-length bispecific antibody can be generated, for example, using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies by: substitutions are introduced at the heavy chain CH3 interface in each half molecule to facilitate heterodimer formation of two antibody halves with different specificities in an in vitro cell-free environment or using co-expression. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine residue of a second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding a different epitope.

As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains with the same CH3 amino acid sequence.

As used herein, "heterodimerization" refers to the interaction of two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with different CH3 amino acid sequences.

The "button-in-hole" strategy (see, e.g., PCT International publication WO 2006/028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the boundary of the CH3 domain in human IgG may be mutated at positions that affect the CH3 domain interaction, thereby promoting heterodimer formation. Amino acids with small side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds a second antigen. Upon co-expression of both antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "button" with the heavy chain with the "button". An exemplary CH3 substitution pair (denoted as modification position in the first CH3 domain of the first heavy chain/modification position in the second CH3 domain of the second heavy chain) that forms a button and clasp is: T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S _ L368A _ Y407V.

Other strategies may also be used, such as promoting heavy chain heterodimerization using electrostatic interactions by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in U.S. patent publication US 2010/0015133; U.S. patent publication US 2009/0182127/U.S. patent publication US2010/028637 or U.S. patent publication US 2011/0123532. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y _ F405A _ Y407V/T394W, T366I _ K392M _ T394W/F405A _ Y407V, T366L _ K392M _ T394W/F405A _ Y407V, L351Y _ Y407A/T366A _ K409F, L351Y _ Y407A/T366V _ K409F, Y407A/T366A _ K409F, or T350V _ L351Y _ F405A _ Y407V/T350V _ T366 _ V _ K V _ T394 363672 as described in US patent publication US 2012/V or US patent publication US 2013/V.

In addition to the above methods, bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming bispecific heterodimeric antibodies from the two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization according to the methods described in international patent publication WO 2011/131746. In the method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; incubating the antibodies together under reducing conditions sufficient to disulfide isomerization of cysteines in the hinge region; thereby generating bispecific antibodies by Fab arm exchange. The incubation conditions are optimally restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), Dithiothreitol (DTT), Dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and β -mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions may be used: incubating at a pH of 5-8, e.g., pH7.0 or pH7.4, in the presence of at least 25mM 2-MEA or in the presence of at least 0.5mM dithiothreitol at a temperature of at least 20 ℃ for at least 90 minutes.

anti-TNF antibodies (also referred to as TNF antibodies) useful in the methods and compositions of the invention can optionally be characterized by high affinity binding to TNF and optionally and preferably low toxicity. In particular, the antibodies, specific fragments or variants of the invention (wherein the individual components, such as the variable, constant and framework regions, individually and/or collectively optionally and preferably have low immunogenicity) may be used in the invention. Antibodies useful in the invention are optionally characterized in that they can be used to treat patients for extended periods of time, measurably alleviate symptoms and have low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, may help achieve a therapeutic result. "Low immunogenicity" is defined herein as producing a significant HAHA, HACA or HAMA response in less than about 75%, or preferably less than about 50%, of treated patients and/or causing low titers (less than about 300, preferably less than about 100, as measured by a dual-antigen enzyme immunoassay) in treated patients (Elliott et al, Lancet 344: 1125-1127(1994), which is incorporated herein by reference in its entirety).

Utility: the isolated nucleic acids of the invention can be used to produce at least one anti-TNF antibody, or a specific variant thereof, which can be used to measure or effect in cells, tissues, organs, or animals (including mammals and humans) to diagnose, monitor, modulate, treat, ameliorate, help prevent the occurrence of, or alleviate symptoms of at least one TNF disorder selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurological disorder or disease.

Such methods may comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention or reduction of symptoms, effects or mechanisms an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody. The effective amount may include an amount of about 0.001mg/kg to 500mg/kg per single administration (e.g., bolus), multiple administrations, or continuous administration, or achieve a serum concentration of 0.01 μ g/ml to 5000 μ g/ml per single administration, multiple administrations, or continuous administration, or any effective range or value therein, which is administered and determined using known methods as described herein or known in the relevant art. And (4) quoted. All publications or patents cited herein are incorporated herein by reference in their entirety as they show the state of the art to which the invention pertains and/or to provide a description and enablement of the present 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 formatsFormula (II) is shown. The following references are incorporated herein by reference in their entirety: edited by Ausubel et al, Current Protocols in Molecular Biology, John Wiley &Sons, Inc., NY, NY (1987-2001); sambrook et al, Molecular Cloning: a Laboratory Manual, 2^ndEdition, Cold Spring Harbor, NY (1989); harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); edited by Colligan et al, 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)。

Antibodies of the invention: comprises the amino acid sequence of SEQ ID NO: 1. 2 and 3 and/or all of the heavy 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, Current Protocols in Molecular Biology, John Wiley&Sons, Inc., NY, NY (1987-2001); sambrook et al, Molecular Cloning: a Laboratory Manual, 2^ndEdition, Cold Spring Harbor, NY (1989); harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); edited by Colligan et al, Current Protocols in Immunology, John Wiley &Sons, Inc., NY (1994-2001); colligan et al, Current Protocols in Protein Science, John Wiley&Sons, NY, (1997-2001), each of which is incorporated herein by reference in its entirety.

Human antibodies specific for human TNF proteins or fragments thereof, such as isolated and/or TNF proteins and/or portions thereof (including synthetic molecules such as synthetic peptides) can be generated against an appropriate immunogenic antigen. Other specific or general mammalian antibodies can be similarly generated. The preparation of immunogenic antigens and the production of monoclonal antibodies can be performed using any suitable technique.

In one method, the hybridoma is produced by a suitable immortalized cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, > 243, P3X63Ag8.653, Sp2 SA3, 2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACTIV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAIWMAA, NEURO 2A, etc., or heteromyeloma (heteroloma), a fusion product thereof, or any cell or fusion cell derived therefrom, or any other cell line suitable therefor, see, e.g., www.atcc.org, www.lifetech.com., fused with antibody-producing cells such as, peripheral cells isolated from such as, lymphocytes or other immune cells, including, tonsil cells or other suitable cell lines known in the art, or any other cell that expresses a heavy or light chain constant or variable sequence or framework or CDR sequence as an endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammalian, rodent, equine, ovine, caprine, ovine, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-, double-or triple-stranded, hybrid, etc., or any combination thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2, which are incorporated by reference herein in their entirety.

The antibody-producing cells may also be obtained from the peripheral blood, or preferably the spleen or lymph nodes, of a human or other suitable animal that has been immunized with the antigen of interest. Any other suitable host cell may also be used to express heterologous or endogenous nucleic acids encoding the antibodies, specific fragments or variants thereof of the present invention. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and can be cloned by limiting dilution or cell sorting or other known methods. Cells producing antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods for generating or isolating antibodies with the requisite specificity may be used, including, but not limited to, methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, phage, ribosome, oligonucleotide, RNA, cDNA, etc. display libraries; e.g., those available from Cambridge antibody Technologies, Cambridge shire, UK; Morphosys, Martinsreid/Planegg, DE; Biovariation, Aberdeen, Scotland, UK; BioInvent, Lund, Sweden; Dyax, Enzon, Affymax/Biosite; Xoma, Berkeley, CA; Ixsys. see, e.g., EP 368,684, PCT/GB 91/01134; PCT/GB 92/01755; PCT/GB 92/GB 3638; PCT/GB 92/00883; PCT/93/GB 6359605; PCT/006 08/350260/01429; PCT/3527; PCT/3527/14424; PCT/366342/14424; PCT/366326/4642; PCT/14424; PCT/366326/14424; PCT/366326/468; PCT/; PCT/14424; PCT/3527; PCT/; PCT/366328/3639; PCT/;) can be used in the methods for example, and/WO 3; PCT/3639; PCT/369; PCT/WO 3; PCT/369/WO 3; PCT/369/11; PCT; WO 3; PCT; WO 11/369/14424; PCT; WO 3; PCT; WO 3; PCT; WO 3; WO 9/369/11/369/14424/11/9/14424; PCT/; PCT/; PCT (ii) a WO 96/07754; (Scripps); EP 614989 (MorphoSys); WO95/16027 (BioInvent); WO 88/06630; WO90/3809 (Dyax); US 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO 89/06283; EP 371998; EP 550400; (Xoma); EP 229046; PCT/US91/07149 (Ixsys); or randomly generated peptides or proteins-US 5723323, 5763192, 5814476, 5817483, 5824514, 5976862, WO 86/05803, EP 590689 (Ixsys, now Applied Molecular Evolution (AME), each herein incorporated by reference in its entirety)) or dependent on immunization of transgenic animals (e.g. SCID mice, Nguyen et al, microbiol. immunol. vol. 41, p. 901-907, 1997; sandhu et al, crit.Rev.Biotechnol. Vol.16, pp.95-118, 1996; eren et al, Immunol.93: 154-161(1998), each incorporated herein by reference and the relevant patents and applications in their entirety) are capable of producing the full functionality of human antibodies as is known in the art and/or as described herein. Such techniques include, but are not limited to, ribosome display (Hanes et al, Proc. Natl. Acad. Sci. USA, 94: 4937-4942 (5 months 1997); Hanes et al, Proc. Natl. Acad. Sci. USA, 95: 14130-14135 (11 months 1998)); single Cell antibody production techniques (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Pat. No. 5,627,052, Wen et al, J.Immunol. Vol.17, pp. 887. 892, 1987; Babcook et al, Proc. Natl. Acad. Sci. USA 93: 7843. sup. 7848(1996)), gel microdroplet and flow cytometry (Powell et al, Biotechnol. 8: 333. sup. 337 (1990); One Systems, Cambrige, MA; Gray et al, J.Imm. Meth. Vol. 182, pp. 155. Vit. 163, 1995; Kenny et al, Bio/Technol. Vol. 13: pp. 787. 790, 1995); B Cell selection (Steenbakkers et al, Biotechnol. 19: 125. Biotechnol. 134; Bomby, Inc. 5. Biotechn. 1988, publication).

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

Known human Ig sequences are disclosed in many publications and websites, for example:

www.ncbi.nlm.nih.gov/entrez/query.fcgi；

www.atcc.org/phage/hdb.html；

www.sciquest.com/；

www.abcam.comm/；

www.antibodyresource.com/onlinecomp.html；

www.public.iastate.edu/～pedro/research_tools.html；

www.mgen.uni-heidelberg.de/SD/IT/IT.html；

www.whffeeman.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.aximtl.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. The subject of Kabat et al,

"Sequences of Proteins of Immunological Interest", U.S. dept.health, 1983, each of which is incorporated herein by reference in its entirety.

Such input sequences may be used to reduce immunogenicity or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic, as is known in the art. Generally, some or all of the non-human or human CDR sequences are retained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids. Antibodies can also optionally be humanized to retain high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can also optionally be made 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 generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. These displayed assays enable analysis of the likely role of residues in the functional performance of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from consensus and import sequences to enable desired antibody characteristics, such as increased affinity for a target antigen. In general, CDR residues are directly and substantially mostly involved in affecting antigen binding. Humanization or engineering of the antibodies of the invention may be performed using any known method, such as, but not limited to, those described in Winter (Jones 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.U.S.A.89: 4285 (1992); Presta et al, J.Immunol.151: 2623 (1993)), U.S. Patents: 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5,766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539, 4816567, PCT/US98/16280, US96/18978, US9I/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755, WO90/14443, WO90/14424, WO90/14430, EP 229246, each of which is incorporated herein by reference in its entirety, including the references cited therein.

anti-TNF antibodies can also optionally be generated by immunizing a transgenic animal (e.g., mouse, rat, hamster, non-human primate, etc.) that can produce a full repertoire of human antibodies, as described herein and/or as 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.

Transgenic mice that can produce a full repertoire of human antibodies that bind to human antigens can be generated by 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, assigned to Lonberg et al; Jakobovits et al, WO 98/50433, Jakobovits et al, WO 98/24893, Lonberg et al, WO 98/24884, Lonberg et al, WO 97/13852, Lonberg et al, WO 94/25585, Kucherlapate et al, WO 96/34096, Kucherlapate et al, EP 0463151B 1, Kucherlapate et al, EP 0710719A 1, Surani et al, U.S. Pat. No. 5,545,807, Bruggemann et al, WO 90/04036, Bruggemann et al, EP 0438474B 1, Lonberg et al, EP 0814259A 2, Lonberg et al, U.S. Pat. No. 5, Bruggemann masculin, Bruggemann et al, Kuuggemann masculin et al, WO 90/04036, Tankyurgy et al, (GB 579, Nature et al, (1994) 1994: 9, Nature et al, (GB 579, Nature et al), (U.4, Nature et al, Nature 579, U.S. Pat. 5, Nature et al, U.S. Pat. No. 5, et al, in which is incorporated by No. 5, U.S. 5, et al, U.S. Nos. 5, mendez et al, Nature Genetics 15: 146- & ltSUB & gt 156(1997), Taylor et al, Nucleic Acids Research 20 (23): 6287-: 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 comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that has undergone or can undergo functional rearrangement. The endogenous immunoglobulin locus in such mice can be disrupted or deleted to eliminate the ability of the animal to produce antibodies encoded by the endogenous gene.

Screening for antibodies that specifically bind to similar proteins or fragments can be conveniently accomplished using peptide display libraries. This method involves screening a large collection of peptides for individual members having a desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences may be 3 to 5000 or more amino acids in length, often 5-100 amino acids in length, and usually about 8 to 25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of peptide sequences on the surface of a phage or cell. Each phage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are described in PCT patent publications 91/17271, 91/18980, 91/19818 and 93/08278. Other systems for generating peptide libraries have aspects of both in vitro chemical synthesis methods and recombinant methods. See PCT patent publications 92/05258, 92/14843, and 96/19256. See also U.S. patent 5,658,754; and 5,643,768. Peptide display libraries, vectors and screening kits are commercially available from suppliers such as Invitrogen (Carlsbad, CA) and Cambridge antibody Technologies (Cambridge, UK). See, e.g., U.S. patents 4704692, 4939666, 4946778, 5260203, 5455030, 5518889, 5534621, 5656730, 5763733, 5767260, 5856456, assigned to Enzon; 5223409, 5403484, 5571698, 5837500, assigned to Dyax, 5427908, 5580717, assigned to Affymax; 5885793, assigned to Cambridge anti Technologies; 5750373, assigned to Genentech, 5618920, 5595898, 5576195, 5698435, 5693493, 5698417, assigned to Xoma, Colligan, supra; ausubel, supra; or Sambrook, supra, each of the above patents and publications is incorporated by reference herein in its entirety.

The antibodies of the invention may also be prepared using at least one anti-TNF antibody-encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that are capable of producing such antibodies in their milk. Such animals may be provided using known methods. See, for example and without limitation, U.S. patent 5,827,690; 5,849,992, respectively; 4,873,316; 5,849,992, respectively; 5,994,616, respectively; 5,565,362, respectively; 5,304,489, et al, each of which is incorporated herein by reference in its entirety.

The antibodies of the invention can also be prepared using at least one anti-TNF antibody-encoding nucleic acid to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco and corn) that produce such antibodies, specific portions or variants thereof in plant parts thereof or cells cultured from plant parts thereof. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large quantities of recombinant proteins, for example using inducible promoters. See, e.g., Cramer et al, curr. top. microb0l.immunol.240: 95-118(1999), and the references cited therein. Likewise, transgenic maize has also been used to express mammalian proteins on a commercial production scale with biological activity equivalent to those 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 the references cited therein. Antibodies, including antibody fragments such as single chain antibodies (scFv), can also be produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers. See, e.g., Conrad et al, Plant mol. biol. 38: 101-109(1998), and references cited therein. Thus, the antibodies of the invention may also be produced according to known methods using transgenic plants. See also, e.g., Fischer et al, biotechnol.appl.biochem.30: 99-108(Oct., 1999); ma et al, Trends Biotechnol.13: 522-7 (1995); ma et al, Plant Physiol, Vol.109, p.341 and 346, 1995; whitellam et al, biochem. Soc. trans. Vol.22, p.940-944, 1994; and references cited therein. For plant expression of antibodies in general, see, but not limited to, each of the above references is also incorporated herein by reference in its entirety.

The antibodies of the invention can have a wide range of affinities (K)_D) Binds to human TNF. In a preferred embodimentIn embodiments, at least one human mAb of the present invention can optionally bind human TNF with high affinity. For example, a human mAb can be equal to or less than about 10^-7M, such as, but not limited to, 0.1-9.9 (or any range or value therein). times.10^-7、10^-8、10^-9、10^-10、10^-11、10^-12、10^-13Or any range or value of K therein_DBinds to human TNF.

The affinity or avidity of an antibody for an antigen may be determined experimentally using any suitable method. (see, e.g., Berzofsky et al, "Antibody-Antibody Interactions", Fundamental Immunology, Paul, edited by W.E, Raven Press: New York, NY (1984); Kuby, Janis Immunology, W.H.Freeman and Company: New York, NY (1992); and methods described herein). The measured affinity of a particular antibody-antigen interaction will be different if measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen binding parameters (e.g., K)_D、K_a、K_d) The measurement of (a) is preferably performed with a standard solution of the antibody and antigen, and a standard buffer, such as the buffer described herein.

A nucleic acid molecule. Using information provided herein, such as encoding SEQ ID NO: 1. 2, 3, 4, 5, 6, 7, 8, or a specific fragment, variant, or consensus sequence thereof, or a deposited vector comprising at least one of these sequences, a nucleotide sequence encoding at least 70% -100% contiguous amino acids of at least one of SEQ ID NOs: i. 2 and 3 and/or all of the heavy chain variable CDR regions of SEQ ID NOs: 4. 5 and 6, at least one anti-TNF antibody of all light chain variable CDR regions.

The nucleic acid molecules of the invention can 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, eDNA and genomic DNA produced by cloning or synthesis, or any combination thereof. The DNA may be triplex, 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 referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

The isolated nucleic acid molecules of the invention can include nucleic acid molecules having an Open Reading Frame (ORF), optionally having one or more introns, such as, but not limited to, at least one designated 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 having a coding sequence for an anti-TNF antibody or variable region (e.g., SEQ ID NOS: 7, 8); and nucleic acid molecules having nucleotide sequences substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode at least one anti-TNF antibody, as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it will be apparent to those skilled in the art that such degenerate nucleic acid variants encoding a specific anti-TNF antibody of the present invention can be routinely produced. See, e.g., Ausubel et al, supra, and such nucleic acid variants are included in the present invention. Non-limiting examples of isolated nucleic acid molecules of the invention include SEQ ID NOs: 10. 11, 12, 13, 14, 15, corresponding to non-limiting examples of nucleic acids encoding HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, HC variable region, and LC variable region, respectively.

As noted herein, the nucleic acid molecules of the invention comprise nucleic acids encoding anti-TNF antibodies, which can include, but are not limited to, those nucleic acids that individually encode the amino acid sequences of antibody fragments; the coding sequence of the entire antibody or a portion thereof; the coding sequence for the antibody, fragment or portion, and additional sequences, such as the coding sequence for at least one signal leader peptide or fusion peptide with or without additional coding sequences as described above; such as at least one intron; also included are additional non-coding sequences, including but not limited to non-coding 5 'and 3' sequences, such as transcribed, non-translated sequences that function in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and stabilization of mRNA); additional coding sequences that encode additional amino acids, such as those that provide additional functions. Thus, the antibody-encoding sequence may be fused to a marker sequence, such as a sequence encoding a peptide that may facilitate purification of a fused antibody comprising an antibody fragment or portion.

A polynucleotide that selectively hybridizes to a polynucleotide described herein. The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of the present embodiments may be used to isolate, detect and/or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides of the invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotide is an isolated genomic sequence or a cDNA sequence, or is complementary to a cDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, more preferably at least 95% of the full-length sequence. cDNA libraries can be normalized to increase the appearance of rare sequences. Low or medium stringency hybridization conditions are generally, but not exclusively, used for sequences having reduced sequence identity relative to the complementary sequence. Medium and high stringency conditions can optionally be used for sequences of greater identity. Low stringency conditions allow for selective hybridization of sequences having about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

Optionally, the polynucleotides of the invention will encode at least a portion of an antibody encoded by a polynucleotide described herein. The polynucleotides of the invention comprise nucleic acid sequences that can be used to selectively hybridize to polynucleotides encoding the antibodies of the invention. See, e.g., Ausubel (supra); colligan (supra), each of which is incorporated by reference herein in its entirety.

And (3) constructing nucleic acid. The isolated nucleic acids of the present invention can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or a combination thereof, as are well known in the art.

The nucleic acid may conveniently comprise a sequence other than a polynucleotide of the invention. For example, a multiple cloning site comprising one or more endonuclease restriction sites can be inserted into a nucleic acid to aid in the isolation of the polynucleotide. In addition, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the invention. For example, a hexahistidine tag sequence provides a convenient means for purifying the proteins of the invention. The nucleic acids of the invention (except for the coding sequences) are optionally vectors, adaptors, or linkers for cloning and/or expressing the polynucleotides of the invention.

Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters and linkers is well known in the art. (see, e.g., Ausubel, supra; or Sambrook, supra).

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 a variety of cloning methods known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to a polynucleotide of the invention are used to identify a desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and the construction of cDNA and genomic libraries, are well known to those of ordinary skill in the art. (see, e.g., Ausubel, supra; or Sambrook, supra).

Nucleic acid screening and isolation methods. cDNA or genomic libraries can be screened using probes based on the sequences of the polynucleotides of the invention, such as those disclosed herein. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that hybridization of various degrees of stringency can be used in the assay; and the hybridization or wash medium may be stringent. As the conditions for hybridization become more stringent, a higher degree of complementarity must exist between the probe and target in order for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of partially denaturing solvents such as formamide. For example, the stringency of hybridization is conveniently varied by varying the polarity of the reactant solution, for example by manipulating the concentration of formamide in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100% or 70% -100% or any range or value therein. It is understood, however, that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.

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

Known methods of DNA or RNA amplification include, but are not limited to, Polymerase Chain Reaction (PCR) and related amplification methods (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al, 4,795,699 and 4,921,794 to Tabor et al, 5,142,033 to Innis, 5,122,464 to Wilson et al, 5,091,310 to Innis, 5,066,584 to Gyllensten et al, 4,889,818 to Gelfand et al, 4,994,370 to Silver et al, 4,766,067 to Biswas, 4,656,134 to Ringold) and RNA-mediated amplification of NASs using target sequence-specific RNA as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al, entitled BA), the entire contents of which are incorporated herein by reference. (see, e.g., Ausubel, supra; or Sambrook, supra.)

For example, the sequences of polynucleotides of the invention and related genes can be amplified directly from genomic DNA or cDNA libraries using Polymerase Chain Reaction (PCR) techniques. For example, PCR and other in vitro amplification methods can also be used to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids for use as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide a skilled artisan in the overall in vitro amplification method can be found in Berger (supra), Sambrook (supra), and Ausubel (supra), and U.S. Pat. No. 4,683,202(1987) to Mullis et al; and Innis et al, PCR Protocols A guides to Methods and Applications, eds., Academic Press Inc., San Diego, CA (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR Kit (Clontech). In addition, (e.g., T4 gene 32 protein (Boehringer Mannheim) can be used to increase the yield of long PCR products.

Synthetic methods for constructing nucleic acids. Isolated nucleic acids of the invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel et al, supra). Chemical synthesis generally results in a single-stranded oligonucleotide that can be converted to double-stranded DNA by hybridization to a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that while chemical synthesis of DNA may be limited to sequences of about 100 or more bases, longer sequences may be obtained by ligating shorter sequences.

A recombinant expression cassette. The invention also provides recombinant expression cassettes comprising a nucleic acid of the invention. Nucleic acid sequences of the invention, such as cDNA or genomic sequences encoding an antibody of the invention, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that will direct transcription of the polynucleotide in a predetermined host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the invention.

In some embodiments, an isolated nucleic acid that acts as a promoter, enhancer, or other element may be introduced at an appropriate location (upstream, downstream, or in an intron) in a non-heterologous form of a polynucleotide of the invention in order to up-or down-regulate expression of the polynucleotide of the invention. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and/or substitution.

Vectors and host cells. The invention also relates to vectors comprising the isolated nucleic acid molecules of the invention, host cells genetically engineered with the 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 incorporated by reference herein in its entirety.

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

The DNA insert should be operably linked to a suitable promoter. The expression construct will also contain a transcription start site, a termination site, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation site at the beginning of the mRNA to be translated and a stop codon (e.g., UAA, UGA or UAG) at the appropriate position at the end of the mRNA, with UAA and UAG being preferred for mammalian or eukaryotic cell expression.

The expression vector will preferably, but optionally, include at least one selectable marker. Such markers include, for example, but are not limited to: for eukaryotic cell cultures, resistance genes for Methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. No. 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 (GS, U.S. Pat. No. 5,122,464; 5,770,359; 5,827,739); and for culture in E.coli (E.coli) and other bacteria or prokaryotes, tetracycline or ampicillin resistance genes (the above patents are hereby incorporated by reference in their entirety). Suitable culture media and conditions for the above-described host cells are known in the art. Suitable vectors will be apparent to the skilled person. Introduction of the vector construct into a host cell may be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other known methods. Such methods have been described in the art, such as Sambrook (supra), chapters 1-4 and chapters 16-18; ausubel (supra), chapters 1, 9, 13, 15, 16.

At least one antibody of the invention may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For example, regions of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the antibody to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Likewise, peptide moieties may be added to the antibodies of the invention to aid in purification. Such regions may be removed prior to the 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 chapters 18.1-18.74; ausubel (supra), chapters 16, 17 and 18.

One skilled in the art will recognize that many expression systems may be used to express nucleic acids encoding proteins of the present invention.

Alternatively, the nucleic acid of the invention may be expressed in a host cell by switching on (by manipulation) in a host cell containing the endogenous DNA encoding the antibody of the invention. Such methods are well known in the art (e.g., as described in U.S. Pat. nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

Cells that can be used to produce antibodies, specific portions or variants thereof include mammalian cells. Mammalian cell systems will typically be cultured in the form of a monolayer of cells, but the cells may also be adapted to grow in suspension, for example in shake flasks or bioreactors. A number of suitable host cell lines capable of expressing the entire glycosylated protein have been developed in the art, including COS-1 (e.g., seeCRL-1650), COS-7 (e.g., CRL-1650)CRL-1651), HEK293, BHK21 (e.g.CCL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC C)RL-26), chinese hamster ovary Cells (CHO), Hep G2, p3x63ag8.653, Sp2/0-Ag14, HeLa cells, and the like, which are readily available from, for example, the american type culture collection (Manassas, VA). In certain embodiments, the host cells include CHO cells and cells of lymphoid origin, such as myeloma and lymphoma cells, e.g., CHO-K1 cells, p3x63ag8.653 cells ((r))CRL-1580) and Sp2/0-Ag14 cell(s) ((R)CRL-1581)。

CHO cell line

Most Recombinant therapeutic proteins produced today are prepared in Chinese Hamster Ovary (CHO) cells (Jayapal KP et al, Recombinant protein therapeutics from CHO cells-20years and counting. chem Eng prog.2007; 103: 40-47; Kunert R, Reinhart D., Advance in Recombinant antibody manufacturing. apple Microbiol.2016, Vol.100, No. 8, p.3451, 3461), although several other mammalian cell lines are available. Their advantages include, for example, robust growth as adherent cells or in suspension, adaptation to serum-free and chemically defined media, high yields, and a history of established regulatory approval for therapeutic recombinant protein production. They are also very easy to genetically modify and the methods used for cell transfection, recombinant protein expression and clone selection are well characterized. CHO cells may also provide human-compatible post-translational modifications. As used herein, "CHO cell" includes, but is not limited to, for example, CHO-DG44, CHO-Kl, CHO-M, CHO-S, CHO GS knockouts, and modifications and derivatives thereof.

The expression vector of these cells may include one or more of the following expression control sequences, such as but not limited to: an origin of replication; promoters (e.g., late or early SV40 promoter, CMV promoter (U.S. Pat. No. 5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1. alpha. promoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulin promoter, enhancers and/or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag poly A addition sites), and transcription terminator sequences see, e.g., Ausubel et al (supra); Sambrook et al (supra.) other cells useful in producing the nucleic acids or proteins of the invention are also known and/or can be obtained, e.g., from the U.S. type culture Collection cell lines and hybridoma catalogues or other known or commercial sources.

When eukaryotic host cells are used, polyadenylation or transcription termination sequences will typically be incorporated into the vector. An example of a termination sequence is a polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of transcripts may 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, gene sequences that control replication in the host cell can be incorporated into the vector, as is known in the art.

And (5) purifying the antibody.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, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") may also be used for purification. See, e.g., Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley&Sons, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

Antibodies of the 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 the recombinant production procedure, the antibodies of the invention may or may not be glycosylated, with glycosylation being 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, Colligan, Protein Science, supra, chapters 12-14, all of which are incorporated herein by reference in their entirety.

anti-TNF antibodies

Comprises the amino acid sequence of SEQ ID NO: 1. 2 and 3 and/or all of the heavy chain variable CDR regions of SEQ ID NOs: 4. 5 and 6 comprises an antibody amino acid sequence encoded by any suitable polynucleotide disclosed herein, or any isolated or prepared antibody. Preferably, the human antibody or antigen-binding fragment binds 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, thereby inhibiting the activity mediated by the binding of TNF to TNF receptors or by other TNF-dependent or mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody that can inhibit TNF-dependent activity by about 20% to 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 as described herein and/or as known in the art. The human antibodies of the invention may be of any type (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may comprise kappa or lambda light chains. In one embodiment, the human antibody comprises an IgG heavy chain or defined fragment, e.g., at least one of isotypes IgG1, IgG2, IgG3, or IgG 4. Such antibodies can be prepared as described herein and/or as known in the art by employing transgenic mice or other transgenic non-human mammals comprising at least one human light chain (e.g., IgG, IgA, and IgM (e.g., γ 1, γ 2, γ 3, γ 4) transgene.

As used herein, the term "antibody" or "antibodies" includes biosimilar antibody molecules approved under the biological product price competition and innovation Act of 2009 (BPCI Act) and similar legal regulations worldwide. According to BPCI Act, if the data show that the antibody is "highly similar" to the reference product, but the clinically inactive components have minor differences and are "expected" to produce the same clinical results as the reference product in terms of safety, purity and potency, it can be confirmed that the antibody is biosimilar (endocrine practice: 2018, month 2, volume 24, phase 2, page 195 to page 204). Provides a simplified approach to approval for these bio-mimetic antibody molecules, enabling applicants to rely on clinical data of innovative drug reference products to ensure regulatory approval. In contrast to the original innovative pharmaceutical reference antibody approved by the FDA based on successful clinical trials, the biostimulant antibody molecule is referred to herein as a "subsequent biologic". As shown herein, the first and second components of the device,(golimumab) is a primary innovative drug reference anti-TNF antibody based on successful clinical trials with FDA approval. Golimumab has been marketed in the united states since 2009.

At least one antibody of the invention binds to at least one specific epitope that is specific for at least one TNF protein, subunit, fragment, moiety, or any combination thereof. The at least one epitope may comprise at least one antibody binding region comprising at least a portion of said protein, which epitope is preferably constituted by at least one extracellular, soluble, hydrophilic, external or cytoplasmic portion of said protein. The at least one specific epitope may comprise any combination of at least one amino acid sequence that is SEO ID NO: 9 to the entire specified portion from at least 1-3 amino acids of contiguous amino acids.

Generally, a human antibody or antigen-binding fragment of the invention will comprise an antigen-binding region comprising at least one human complementarity determining region (CDR1, CDR2 and CDR3) or a variant of at least one heavy chain variable region and at least one human complementarity determining region (CDR1, CDR2 and CDR3) or a variant of at least one light chain variable region. As a non-limiting example, an antibody or antigen-binding portion or variant may comprise a polypeptide having the sequence of SEQ ID NO: 3 and/or a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 6, and at least one of the light chain CDRs 3 of the amino acid sequence of claim 6. In a particular embodiment, the antibody or antigen-binding fragment can have an antigen-binding region that comprises at least a portion of at least one heavy chain CDR (i.e., CDR1, CDR2, and/or CDR3) having an amino acid sequence corresponding to CDR1, CDR2, and/or CDR3 (e.g., SEQ id nos: 1, 2, and/or 3). In another specific embodiment, the antibody or antigen-binding portion or variant may 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 an amino acid sequence corresponding to CDR1, CDR2, and/or CDR3 (e.g., SEO ID NOs: 4, 5, and/or 6). In a preferred embodiment, the three heavy chain CDRs and the three light chain CDRs of the antibody or antigen-binding fragment have the amino acid sequences of the corresponding CDRs of at least one of mabs TNV148, TNV14, TNV15, TNV196, TNV118, TNV32, TNV86 as described herein. Such antibodies can be prepared by the following method: the various portions (e.g., CDRs, framework) of an antibody are chemically linked together using conventional techniques, a nucleic acid molecule (i.e., one or more) encoding the antibody is prepared and expressed using conventional techniques of recombinant DNA technology or by using any other suitable method.

The anti-TNF antibody may comprise at least one of a heavy chain or light chain variable region having a defined amino acid sequence. For example, in a preferred embodiment, a polypeptide optionally having the amino acid sequence of SEQ ID NO: 7, and/or optionally at least one heavy chain variable region having the amino acid sequence of SEQ ID NO: 8, an antibody that binds to human TNF and comprises a defined heavy or light chain variable region can be prepared using a suitable method, such as phage display (Katsube, Y. et al, Int J mol. Med, 1 (5): 863-868(1998)) or using transgenic animals. 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 that can undergo functional rearrangement can be immunized with human TNF or a fragment thereof to elicit the production of antibodies. 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, the encoding nucleic acid or portion thereof may be used to express the antibody, specified portion or variant in a suitable host cell.

The invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains, and CDRs comprising amino acid sequences substantially identical to the amino acid sequences described herein. Preferably, such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs can have high affinity (e.g., less than or equal to about 10)^-9K of M_D) Binds to human TNF. Amino acid sequences that are substantially identical to the sequences described herein include sequences having conservative amino acid substitutions as well as amino acid deletions and/or insertions. Conservative amino acid substitutions are those that replace a first amino acid with a second amino acid that has similar chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) as the first amino acid. Conservative substitutions include the substitution of one amino acid for another within the following groups: lysine (K), arginine (R) and histidine (H); aspartic acid (D) and glutamic acid (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.

Amino acid code.The amino acids that constitute the anti-TNF antibodies of the present invention are generally abbreviated. Amino acids can be represented by their single letter code, three letter code, name, or trinucleotide codons, thereby indicating the amino acid name, as is well known in the art (see Alberts)Et al, Molecular Biology of The Cell, third edition, Garland Publishing, inc., New York, 1994):

as illustrated herein, an anti-TNF antibody of the present invention can include one or more amino acid substitutions, deletions, or additions from natural mutations or from artificial manipulation.

Of course, the number of amino acid substitutions that can be made by the skilled person depends on many factors, including those described above. As illustrated herein, generally, the number of amino acid substitutions, insertions, or deletions of 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, such as 1-30, or any range or value therein.

Amino acids essential for function in the anti-TNF antibodies of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue of 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 of crucial importance 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)).

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

The anti-TNF antibody can also optionally comprise SEQ ID NO: 7. 8 from 70% to 100% of at least one of the contiguous amino acids.

In one embodiment, the amino acid sequence of the immunoglobulin chain or portion thereof (e.g., variable region, CDR) is identical to SEQ ID NOS: 7. 8 (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). For example, the amino acid sequence of the light chain variable region may be identical to SEQ ID NO: 8, or the amino acid sequence of heavy chain CDR3 can be compared to the amino acid sequence of SEQ ID NO: 7, a comparison is made. 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 known in the art.

In SEQ ID NO: 7. exemplary heavy and light chain variable region sequences are provided in 8. An antibody of the invention, or a particular variant thereof, can comprise any number of contiguous amino acid residues from an antibody of the invention, wherein the number is selected from an integer from 10% to 100% of the number of contiguous residues in an anti-TNF antibody. Optionally, the contiguous amino acid subsequence 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 subsequences may be any integer selected from 1 to 20, such as at least 2, 3, 4 or 5.

The skilled artisan will appreciate that the invention includes at least one biologically active antibody of the invention. The specific activity of a biologically active antibody is at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% -1000% of the specific activity of the natural (non-synthetic), endogenous or related and known antibody. Methods for determining and quantifying measures of enzymatic activity and substrate specificity are well known to those skilled in the art.

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

The modified antibodies and antigen-binding fragments of the invention may comprise one or more organic moieties covalently bonded, directly or indirectly, to the antibody. Each organic moiety bonded to an antibody or antigen-binding fragment of the 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" encompasses monocarboxylic acids and dicarboxylic acids. "hydrophilic polymer group," as that term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, antibodies modified by covalent attachment of polylysine are included in the present invention. Hydrophilic polymers suitable for modifying the antibodies of the invention may be linear or branched and include, for example, polyalkanediols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), hydrophilic amino acid polymers (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkylene oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer modifying the antibody of the invention has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG may be used ₅₀₀₀And PEG_20,000Where the subscript is the average molecular weight (in daltons) of the polymer. The hydrophilic polymer group may be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acids or fatty acid ester groups can be prepared by employing suitable methods. For example, amines may be includedThe polymer of groups is coupled to carboxylate groups of fatty acids or fatty acid esters, and activated carboxylate groups on fatty acids or fatty acid esters (e.g., activated with N, N-carbonyldiimidazole) can be coupled to hydroxyl groups on the polymer.

Fatty acids and fatty acid esters suitable for modifying the antibodies of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying the antibodies of the invention include, for example, n-dodecanoate (C)₁₂Laurate), n-tetradecanoate (C)₁₄Myristic acid ester), n-octadecanoic acid ester (C)₁₈Stearic acid ester), n-eicosanoic acid ester (C)₂₀Arachidic acid ester), n-behenic acid ester (C)₂₂Behenate), n-triacontanoic acid ester (C)₃₀) N-tetraalkanoic acid ester (C)₄₀) Cis-delta 9-octadecanoic acid ester (C)₁₈Oleate), all-cis-. DELTA.5, 8, 11, 14-eicosanoate (C)₂₀Arachidonate), suberic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

Modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. The term "modifying agent" as used herein refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid ester) that comprises 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, halogen (chloro, bromo, fluoro, iodo), N-hydroxysuccinimide ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. The aldehyde functional group can be coupled to an amine-or hydrazide-containing molecule, and the azide group can be reacted with a trivalent phosphorus group to form a phosphoramidate orA phosphoryl imine bond. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). The activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid ester), or through a linker moiety, such as divalent C ₁-C₁₂Groups in which one or more carbon atoms may be substituted with a heteroatom such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethyleneglycol, - (CH)₂)₃-、-NH-(CH₂)₆-NH-、-(CH₂)₂-NH-and-CH₂-O-CH₂-CH₂-O-CH₂-CH₂-O-CH-NH-. A modifying agent comprising a linking moiety can be generated, for example, by: mono-Boc-alkyldiamines (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) are reacted with fatty acids in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form amide bonds between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine, which can be coupled to another carboxylic acid ester (as described), or can be reacted with maleic anhydride and the resulting product cyclized to yield an activated maleimide-based derivative of the fatty acid. (see, e.g., WO 92/16221 to Thompson et al, the entire teachings of which are incorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, the organic moiety can be bound to the antibody in a non-site specific manner by using an amine-reactive modifier (e.g., a 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 an antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce a modified antibody of the invention. Modified human antibodies and antigen-binding fragments comprising an organic moiety bonded to a specific site of an antibody of the invention may 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); Capella et al, Biotechnol.Bioeng.56 (4): 456-463(1997)), and in Hermanson, G.T., Bioconjugate Techniques, Academic Press: the method described in San Diego, CA (1996).

An anti-idiotype antibody directed against the anti-Tnf antibody composition. In addition to monoclonal or chimeric anti-TNF antibodies, the invention also relates to anti-idiotypic (anti-Id) antibodies specific for such antibodies of the invention. An anti-Id antibody is an antibody that recognizes a unique determinant that is normally associated with the antigen binding region of another antibody. anti-Id can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the Id antibody source with the antibody or CDR-containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody, thereby producing an anti-Id antibody. The anti-Id antibody may also be used as an "immunogen" to induce an immune response in another animal, thereby generating what is known as an anti-Id antibody.

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 anti-TNF antibodies as described herein and/or as known in the art, provided in a non-naturally occurring composition, mixture, or form. Such compositions include non-naturally occurring compositions comprising at least one or two full-length sequences, C-terminal and/or N-terminal deleted variants, domains, fragments or specified variants of an anti-TNF antibody amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 2, 3, 4, 5, 6, 7, 8, or a specific fragment, domain or variant thereof. Preferred anti-TNF antibody compositions comprise at least one or two full-length sequences, fragments, domains or variants as at least one CDR-containing or LBR moiety from SEQ ID NO: 1. 2, 3, 4, 5, 6 or a specific fragment, domain or variant thereof. More preferred compositions comprise SEQ ID NO: 1. 2, 3, 4, 5, 6 or 40% -99% of at least one of the specified fragments, domains or variants thereof. Such composition percentages are calculated as weight, volume, concentration, molarity, or molarity of a liquid or anhydrous solution, mixture, suspension, emulsion, or colloid, as known in the art or as described herein.

The anti-TNF antibody compositions of the present invention can further comprise any suitable and effective amount of at least one of a composition or a pharmaceutical composition comprising at least one anti-TNF antibody administered to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one agent selected from the group consisting of: 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 antirheumatic drug (such as methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic (anestetic), a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial (such as an aminoglycoside, an antifungal, an antiparasitic, an antiviral, a penicillcarbapenem, a cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteroid, an anabolic steroid, a diabetes-related agent, a pharmaceutical, Minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietins (e.g., erythropoietin α), filgrastims (e.g., G-CSF, Youjin), sargrastim (GM-CSF, Leukine), vaccinants, immunoglobulins, immunosuppressive agents (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatic agents, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetic agents, stimulants, donepezil, tacrine, asthma medications, beta agonists, inhaled steroids, leukotriene inhibitors, anti-inflammatory agents, anti-anxiety agents, hypnotics, sympathomimetic agents, stimulants, donepezil, anti-asthmatic drugs, beta agonists, inhaled steroids, anti-inflammatory agents, anti-tussives, anti-drugs, anti-tussives, anti-drugs, Methylxanthine, cromolyn, epinephrine or the like, alpha-streptokinase (in bermuda), a cytokine or cytokine antagonist. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 to IL-23. Suitable dosages are well known in the art. See, e.g., Wells et al, editors, Pharmacotherapy Handbook, second edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, edited by Deluxe, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

Such anti-cancer or anti-infective agents may also include a toxin molecule associated, bound, co-formulated, or co-administered with at least one antibody of the invention. The toxin may optionally act to selectively kill pathological cells or tissues. The pathological cells may be cancer cells or other cells. Such toxins may be, but are not limited to, purified or recombinant toxins or toxin fragments comprising at least one functional cytotoxic domain of a toxin, for example selected from at least one of ricin, diphtheria toxin, venom toxin, or bacterial toxin. The term toxin also includes endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacterium or virus, which can cause any pathological condition in humans and other mammals, including toxin shock, which can lead to death. Such toxins may include, but are not limited to, enterotoxigenic e.coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin (Shigella), Aeromonas enterotoxin (Aeromonas) enterotoxin, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin (staphyloccal) a (sea), b (seb), or c (sec), Streptococcal enterotoxin (streptococcus), and the like. Such bacteria include, but are not limited to, strains of: enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (e.g., serotype 0157: H7 strain), and staphylococus Staphylococci (e.g., Staphylococcus aureus (staphyloccus aureus), Staphylococcus pyogenes (staphyloccus pyogenenes)), Shigella (e.g., Shigella dysenteriae, Shigella flexneri), Shigella baumannii (Shigella dysenteriae), Salmonella (Salmonella) genus (e.g., Salmonella typhi, Salmonella choleraesuis (Salmonella-suis), Salmonella enteritidis (salmonellae), Clostridium (Clostridium) genus (e.g., Clostridium capsulatum (Clostridium perfringens), Clostridium difficile (Clostridium difficile), Clostridium botulinum (Clostridium botulinum), Clostridium perfringens (Clostridium), campylobacter (Clostridium perfringens)), campylobacter (e.g., campylobacter), campylobacter (Clostridium monobacter), Clostridium (campylobacter), Clostridium sp (e.g., campylobacter coli (campylobacter), Clostridium sp. chrysosporium (e.g., campylobacter coli), campylobacter coli (e.g., campylobacter coli (campylobacter coli), campylobacter strains (e.g., campylobacter coli (campylobacter coli) strain (campylobacter coli (e.g., campylobacter coli (campylobacter coli strain, aeromonas sobria (Aeromonas sobria), Aeromonas hydrophila (Aeromonas hydrophila), Aeromonas caviae (Aeromonas caviae), Pleiosomonas shigelloides (Pleiosomonas shigelloides), Yersinia enterocolitica (Yersinia enterocolitica), Vibrio (Vibrio) genus (e.g., Vibrio (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahaemolyticus)), Klebsiella (Klebsiella) genus, Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Streptococcus (Streptococcus). See, e.g., Stein editions, NTERNAL MEDICINE, 3 rd edition, pages 1-13, Little, Brown and co., Boston, (1990); evans et al, edited by Bacterial Infections of Humans: epidemic and Control, 2 nd edition, page 239-; the result of the Mandell et al, Principles and Practice of Infectious Diseases, 3 rd edition, Churchill Livingstone, New York (1990); edited by Berkow et al, The Merck Manual, 16 th edition, Merck and Co., Rahway, N.J., 1992; wood et al, FEMS Microbiology Immunology, 76: 121-134 (1991); marrack et al, Science, 248: 705-711(1990)), the entire contents of which are incorporated herein by reference.

The anti-TNF antibody compound, composition, or combination of the present invention may further comprise at least one of any suitable adjuvants, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro's eds, Remington's Pharmaceutical Sciences, 18 th edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and/or stability of the anti-TNF antibody, fragment or variant composition may be selected in a conventional manner, as is known in the art or as described herein.

Pharmaceutical excipients and additives useful in the compositions of the present invention include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including mono-, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, having 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumin such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components that may also play a role in 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.

Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), inositol, and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

The anti-TNF antibody composition can further comprise a buffer or a pH modifier; typically, the buffer is 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 hydrochloride or phosphate buffer. Preferred buffering agents for use in the compositions of the present invention are organic acid salts, such as citrate.

In addition, the anti-TNF antibody compositions of the present invention may comprise polymeric excipients/additives such as polyvinylpyrrolidone, polysucrose (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).

These and additional known pharmaceutical excipients and/or additives suitable for use in the anti-TNF antibody, partial or variant compositions according to the present invention are known in the art, for example, as listed in the following documents: "Remington: the Science & Practice of Pharmacy, 19 th edition, Williams & Williams, (1995) and The "Physician's Desk Reference", 52 th edition, Medical Economics, Montvale, NJ (1998), The disclosures of which are incorporated herein by Reference in their entirety. Preferred carrier or excipient materials are carbohydrates (e.g. sugars and alditols) and buffers (e.g. citrate) or polymeric agents.

And (4) preparing the preparation. As indicated above, the present invention provides stable formulations suitable for pharmaceutical or veterinary use, preferably phosphate buffered saline or selected salts, as well as preservative solutions and formulations containing a preservative, and multi-purpose preserved formulations comprising at least one anti-TNF antibody in a pharmaceutically acceptable formulation. The preservative formulation comprises at least one known preservative or is optionally selected from at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g. hexahydrate), alkyl benzoate (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof dissolved in an aqueous diluent. Any suitable concentration or mixture as known in the art may be used, such as 0.001% -5% or any range or value therein, such as but not limited to: 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 value or range therein. Non-limiting examples include: no preservative, 0.1% -2% m-cresol (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.9%, 1.0%), 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., 0.05%, 0.25%, 0.28%, 0.5%, 0.9%, 1.0%), 0.0005% -1.0% alkyl parabens (e.g., 0.00075%, 0.0009%, 0.001%, 0.002%, 0.005%, 0.0075%, 0.01%, 0.02%, 0.05%, 0.075%, 0.09%, 0.009%, 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 0.9%, 1.9%, etc.).

As indicated above, the present invention provides an article of manufacture comprising packaging material and at least one vial containing a solution of at least one anti-TNF antibody with a defined buffer and/or preservative (optionally dissolved in an aqueous diluent), wherein the packaging material comprises a label indicating that such solution can be stored for a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or longer. The invention also includes an article of manufacture comprising a packaging material, a first vial comprising lyophilized at least one anti-TNF antibody, and a second vial comprising an aqueous diluent that defines a buffer or preservative, wherein the packaging material comprises a label that directs a patient to reconstitute the at least one anti-TNF antibody in the aqueous diluent to form a solution that can be stored for a period of 24 hours or more.

The at least one anti-TNF antibody used according to the present invention may be prepared by recombinant means, including from mammalian cells or transgenic preparations, or may be purified from other biological sources, as described herein or as known in the art.

The range of at least one anti-TNF antibody in the product of the invention includes amounts that yield a concentration of about 1.0 μ g/ml to about 1000mg/ml upon reconstitution if in a wet/dry system, but lower and higher concentrations are possible and will be different from transdermal patch, transpulmonary, transmucosal or osmotic or micropump methods depending on the intended delivery vehicle, e.g., solution formulation.

Preferably, the aqueous diluent also optionally comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from: phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. The concentration depends on the preservative selected and is readily determined by the skilled person.

Other excipients such as isotonic agents, buffers, antioxidants, preservatives, enhancers may optionally and preferably be added to the diluent. Isotonic agents such as glycerol are often used in known concentrations. Physiologically tolerated buffers are preferably added to provide improved pH control. The formulation may cover a wide pH range, such as from about pH 4 to about pH 10, with a preferred range of from about pH 5 to about pH 9, and a most preferred range of from about 6.0 to about 8.0. Preferably the formulations of the present invention have a pH between about 6.8 and about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, especially Phosphate Buffered Saline (PBS).

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 non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188,Polyols, other block copolymers, and chelates such as EDTA and EGTA, may optionally be added to the formulation or composition to reduce aggregation. These additives are particularly useful if the formulation is to be administered using a pump or a plastic container. The presence of the pharmaceutically acceptable surfactant reduces the tendency of the protein to aggregate.

The formulations of the present invention may be prepared by a method comprising mixing at least one anti-TNF antibody and a preservative selected from phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. The at least one anti-TNF antibody and preservative are mixed in an aqueous diluent using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one anti-TNF antibody in a buffer is combined with a desired preservative in the buffer in an amount sufficient to provide the desired concentration of protein and preservative. Variations of this method will be recognized by those of ordinary skill in the art. For example, the order of addition of the ingredients, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of application used.

The claimed formulation may be provided to a patient in the form of a clear solution or in the form of a double vial comprising one vial of lyophilized at least one anti-TNF antibody reconstituted with a second vial containing an aqueous diluent, said second vial containing water, preservatives and/or excipients, preferably phosphate buffer and/or saline and selected salts. A single solution vial or double vial requiring reconstitution can be reused multiple times and can satisfy a single or multiple cycles of patient treatment and thus can provide a more convenient treatment regimen than currently available.

The presently claimed articles may be used for applications over a period of time ranging from immediately to 24 hours or more. Thus, the claimed articles of the present invention provide significant advantages to the patient. The formulations of the present invention can optionally be safely stored at temperatures of about 2 ℃ to about 40 ℃ and retain the biological activity of the protein for extended periods of time, thereby allowing the package label indicating that the solution can be maintained and/or used for periods of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more. Such labels may include a use period of up to 1-12 months, half a year, and/or 2 years if a preservative diluent is used.

The solution of at least one anti-TNF antibody of the present invention can be prepared by a method comprising mixing the 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 measured amount of at least one antibody in water or buffer is combined in an amount sufficient to provide the protein and optional preservative or buffer to the desired concentration. Variations of this method will be recognized by those of ordinary skill in the art. For example, the order of addition of the ingredients, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of application used.

The claimed product may be provided to a patient in the form of a clear solution or in the form of a double vial comprising one vial of lyophilized at least one anti-TNF antibody reconstituted with a second vial containing an aqueous diluent. Either a single solution vial or a double vial requiring reconstitution can be reused multiple times and can satisfy a single or multiple cycles of patient treatment and thus provide a more convenient treatment regimen than currently available.

The claimed product may be provided to a patient indirectly by providing a clear solution or a double vial comprising one vial of lyophilized at least one anti-TNF antibody reconstituted with a second vial containing an aqueous diluent by a dosing room, clinic or other such facility and unit. The clear solutions in this case may have a volumetric size of at most one liter or even more, thereby providing a large reservoir from which smaller portions of the at least one antibody solution may be removed one or more times for transfer into smaller vials and provided to their customers and/or patients by pharmacies or clinics.

Identification devices including these single vial systems include those pen injector devices used to deliver solutions, such as BD Pens, BD Pen、AndGenotronormHumatroRoferonJ-tip Needle-Freefor example, as made or developed by:

Becton Dickensen(Franklin Lakes，NJ，www.bectondickenson.com)；

Disetronic(Burgdorf，Switzerland，www.disetronic.com)；

Bioject，Portland，Oregon(www.bioject.com)；

Weston Medical(Peterborough，UK，www.weston-medical.com)；

Medi-Ject Corp(Minneapolis，MN，www.mediject.com)。

recognized devices that include dual vial systems include those pen injector systems for reconstituting lyophilized drugs in a cartridge for delivering the reconstitution solution, such as

The claimed product of the present invention includes a packaging material. The packaging material provides the conditions under which the product can be used, in addition to the information required by the regulatory agency. For a two-vial, wet/dry product, the packaging material of the present invention provides instructions directing the patient to reconstitute at least one anti-TNF antibody in an aqueous diluent to form a solution, and to use the solution over a period of 2-24 hours or more. For single vial solution products, the label indicates that such solutions can be used over a period of 2-24 hours or more. The claimed product of the present invention is useful for human pharmaceutical product applications.

The formulations of the invention may be prepared by a method comprising mixing at least one anti-TNF antibody with a selected buffer, preferably a phosphate buffer containing saline or a selected salt. The at least one antibody and the buffer are mixed in an aqueous diluent using conventional solubilization and mixing procedures. For example, to prepare a suitable formulation, a measured amount of at least one antibody in water or buffer is mixed with a desired buffer in an amount of water sufficient to provide the protein and buffer at the desired concentrations. Variations of this method will be recognized by those of ordinary skill in the art. For example, the order of addition of the ingredients, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of application used.

The claimed stable or preserved formulation may be provided to a patient in the form of a clear solution or in a dual vial comprising one vial of lyophilized at least one anti-TNF antibody reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent. Either a single solution vial or a double vial requiring reconstitution can be reused multiple times and can satisfy a single or multiple cycles of patient treatment and thus provide a more convenient treatment regimen than currently available.

At least one anti-TNF antibody in the stable or preserved formulations or solutions described herein can be administered to a patient according to the present invention via a variety of delivery methods, including SC or IM injections; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micropump, or other means known to those skilled in the art, as is well known in the art.

And (4) application in treatment. The invention also provides methods of using at least one dual integrin antibody of the invention for modulating or treating at least one TNF-related disease in a cell, tissue, organ, animal or patient as known in the art or described herein.

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 disorder, a malignant disorder, or a neurological disorder.

The present invention also provides methods for modulating or treating at least one immune-related disorder in a cell, tissue, organ, animal or patient, including but not limited to at least one of the following: rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, Ankylosing Spondylitis (angiosing Spondylitis), 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/reverse procedure of vasectomy (vasectomy reverse procedure), allergic/atopic disease, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplantation, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, inflammatory bowel disease, inflammatory bowel syndrome, asthma, inflammatory bowel disease, asthma, inflammatory bowel disease, inflammatory bowel syndrome, inflammatory bowel disease, asthma, inflammatory bowel disease, asthma, inflammatory bowel disease, 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, alcohol-induced hepatitis, chronic inflammatory pathological conditions, sarcoidosis, Crohn's disease conditions, sickle cell anemia, diabetes, nephropathy, atopic disorders, hypersensitivity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, leukemia, thrombocytopenia, transplant rejection of any organ or tissue, kidney transplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, Bone Marrow Transplant (BMT) rejection, kidney transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant rejection, and the like, Skin allograft rejection, cartilage transplant rejection, bone transplant rejection, small intestine transplant rejection, fetal thymus implant rejection, parathyroid transplant rejection, xenograft rejection of any organ or tissue, allograft rejection, anti-receptor hypersensitivity, Graves ' disease, Raynaud's disease, insulin resistant diabetes B, asthma, myasthenia gravis, antibody mediated cytotoxicity, hypersensitivity III, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and cutaneous change syndrome), polyneuropathy, megarchitemia, endocrinopathy, monoclonal gammopathy, skin change syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes, chronic active hepatitis, chronic kidney disease, and kidney disease, Primary biliary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV hypersensitivity, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, intracellular organism-induced granuloma, drug sensitivity, metabolism/idiopathic, wilson's disease, hemochromatosis, alpha-1 antitrypsin deficiency, diabetic retinopathy, hashimoto's thyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, Chronic Obstructive Pulmonary Disease (COPD), familial hemophagocytic lymphohistiocytosis, skin disorders, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy, anti-cd 3 therapy, chronic inflammatory bowel disease, Chronic Obstructive Pulmonary Disease (COPD), chronic inflammatory bowel disease (inflammatory bowel disease), chronic obstructive pulmonary disease (chronic obstructive pulmonary disease), chronic obstructive pulmonary disease, chronic inflammatory bowel disease, chronic obstructive pulmonary disease, chronic, Cytokine therapy, chemotherapy, radiation therapy (including, for example, but not limited to, asthenia, anemia, cachexia, etc.), chronic salicylic acidosis, and the like. See, e.g., Merck Manual, 12-17 th edition, Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, edited by Wells et al, second edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each of which is incorporated herein by reference in its entirety.

The present invention also provides methods of modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal or patient, including but not limited to at least one of the following: myocardial stunning syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic arteriosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, fainting, shock, syphilis of the cardiovascular system, heart failure, pulmonary heart disease, primary pulmonary hypertension, arrhythmia, atrial ectopic beat, atrial flutter, atrial fibrillation (persistent or paroxysmal), post-perfusion syndrome, cardiopulmonary bypass inflammatory response, mixed or polytropic atrial tachycardia, regular narrow QRS tachycardia, specific arrhythmia, ventricular fibrillation, bundle of His arrhythmia, atrioventricular block, bundle branch block, myocardial ischemic disease, coronary heart disease, angina, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvular heart disease, Endocarditis, pericardial disease, cardiac tumors, aortic and peripheral aneurysms, aortic dissection, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular disease, arterial occlusive disease, peripheral arteriosclerotic disease, thromboangiitis obliterans, functional peripheral arterial disease, raynaud's phenomenon and disease, cyanosis of hands and feet, erythromelalgia, venous disease, venous thrombosis, varicose veins, arteriovenous fistula, lymphedema, lipoedema, unstable angina, reperfusion injury, post-pump syndrome, ischemia reperfusion injury, and the like. Such methods can optionally comprise 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.

The present invention also provides 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 the following: acute or chronic bacterial infections, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (type a, type b or type c, etc.), septic arthritis, peritonitis, pneumonia, epiglottitis, e.coli 0157: h7, hemolytic uremic syndrome/thrombolytical thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium intracellulare, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epididymitis, legionella, lyme disease, influenza a, EB virus, viral encephalitis/aseptic meningitis, and the like.

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 the following diseases: leukemia, Acute Lymphoblastic Leukemia (ALL), B-cell, T-cell or FAB ALL, Acute Myeloid Leukemia (AML), Chronic Myelogenous 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 cancer, histiocytosis, tumor-related syndrome/hypercalcemia of malignancy, solid tumor, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and the like.

The present invention also provides 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: neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia syndrome, demyelinating diseases such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as corticospinal system lesions; basal ganglia disorders or cerebellar disorders; hyperkinetic movement disorders such as Huntington's chorea and senile chorea; drug-induced movement disorders, such as drug-induced disorders that block CNS dopamine receptors; motor reducing disorders such as parkinson's disease; progressive supranuclear palsy; structural lesions of the cerebellum; degeneration of the spinocerebellum, such as spinocerebellar ataxia, Friedreich's ataxia, cerebellar cortical degeneration, multiple systemic degeneration (Mencel, Dejerine-Thomas, Shi-Drager and Machado-Joseph); systemic disorders (refsum's disease, abetalipoproteinemia, ataxia, telangiectasia and mitochondrial multisystem disorders); demyelinating nuclear disorders such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit, such as neurogenic muscular atrophy (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile myelogenous atrophy, and juvenile myelogenous atrophy); alzheimer's disease; middle-aged Down syndrome; diffuse lewy body disease; senile dementia with lewy body type; Wernike-Korsakov syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; subacute sclerosing panencephalitis, hallowden-schartz disease (halllerrorden-Spatz disease); and dementia pugilistica, and the like. Such methods may optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. See, e.g., Merck Manual, 16 th edition, Merck & Company, Rahway, NJ (1992)

Any of the methods of the invention can comprise 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. Such methods may optionally further comprise co-administration or combination therapy to treat such immune disorders, wherein administration of the at least one anti-TNF antibody, specific portions or variants thereof further comprises administering prior to, concurrently with, and/or after its administration at least one agent selected from the group consisting of: at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment thereof, a fusion protein or a small molecule TNF antagonist), nereimumab, infliximab, enteracept, CDP-571, CDP-870, Aframomuzumab, lenacicept, and the like), antirheumatic (such as methotrexate, auranofin, thioglucoside, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxant, anesthetic, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocking agent, antimicrobial (such as aminoglycosides, antifungal, antiparasitic, antiviral, carbapenem, cephalosporin, fluoroquinolone, macrolides, penicillin, sulfa, tetracycline, or a small molecule TNF antagonist, Other antimicrobial agents), antipsoriatic agents, corticosteroids, anabolic steroids, diabetes-related agents, minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietins (e.g., alfa-eptins), filgrastimes (e.g., G-CSF, oxyphosphan), sarmostim (GM-CSF, Leukine), vaccinants, immunoglobulins, immunosuppressive agents (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement agents, estrogen receptor modulators, mydriatic agents, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetics, Agonists, donepezil, tacrine, asthma drugs, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogs, alpha-streptokinase (Pulmozyme), cytokines, or cytokine antagonists. Suitable dosages are well known in the art. See, e.g., Wells et al, editors, Pharmacotherapy Handbook, second edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, edited by Deluxe, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

TNF antagonists (further comprising at least one antibody, specified portions and variants thereof of the present invention) suitable for use in the compositions, combination therapies, co-administrations, devices and/or methods of the present invention include, but are not limited to, anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules that specifically bind to TNF; compounds that prevent and/or inhibit TNF synthesis, TNF release or its effect on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds that prevent 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.

As used herein, "tumor necrosis factor antibody", "TNF α antibody" or fragment and the like can reduce, block, inhibit, eliminate or interfere with TNF α activity in vitro, in situ, and/or preferably in vivo. For example, suitable TNF human antibodies of the present invention can bind TNF α and include anti-TNF antibodies, antigen-binding fragments thereof, and specific mutants or domains thereof that specifically bind TNF α. 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.

The chimeric antibody cA2 consists of a high affinity neutralizing antigen-binding variable region of mouse anti-human TNF α IgG1 antibody (designated a2) and a constant region of human IgG1 κ immunoglobulin. The human IgG1 Fc region can improve the allogeneic antibody effector function, increase circulating serum half-life and reduce the immunogenicity of the antibody. The avidity and epitope specificity of chimeric antibody cA2 was derived from the variable region of murine antibody a 2. In one embodiment, a preferred source of nucleic acid encoding the variable region of murine antibody a2 is the a2 hybridoma cell line.

Chimeric a2(cA2) neutralized the cytotoxic effects of native and recombinant human TNF α in a dose-dependent manner. The affinity constant of chimeric antibody cA2 was calculated to be 1.04X 10 based on the binding assay of chimeric antibody cA2 and recombinant human TNF α¹⁰M^-1. Preferred methods for determining monoclonal antibody specificity and affinity 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; edited by Colligan et al, Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992-; kozbor et al, immunol. today, 4: 72-79 (1983); edited by Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-; and Muller, meth.enzymol, 92: 589 (1983), which are incorporated herein by reference in their entirety.

In one embodiment, murine monoclonal antibody A2 is produced by the cell line designated c 134A. Chimeric antibody cA2 was generated from the cell line numbered c 168A.

Addition of monoclonal anti-TNF antibodies useful in the inventionExamples are described in the art (see, e.g., U.S. Pat. nos. 5,231,024;A. et al, Cytokine 2 (3): 162-169 (1990); U.S. application 07/943,852 (filed on 9/11/1992); rathjen et al, International publication WO 91/02078 (published on 21/2/1991); rubin et al, EPO patent publication 0218868 (published on 22/4/1987); yone et al, EPO patent publication 0288088 (26/10/1988); liang et al, biochem. biophysis. 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), which references are incorporated herein by reference in their entirety).

A TNF receptor molecule. Preferred TNF receptor molecules useful in the present invention are those that bind TNF α with high affinity (see, e.g., Feldmann et al, International publication WO 92/07076 (published at 1992, 30.4.; Schall et al, Cell 61: 361-. In particular, 55kDa (p55 TNF-R) and 75kDa (p75 TNF-R) TNF cell surface receptors may be used in the present invention. Truncated forms of the extracellular domain (ECD) or functional portions thereof comprising the receptor of these receptors (see, e.g., Corcoran et al, Eur. J. biochem. 223: 831-840(1994)) are also useful in the present invention. TNF receptor truncated forms containing ECD of the 30kDa and 40kDa TNF α inhibitory binding proteins have been detected in urine and serum (Engelmann, H. et al, J.biol.chem.265: 1531-1536 (1990)). TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are additional examples of TNF receptor molecules that may be used in the methods and compositions of the present invention. The TNF receptor molecules useful in the present invention are characterized in that they can treat patients for a long period of time, provide good or excellent relief from symptoms, and have low toxicity. Low immunogenicity and/or high affinity, among other undetermined characteristics, may contribute to the therapeutic outcome achieved.

TNF receptor multimeric molecules useful in the invention comprise all or a functional portion of the ECD 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 also comprise a signal peptide for secretion of the protein to direct expression of the multimeric molecule. These multimeric molecules and their methods of preparation have been described in U.S. patent application 08/437,533 (filed 5/9/1995), the contents of which are incorporated herein by reference in their entirety.

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. The immunoreceptor fusion molecule may also 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 preparation have been described in the art (Lesslauer et al, Eur. J. Immunol. Vol.27, pp.2883-2886, 1991; Ashkenazi et al, Proc. Natl. Acad. Sci.USA 88: 10535-. Methods for preparing immunoreceptor fusion molecules can also be found in Capon et al, U.S. Pat. nos. 5,116,964; capon et al, U.S. patent 5,225,538; and Capon et al, Nature 337: 525-531(1989), which are incorporated herein by reference in their entirety.

Functional equivalents, derivatives, fragments or regions of a TNF receptor molecule refer to portions of a TNF receptor molecule or portions of the sequence of a TNF receptor molecule encoding a TNF receptor molecule that are of sufficient size and sequence to be functionally similar to TNF receptor molecules useful in the present invention (e.g., bind TNF α with high affinity and have low immunogenicity). Functional equivalents of TNF receptor molecules also include modified TNF receptor molecules that are functionally similar to the TNF receptor molecules useful in the present invention (e.g., bind TNF α with high affinity and have low immunogenicity). For example, functional equivalents of TNF receptor molecules can include "silent" codons or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or a 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-.

Cytokines include any known cytokines. See, e.g., copew cytokines. 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.

Medical treatment. Any of the methods of the present invention may include a method for treating a TNF-mediated disorder comprising 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 to treat such immune disorders, wherein administration of the at least one anti-TNF antibody, specific portions or variants thereof further comprises administering prior to, concurrently with, and/or after its administration at least one agent selected from the group consisting of: at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment thereof, a fusion protein or a small molecule TNF antagonist), nereimumab, infliximab, enteracept, CDP-571, CDP-870, Aframomuzumab, lenacicept, and the like), antirheumatic (such as methotrexate, auranofin, thioglucoside, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxant, anesthetic, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocking agent, antimicrobial (such as aminoglycosides, antifungal, antiparasitic, antiviral, carbapenem, cephalosporin, fluoroquinolone, macrolides, penicillin, sulfa, tetracycline, or a small molecule TNF antagonist, Other antimicrobial agents), antipsoriatic agents, corticosteroids, anabolic steroids, diabetes-related agents, minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietins (e.g., alfa-eptins), filgrastimes (e.g., G-CSF, oxyphosphan), sarmostim (GM-CSF, Leukine), vaccinants, immunoglobulins, immunosuppressive agents (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement agents, estrogen receptor modulators, mydriatic agents, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetics, Agonists, donepezil, tacrine, asthma drugs, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogs, alpha-streptokinase (Pulmozyme), cytokines, or cytokine antagonists.

Typically, treatment of a pathological condition is achieved by administering an effective amount or dose of at least one anti-TNF antibody composition, which amounts to at least about 0.01 to 500 milligrams of at least one anti-TNF antibody per kilogram of patient, on average per dose, depending on the specific activity present in the composition, preferably at least about 0.1 to 100 milligrams of antibody per kilogram of patient per single or multiple administrations. Alternatively, effective serum concentrations may include 0.1 μ g/ml to 5000 μ g/ml serum concentration per single or multiple administrations. Suitable dosages are known to medical practitioners and will, of course, depend on the particular disease state, the specific activity of the composition to be administered, and the particular patient undergoing treatment. In some cases, to achieve a desired therapeutic amount, it may be necessary to provide for repeated administration, i.e., repeated administration of a particular monitored or metered dose alone, wherein the individual administration may be repeated until a desired daily dose or effect is achieved.

Preferred dosages may optionally include 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, 94, 96, 97, 98, 99, 95, 99, 100 mg/kg and/or any range thereof, or to achieve the following serum concentrations: 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 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, 20, 15.5, 16, 16.5, 17, 17.5, 17.9, 18, 18.9, 19, 20, 25, 20, 500, or 2500 g/500, or 45080, or more times of the concentration of a single administration of a sample of the blood.

Alternatively, the dosage administered may vary according to known factors, such as the pharmacodynamic properties of the particular agent and its mode and route of administration; age, health, and weight of the recipient; the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Generally, the dosage of the active ingredient may be about 0.1 to 100mg/kg body weight. Generally, 0.1mg/kg to 50 mg/kg, preferably 0.1mg/kg to 10 mg/kg, can be administered at one time or in a sustained release form, effective to achieve the desired result.

As one non-limiting example, treatment of a human or animal may be at least one day, or alternatively or additionally, at least one week, or alternatively, at least one week, or 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, or 40 days, or alternatively or additionally, at least one week, or alternatively, at least one week, or 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, or 52 weeks, or alternatively, or additionally, 1 week, 2, 3, 6, 2, 6, 12, 13, 3, or additionally, 7. At least one of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 years, or any combination thereof, provided as one or regular administrations of 0.1mg/kg to 100mg/kg of at least one antibody of the invention, such as 0.5, 0.9, 1.0, 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 100mg/kg, using single, infusion or repeated administration.

Dosage forms (compositions) suitable for internal administration typically contain from about 0.1 to about 500 milligrams of active ingredient per unit or container. In such pharmaceutical compositions, the active ingredient will generally be present in an amount of from about 0.5% to 99.999% by weight, based on the total weight of the composition.

For parenteral administration, the antibodies can be formulated as solutions, suspensions, emulsions, or lyophilized powders, provided in combination or separately with a pharmaceutically acceptable parenteral vehicle. Examples of such media are water, saline, ringer's solution, dextrose solution, and 1% -10% human serum albumin. Liposomes and non-aqueous media, such as fixed oils, can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation may be sterilized by known or suitable techniques.

Suitable Pharmaceutical carriers are described in the recent version of Remington's Pharmaceutical Sciences, a.osol (standard reference text in the art).

Alternative administration. A number of known and developed modes of administration may be used according to 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 be used in accordance with the present invention with suitable results.

The TNF antibodies of the present invention can be delivered in a vehicle 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 other means described herein or known in the art.

Parenteral formulations and administration. Formulations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection may be formulated according to known methods using suitable emulsifying or wetting agents and suspending agents. Injectable preparations may be nontoxic, parenterally administrable diluents, such as aqueous solutions in solvents or sterile injectable solutions or suspensions. As a usable medium or solvent, water, ringer's solution, isotonic saline, or the like is allowed to be used; as a common solvent or suspending solvent, sterile fixed oils may be used. For these purposes, any kind of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono-or diglycerides or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional forms of injection, pneumatic needle-free injection devices as described in U.S. patent 5,851,198, and laser perforator devices as described in U.S. patent 5,839,446, which are incorporated herein by reference in their entirety.

Alternative delivery means. The invention also relates to the administration of at least one anti-TNF antibody by: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavity, intracavitary, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus injection, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. At least one anti-TNF antibody composition can be prepared for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, in particular in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, such as but not limited to tablet or capsule form; or intranasally, such as, but not limited to, in the form of a powder, nasal drops or aerosol or certain medicaments; or transdermally, such as, but not limited to, a gel, ointment, lotion, suspension, or patch delivery system containing a chemical enhancer such as dimethyl sulfoxide to alter the structure of the skin or increase the concentration of a Drug in a transdermal patch (juninger et al, "Drug approval Enhancement"; Hsieh, d.s. editions, pages 59-90, (Marcel Dekker, inc. new York 1994, incorporated herein by reference in its entirety), or an oxidizing agent that enables formulations containing proteins and peptides to be applied to the skin (WO 98/53847), or an electric field to create an instantaneous transport pathway, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or ultrasound, such as transdermal ultrasound (U.S. patents 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

Pulmonary/nasal administration. For pulmonary administration, it is preferred that the at least one anti-TNF antibody composition is 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 devices or nasal devices known in the art for administering therapeutic agents by inhalation. These devices capable of depositing an aerosolized formulation in the sinus cavities or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, nebulizers, and the like. Other devices suitable for guiding transpulmonary or nasal administration of antibodiesAlso known in the art. All such devices may use formulations suitable for administration by dispensing the antibody in aerosol form. Such aerosols may be comprised of solutions (aqueous or non-aqueous) or solid particles. Metered dose inhalers such asMetered dose inhalers typically utilize a propellant gas and require actuation during inhalation (see, e.g., WO 94/16970, WO 98/35888). Dry powder inhalers such as Turbuhaler^TM(Astra)、(Glaxo)、(Glaxo)、Spiros^TMInhaler (Dura), device sold by the company Inhale Therapeutics andpowder inhalers (Fisons), all using breath to drive a mixed powder (US 4668218(Astra), EP 237507(Astra), WO 97/25086(Glaxo), WO 94/08552(Dura), US 5458135 (inlae), WO 94/06498(Fisons), all of which are incorporated herein by reference in their entirety). Atomizers, e.g. AERx ^TM Aradigm、Atomizer (Mallinckrodt) and AcornNebulizers (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), all of which are incorporated herein by reference in their entirety, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, and the like produce small particle aerosols. These specific examples of commercially available inhalation devices are intended to be representative of specific devices suitable for use in the practice of the present invention and are not intended to limit the scope of the present invention. Preferably, the composition comprising at least one anti-TNF antibody is administered by dry powder inhaler or sprayAnd (4) delivering. For administration of at least one antibody of the invention, the inhalation device needs to have several desirable characteristics. For example, advantageously, delivery by inhalation devices is reliable, reproducible and accurate. The inhalation device may optionally deliver small dry particles, for example less than about 10 μm, preferably about 1 μm to 5 μm, for ease of breathing.

The TNF antibody composition is administered as a spray. A spray comprising the TNF antibody composition protein may 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 the desired output and particle size. Electrospray can be generated, for example, by electric field in combination with capillary or nozzle feed. Advantageously, the particles of the at least one anti-TNF antibody composition protein delivered by the nebulizer have a particle size of less than about 10 μm, preferably a particle size in the range of about 1 μm to about 5 μm, most preferably in the range of about 2 μm to about 3 μm.

Formulations of at least one anti-TNF antibody composition protein suitable for use with a nebulizer typically comprise the antibody composition in an aqueous solution at concentrations of: about 0.1mg to about 100mg of at least one anti-TNF antibody composition protein/ml solution or mg/gm, or any range or value therein, such as, 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 100mg/ml or mg/gm. The formulation may contain agents such as excipients, buffers, isotonicity agents, preservatives, surfactants and preferably includes zinc. The formulation may also contain excipients or agents for stabilizing the proteins of the antibody composition, such as buffers, reducing agents, bulk proteins (bulk proteins) or carbohydrates. Filler proteins that may be used to formulate the proteins of the antibody composition include albumin, protamine, and the like. Common carbohydrates that can be used to formulate the proteins of the antibody composition include sucrose, mannitol, lactose, trehalose, glucose, and the like. The antibody composition protein formulation may further comprise a surfactant that reduces or prevents surface-induced antibody composition protein aggregation caused by solution atomization during aerosol formation. A variety of conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount will generally be in the range between 0.001% and 14% 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. Additional agents known in the art for the formulation of proteins, such as TNF antibodies, or specific portions or variants may also be included in the formulation.

The TNF antibody composition is administered by nebulizer. The antibody composition protein may be administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, in jet atomizers, a high velocity air jet is generated through an orifice with a compressed air source. As the gas expands through the nozzle, a low pressure zone 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 or droplets as it exits the tube, thereby generating an aerosol. A range of configurations, flow rates and baffle types may be employed to produce the desired performance characteristics from a given spray atomizer. In ultrasonic atomizers, high frequency electrical energy is used to generate vibrational mechanical energy, usually with piezoelectric transducers. The energy is transferred to the formulation of the antibody composition protein, either directly or through a coupling fluid, thereby generating an aerosol comprising the antibody composition protein. Advantageously, the particles of antibody composition protein delivered by the nebulizer have a particle size of less than about 10 μm, preferably a particle size in the range of about 1 μm to about 5 μm, most preferably about 2 μm to about 3 μm.

Formulations of at least one anti-TNF antibody suitable for use with a nebulizer (jet nebulizer or ultrasonic nebulizer) typically include a concentration of about 0.1mg to about 100mg of the at least one anti-TNF antibody protein per ml of solution. The formulation may contain agents such as excipients, buffers, isotonicity agents, preservatives, surfactants and preferably includes zinc. The formulation may further comprise excipients or agents for protein stabilization of the at least one anti-TNF antibody composition, such as buffers, reducing agents, bulking proteins, or carbohydrates. Filler proteins that may be used to formulate at least one anti-TNF antibody composition protein include albumin, protamine, and the like. Common carbohydrates that may be used to formulate the at least one anti-TNF antibody include sucrose, mannitol, lactose, trehalose, glucose, and the like. The at least one anti-TNF antibody formulation can further comprise a surfactant that can reduce or prevent surface-induced aggregation of the at least one anti-TNF antibody by nebulization of the solution during formation of the aerosol. A variety of conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount will generally be between 0.001% and 4% by weight of the formulation. Particularly preferred surfactants for the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20 and the like. Additional agents known in the art for the formulation of proteins, such as antibody proteins, may also be included in the formulation.

The TNF antibody composition is administered by a metered dose inhaler. In a Metered Dose Inhaler (MDI), the propellant, at least one anti-TNF antibody, and any excipients or other additives are contained in a canister as a mixture that includes a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles ranging in size from less than about 10 μm, preferably from about 1 μm to about 5 μm, most preferably from about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by employing a formulation of the antibody composition protein prepared by various 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 which employ hydrofluorocarbon propellants.

The formulation of the at least one anti-TNF antibody for use in a metered dose inhaler device will typically comprise a fine powder containing the at least one anti-TNF antibody as a suspension in a non-aqueous medium, for example, in a propellant with the aid of a surfactant. The propellant may be any conventional material used for this purpose such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or hydrocarbons including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1, 1, 1, 2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134 a), HFA-227 (hydrofluoroalkane-227). Preferably, the propellant is a hydrofluorocarbon. The surfactant may be selected to stabilize the at least one anti-TNF antibody as a suspension in the propellant, to protect the active agent from chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid and the like. In some cases, solution aerosols using solvents such as ethanol are preferred. Additional agents known in the art for formulating proteins may also be included in the formulation.

One of ordinary skill in the art will recognize that the methods of the invention can be accomplished by pulmonary administration of at least one anti-TNF antibody composition via a device not described herein.

Oral preparation and administration. Oral formulations rely on co-administration of adjuvants (e.g., resorcinol and non-ionic 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., trypsin inhibitor, diisopropyl fluorophosphate (DFF) and aprotinin (trasylol)) to inhibit enzymatic degradation. The active ingredient compounds in solid dosage forms for oral administration may be mixed with at least one additive selected from the group consisting of sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginates, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers and glycerides. These dosage forms may also contain other types of additives, for example inactive diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffering agents, sweeteners, flavoring agents, fragrances and the like.

Tablets and pills can be further processed into enteric-coated formulations. Liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations which are permissible for medical use. These formulations may contain inactive diluents commonly used in the art, such as water. Liposomes have been described as drug delivery systems for insulin and heparin (us patent 4,239,754). Recently, microspheres of artificial polymers (proteinoid) of mixed amino acids have been used to deliver drugs (U.S. Pat. No. 4,925,673). In addition, the carrier compounds described in U.S. patent 5,879,681 and U.S. patent 5,5,871,753 are known in the art for oral delivery of bioactive agents.

Mucosal preparations and administration. For absorption across mucosal surfaces, compositions and methods of administering at least one anti-TNF antibody include an emulsion comprising a plurality of submicron particles, mucoadhesive macromolecules, bioactive peptides, and an aqueous continuous phase that facilitates absorption across mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. patent 5,514,670). Mucosal surfaces suitable for administration of the emulsions of the present invention may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal and rectal routes of administration. Formulations for vaginal and rectal administration, such as suppositories, may contain, for example, polyalkylene glycols, petrolatum, cocoa butter and the like as excipients. Formulations for intranasal administration may be solid and contain, for example, lactose as a vehicle, or may be aqueous or oily solutions of nasal drops. For oral administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. patent 5,849,695).

Transdermal preparation and application. For transdermal administration, the at least one anti-TNF antibody is encapsulated in a delivery device such as a liposome or polymeric nanoparticle, microparticle, microcapsule, or microsphere (collectively microparticles, unless specifically indicated). A variety of suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids (such as polylactic acid, polyglycolic acid, and copolymers thereof), polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (U.S. patent 5,814,599).

Long-term application and preparation. Delivery of a compound of the invention to a subject by one administration over a prolonged period of time, for example, over a period of one week to one year, may sometimes be desirable. A variety of sustained release, depot or implant dosage forms may be utilized. For example, the dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has low solubility in body fluids, e.g., (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene monosulfonic or disulfonic acid, polygalacturonic acid, or the like; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or the like, or salts with organic cations formed from, for example, N' -dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as a zinc tannate salt. In addition, the compounds of the present invention or preferably relatively insoluble salts such as those described above may be formulated in a gel suitable for injection, for example, in an aluminum monostearate gel with, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of sustained release depot formulation for injection contains a compound or salt dispersed to be encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as the polylactic acid/polyglycolic acid polymer described in U.S. patent No. 3,773,919. The compounds or preferably relatively insoluble salts such as those described above may also be formulated into cholesterol-based silicone rubber pellets, especially 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 "suspended and Controlled Release Drug Delivery Systems", edited by J.R. Robinson, Marcel Dekker, Inc., N.Y., 1978).

Having generally described the present invention, the same will be more readily understood through reference to the following examples, which are given by way of illustration only and are not intended to be limiting.

Example 1: cloning and expression of TNF antibodies in mammalian cells.

Typical mammalian expression vectors contain at least one promoter element that mediates the initiation of transcription of the mRNA and antibody coding sequences, and signals required for the termination and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanked by donor and acceptor sites for RNA splicing. High efficiency of transcription can be achieved using the following sequences: early and late promoters from SV40, Long Terminal Repeats (LTRS) from retroviruses such as RSV, HTLVI, HIVI, and early promoters of Cytomegalovirus (CMV). However, cellular elements (e.g., the human actin promoter) may also be used. Suitable expression vectors for use in the practice of the present invention include, for example, vectors such as: 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 (pRSVcat), (C: (pR X) 37152)、pSV2dhfr(37146) And pBC12 MI. Mammalian host cells that may be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, qual QC1-3 cells, mouse L cells, and Chinese Hamster Ovary (CHO) cells.

Alternatively, the gene may be expressed in a stable cell line containing the gene integrated into the chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows for the identification and isolation of transfected cells.

The transfected gene can also be amplified to express the encoded antibody in large quantities. The DHFR (dihydrofolate reductase) marker can be used to develop cell lines carrying hundreds or even thousands of copies of the gene of interest. Another selectable marker that may be used is 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 selection medium and the cells with the highest resistance are selected. These cell lines contain an amplifiable gene integrated into the chromosome. Chinese Hamster Ovary (CHO) cells and NSO cells are commonly used for the production of antibodies.

Expression vectors pC1 and pC4 contain strong promoters (LTR) of Rous sarcoma virus (Cullen et al, molecular. Cell. biol.5: 438-447(1985)) and fragments of the CMV-enhancer (Boshart et al, Cell 41: 521-530 (1985)). Multiple cloning sites, for example, with restriction endonuclease cleavage sites BamHI, XbaI and Asp718, facilitate cloning of the gene of interest. The vector additionally contains the 3' intron of the rat preproinsulin gene, polyadenylation and termination signals.

Cloning and expression in CHO cells

One vector commonly used for expression in CHO cells is pC 4. Plasmid pC4 is plasmid pSV2-dhfr (37146) A derivative of (1). 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 selection media (e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic methotrexate. The amplification of the DHFR gene in cells resistant to Methotrexate (MTX) is well documented (see, e.g., F.W.Alt et al, J.biol.chem.253, pp. 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 concentrations of MTX developed resistance to the drug due to the overproduction of the target enzyme DHFR as a result of DHFR gene amplification. If the second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this method can be used to develop cell lines carrying more than 1,000 copies of the amplified gene. Subsequently, when methotrexate is removed, obtain Resulting in a cell line containing the amplifiable gene integrated into one or more chromosomes of the host cell.

For expression of the gene of interest, plasmid pC4 contains the strong promoter of the Rous sarcoma virus Long Terminal Repeat (LTR) (Cullen et al, molecular. Cell. biol. 5: 438-447(1985)) and a fragment isolated from the enhancer of the human Cytomegalovirus (CMV) immediate early gene (Boshart et al, Cell 41: 521-530 (1985)). Downstream of the promoter are BamHI, XbaI and Asp718 restriction enzyme cleavage sites that allow gene integration. Following these cloning sites, the plasmid contains the 3' intron of the rat preproinsulin gene and a polyadenylation site. Other highly efficient promoters may also be used for expression, such as the human β -actin promoter, the SV40 early or late promoter, or long terminal repeats from other retroviruses such as HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express TNF in a regulated manner in mammalian cells (M.Gossen and H.Bujard, Proc.Natl.Acad.Sci.USA 89: 5547-. For polyadenylation of mRNA, other signals from, for example, human growth hormone or globin genes may also be used. Stable cell lines carrying the gene of interest integrated into the chromosome may also be selected when co-transfected with a selectable marker such as gpt, G418 or hygromycin. It may be advantageous to use more than one selectable marker at the beginning, e.g., G418 plus methotrexate.

Plasmid pC4 was digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector was then separated from a 1% agarose gel.

The DNA encoding the isolated variable and constant regions was then ligated to the dephosphorylated vector using T4 DNA ligase. Coli HB101 or XL-1Blue cells are then transformed and bacteria containing the fragment inserted into plasmid pC4 are identified using, for example, restriction enzyme analysis.

Chinese Hamster Ovary (CHO) cells lacking an active DHFR gene were used for transfection. 5 μ g of expression plasmid pC4 was co-transfected with 0.5 μ g of plasmid pSV2-neo using liposomes. Plasmid pSV2-neo contains a dominant selection marker, the neo gene from Tn5, which encodes an enzyme that confers resistance to a group of antibiotics including G418. Cells were seeded in alpha minus MEM supplemented with 1. mu.g/ml G418. After 2 days, cells were trypsinized and seeded in alpha minus MEM supplemented with 10ng/ml, 25ng/ml, or 50ng/ml methotrexate plus 1. mu.g/ml G418 in hybridoma clone plates (Greiner, Germany). After about 10-14 days, single clones were trypsinized and then seeded in 6-well dishes or 10ml flasks with different concentrations of methotrexate (50nM, 100nM, 200nM, 400nM, 800 nM). Clones grown at the highest concentration of methotrexate were then transferred to new 6-well plates containing higher concentrations of methotrexate (1mM, 2mM, 5mM, 10mM, 20 mM). The same procedure was repeated until clones grown at a concentration of 100mM-200mM were obtained. For example, expression of the desired gene product can be analyzed by SDS-PAGE and Western blotting or by reverse phase HPLC analysis.

Example 2: transgenic mice were used to generate high affinity human IgG monoclonal antibodies reactive with human TNF.

And (6) summarizing. Transgenic mice containing human heavy and light chain immunoglobulin genes have been used to generate high affinity, fully human monoclonal antibodies that can be used therapeutically to inhibit the effects of TNF in the treatment of one or more TNF-mediated diseases. (CBA/J x C57/BL6/J) F transgenic for human variable and constant region antibodies comprising heavy and light chains₂The hybrid mice were immunized with human recombinant TNF (Taylor et al, Intl. Immunol.6: 579-. Several fusions produced one or more sets of fully human TNF reactive IgG monoclonal antibodies. The fully human anti-TNF antibody was further characterized. All were IgG1 κ. Such antibodies were found to have a size of between 1X 10⁹And 9X 10¹²Affinity constant between. The unexpectedly high affinity of these fully human monoclonal antibodies makes them suitable candidates for therapeutic applications in TNF-related diseases, pathologies or disorders.

Abbreviations. BSA-bovine serum albumin; CO 2₂-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 (IP-IP); IV-intravenous; mab or 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/volume; w/v-weight/volume.

Materials and methods

An animal. Transgenic mice that can express human antibodies are known in the art (and are commercially available (e.g., from GenPharm International, San Jose, CA; abgenix, Freemont, CA, etc.), which express human immunoglobulins other than mouse IgM or Ig kappa, for example, such transgenic mice contain a human sequence transgene, the human sequence transgene is subjected to V (D) J ligation, heavy chain class conversion and somatic mutation to produce a full complement of human sequence immunoglobulins (Lonberg et al, Nature 368: 856-, in addition, the heavy chain transgene can encode both human μ and human γ 1(Fishwild et al, Nature Biotechnology 14: 845-851(1996)) and/or γ 3 constant regions mice derived from the appropriate genotypic lineage can be used in immunization and fusion procedures to generate fully human monoclonal antibodies against TNF.

And (4) immunization. One or more immunization programs can be used to generate anti-TNF human hybridomas. The first few fusions may be performed after the following exemplary immunization protocol, but other similar known protocols may also be used. Several 14-20 week old females and/or surgically castrated transgenic male mice were immunized IP and/or ID with 1. mu.g to 1000. mu.g of recombinant human TNF emulsified with an equal volume of TITERMAX or complete Freund's adjuvant to a final volume of 100. mu.L to 400. mu.L (e.g., 200). Each mouse can also optionally receive 1 μ g-10 μ g in 100 μ L of saline at each of the 2 SQ loci. Mice can then be immunized IP (1. mu.g-400. mu.g) and SQ (1. mu.g-400. mu.g. times.2) with TNF emulsified with an equal volume of TITERMAX or incomplete Freund's adjuvant after 1-7, 5-12, 10-18, 17-25, and/or 21-34 days. Mice can be bled after 12 to 25 days and 25 to 40 days by retroorbital puncture without the use of anticoagulants. The blood was then allowed to clot at room temperature for 1 hour, and serum was collected and titrated according to known methods using the TNF EIA assay. Fusion was performed when repeated injections did not result in an increase in titer. At this point, mice can be provided with a final IV booster injection of 1 μ g-400 μ g TNF diluted in 100 μ L of physiological saline. Three days later, mice were euthanized by cervical dislocation, and the spleens were aseptically removed and immersed in 10mL cold Phosphate Buffered Saline (PBS) containing 100U/mL penicillin, 100. mu.g/mL streptomycin, and 0.25. mu.g/mL amphotericin B (PSA). Splenocytes were harvested by sterile perfusion of the spleen with PSA-PBS. Cells were washed once with cold PSA-PBS, counted using trypan blue dye exclusion, and resuspended in RPMI1640 medium containing 25mM Hepes.

And (4) fusing the cells. Mouse myeloma cells and live spleen cells can be fused in a ratio of 1: 1 to 1: 10 according to known methods, for example, methods known in the art. As a non-limiting example, spleen cells and myeloma cells may be pelleted together. The pellet was then slowly resuspended in 1mL of a 50% (w/v) PEG/PBS solution (PEG molecular weight 1,450, Sigma) at 37 ℃ over 30 seconds. Fusion was then terminated by slow addition of 10.5mL RPMI 1640 medium containing 25mM Hepes (37 ℃) over 1 minute. The fused cells were centrifuged at 500rpm to 1500rpm for 5 minutes. The cells were then resuspended in HAT medium (RPMI 1640 medium containing 25mM Hepes, 10% fetal clone I serum (Hyclone), 1mM sodium pyruvate, 4mM L-glutamine, 10. mu.g/mL gentamicin, 2.5% origin culture supplement (Fisher), 10% 653 conditions RPMI 1640/Hepes medium, 50. mu.M 2-mercaptoethanol, 100. mu.M hypoxanthine, 0.4. mu.M aminopterin, and 16. mu.M thymidine) and then seeded at 200. mu.L/well in 15 96-well flat-bottom tissue culture plates. The panels were then placed in a 5% CO holding chamber₂And 95% air in a 37 ℃ humidified incubator, and maintained for 7-10 days.

Detection of human IgG anti-TNF antibodies in mouse serum. Solid phase EIA can be used in sieve Human IgG antibodies specific to human TNF in mouse sera were selected. Briefly, plates can be coated overnight with 2. mu.g/mL TNF in PBS. After washing in 0.15M saline containing 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v) BSA in PBS at 200. mu.L/well for 1 hour at room temperature. Immediately use the plate or freeze it at-20 ℃ for future use. Mouse serum dilutions were incubated at 50L/well on TNF-coated plates for 1 hour at room temperature. The plates were washed and then probed with 50 μ L/well HRP-labeled goat anti-human IgG (Fc specific) diluted 1: 30,000 in 1% BSA-PBS for 1 hour at room temperature. The plate can be washed again and 100. mu.L/well of citrate-phosphate substrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H) added at room temperature₂O₂And 1mg/mL OPD) and held for 15 minutes. Stop solution (4N sulfuric acid) was then added at 25 μ L/well and the OD read via an automated plate spectrophotometer at 490 nm.

Detection of fully human immunoglobulin in hybridoma supernatants. Growth positive hybridomas secreting fully human immunoglobulin can be detected using appropriate EIAs. Briefly, 96-well eject plates (VWR, 610744) can be coated with 10. mu.g/mL goat anti-human IgG Fc overnight in sodium carbonate buffer at 4 ℃. Plates were washed and blocked with 1% BSA-PBS at 37 ℃ for 1 hour, then used immediately or frozen at-20 ℃. Undiluted hybridoma supernatant was incubated for 1 hour at 37 ℃ on the plate. Plates were washed and then probed with HRP-labeled goat anti-human kappa diluted 1: 10,000 in 1% BSA-PBS for 1 hour at 37 ℃. The plate was then incubated with the substrate solution as described above.

Determination of fully human anti-TNF reactivity. As described above, the reactivity of hybridomas to TNF can be simultaneously determined using a suitable RIA or other assay. For example, supernatants were incubated on goat anti-human IgG Fc plates, washed, as described above, and then probed with radiolabeled TNF at appropriate counts/well for 1 hour at room temperature. Wells were washed twice with PBS and bound radiolabeled TNF quantified using a suitable counter.

Human IgG1 κ anti-TNF secreting hybridomas were expanded in cell culture and serially subcloned by limiting dilution. The resulting clonal populations can be expanded and cryopreserved in a freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen.

Isoforms. Isotype determination of antibodies can be accomplished using EIA in a format similar to that used to screen for specific titers of mouse immune sera. As described above, TNF can be coated in 96-well plates, and 2 u g/mL purified antibody at room temperature in a plate temperature in one hour. Plates were washed and HRP-labeled goat anti-human IgG diluted 1: 4000 in 1% BSA-PBS₁Or goat anti-human IgG labeled with HRP₃Probing was done at room temperature for 1 hour. The plate was washed again and incubated with substrate solution as described above.

Binding kinetics of human anti-human TNF antibodies to human TNF. For example, the binding characteristics of antibodies can be suitably assessed using TNF capture EIA and BIAcore techniques. In the assay described above, the fractional concentration of purified human TNF antibody used to bind to EIA plates coated with 2 μ g/mL TNF can be assessed. The OD can then be expressed as a semi-logarithmic graph showing relative binding efficiency.

The quantitative binding constant may be obtained, for example, as follows, or by any other known suitable method. The BIAcore CM-5 (carboxymethyl) chip was placed in a BIAcore 2000 unit. HBS buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) was flowed through the flow cell of the chip at 5. mu.L/min until a stable baseline was obtained. A solution of 15mg EDC (N-ethyl-N' - (3-dimethyl-aminopropyl) -carbodiimide hydrochloride) in 200. mu.L of water (100. mu.L) was added to a solution of 100. mu.L NHS (N-hydroxysuccinimide) in 200. mu.L of water. Forty (40) μ L of the resulting solution was injected onto the chip. mu.L of human TNF solution (15. mu.g/mL in 10mM sodium acetate, pH4.8) was injected onto the chip, resulting in an increase of about 500 RU. The buffer was changed to TBS/Ca/Mg/BSA running buffer (20mM Tris, 0.15M sodium chloride, 2mM calcium chloride, 2mM magnesium acetate, 0.5% Triton X-100, 25. mu.g/mL BSA, pH 7.4) and flowed on the chip overnight to allow equilibration and to hydrolyze or block any unreacted succinimide esters.

Antibodies were dissolved in running buffer at 33.33nM, 16.67nM, 8.33nM and 4.17 nM. The flow rate was adjusted to 30 μ L/min and the instrument temperature was adjusted to 25 ℃. Two flow cells were used for kinetic runs, one flow cell with immobilized TNF (sample) and the other flow cell with underivatized flow cell (blank). 120 μ L of each antibody concentration was injected at 30 μ L/min onto the flow cell (association phase) followed by an uninterrupted 360 second buffer flow (dissociation phase). The chip surface was regenerated by two consecutive injections of 30 μ L of 2M guanidinium thiocyanate (tissue necrosis factor α/antibody complex dissociation).

Data analysis was performed using BIA evaluation 3.0 or CLAMP 2.0 as known in the art. For each antibody concentration, blank sensorgrams were subtracted from the sample sensorgram. For dissociation (k)_d，sec^-1) And association (k)_a，mol^-1sec^-1) And calculating (k)_d/k_a) Dissociation constant (K) of_DMol) was fit overall. If the antibody affinity is high enough that the RU > 100 of the captured antibody, additional dilutions of the antibody are made.

Results and discussion

Monoclonal antibodies against human TNF were produced. Several fusions were performed and each fusion was seeded in 15 plates (1440 wells/fusion) to generate tens of antibodies specific for human TNF. Some of these were found to consist of a combination of human and mouse Ig chains. The remaining hybridomas secrete anti-TNF antibodies consisting of only human heavy and light chains. Among the human hybridomas, all were expected to be IgG1 κ.

Binding kinetics of human anti-human TNF antibodies. ELISA analysis confirmed that purified antibodies from most or all of these hybridomas bound TNF in a concentration-dependent manner. The results of the relative binding efficiency of these antibodies are shown in fig. 1 and 2. In this case, the affinity of the antibody for its cognate antigen (epitope) is measured. It should be noted that direct binding of TNF to EIA plates can cause protein denaturation, and the apparent binding affinity cannot reflect binding to non-denatured proteins. 50% binding was found over a range of concentrations.

Quantitative binding constants were obtained using BIAcore analysis of the human antibody, revealing that several human monoclonal antibodies have very high affinity, K_DAt 1X 10^-9To 7X 10^-12Within the range of (1).

Conclusion。

Several fusions were performed using splenocytes from hybrid mice containing human variable and constant region antibody transgenes immunized with human TNF. Several fully human TNF-reactive IgG monoclonal antibodies of a panel of IgG1 kappa isotype were generated. The fully human anti-TNF antibody was further characterized. Several antibodies were generated with a range of 1X 10⁹And 9X 10¹²Affinity constant between. The unexpectedly high affinity of these fully human monoclonal antibodies makes them suitable for therapeutic applications in TNF-dependent diseases, pathologies, or related disorders.

Example 3: human IgG monoclonal antibodies reactive to human TNF α were generated.

And (6) summarizing. (CBA/J x C57BL/6J) F transgenic for human variable and constant region antibodies comprising heavy and light chains₂Hybrid mice (1-4) were immunized with recombinant human TNF α. One fusion, designated GenTNV, produced eight fully human IgG1 kappa monoclonal antibodies that bound to immobilized recombinant human TNF α. Shortly after identification, the eight cell lines were handed over to Molecular Biology institute (Molecular Biology) for further characterization. Since these mabs are completely human in sequence, their immunogenicity in humans is expected to be lower than cA2 (class gram).

Abbreviations. BSA-bovine serum albumin; CO 2₂-carbon dioxide; DMSO-dimethyl sulfoxide; EIA-enzyme immunoassay; FBS-fetal bovine serum; h₂O₂-hydrogen peroxide; an H-heavy chain; HRP-horseradish peroxidase; ID-intradermal; ig-immunoglobulin; TNF-tissue necrosis factor alpha; IP-intraperitoneal (IP-IP); IV-intravenous; mab-monoclonal antibody; OD-optical density; OPD-o-phenylenediamine dihydrochloride; PEG-polyethylene glycol; PSA-penicillin, streptomycin, amphotericin; RT-room temperature; SQ-subcutaneous; TNF α -tumor necrosis factor α; v/v-volume/volume; w/v-weight/volume.

Brief introduction of the drawing. Transgenic mice containing human heavy and light chain immunoglobulin genes were used to produce fully human monoclonal antibodies specific for recombinant human TNF α. It is desirable to use these unique antibodies because cA2 (remikaide) is used therapeutically to inhibit inflammatory processes involved in TNF α -mediated diseases, with the beneficial effects of increased serum half-life and reduced side effects associated with immunogenicity.

As defined herein, the term "half-life" means that the plasma concentration of a drug (e.g., a therapeutic anti-TNF α antibody) is halved after an elimination half-life. Thus, in each subsequent half-life, less drug is eliminated. After one half-life, the amount of drug remaining in the body is 50%, after two half-lives 25%, and so on. The half-life of a drug depends on its clearance and volume of distribution. The elimination half-life is believed to be independent of the amount of drug in the body.

Materials and methods.

An animal. Transgenic mice expressing human immunoglobulin but not mouse IgM or Ig κ have been developed by GenPharm International. These mice contain functional human antibody transgenes that undergo v (d) J-junction, heavy chain-like switching, and somatic mutation to produce a repertoire of antigen-specific human immunoglobulins (1). The light chain transgene portion is derived from a yeast artificial chromosome clone, which includes almost half of the germline human vk locus. In addition to several VH genes, the Heavy Chain (HC) transgene encodes human μ and human γ 1(2) and/or γ 3 constant regions. Mice derived from the HCo12/KCo5 genotype lineage were used in the immunization and fusion process to produce the monoclonal antibodies described herein.

Purification of human TNF α. Human TNF α was purified from the tissue culture supernatant of C237A cells by affinity chromatography using a column packed with TNF α receptor-Fc fusion protein (p55-sf2) (5) coupled to Sepharose 4b (pharmacia). The cell supernatant was mixed with one ninth of its volume of 10 × Dulbecco PBS (D-PBS) and passed through the column at 4 deg.C at 4 mL/min. The column was then washed with PBS, eluting TNF α with 0.1M sodium citrate, pH 3.5, and neutralized with 2M Tris-HCl pH 8.5. Purified TNF α buffer was exchanged into 10mM Tris, 0.12M sodium chloride pH 7.5 and filtered through a 0.2 μ M syringe filter.

And (4) immunization. On days 0, 12 and 28, female GenPharm mice, about 16 weeks old, were immunized with IP (200 μ L) and ID (100 μ L bottom of tail), for a total of 100 μ g TNF α (batches JG102298 or JG102098) emulsified with an equal volume of Titermax adjuvant. Mice were bled on days 21 and 35 by retroorbital puncture without anticoagulant. Blood was allowed to clot for one hour at room temperature, sera were collected and titrated using the TNF α solid phase EIA assay. The fusion called GenTNV was performed after allowing the mice to rest for seven weeks following injection on day 28. Mice with a specific human IgG titer to TNF α of 1: 160 were then given a final IV boost injection of 50 μ g TNF α diluted in 100 μ L of physiological saline. Three days later, mice were euthanized by cervical dislocation, spleens were removed aseptically, and immersed in 10mL of cold Phosphate Buffered Saline (PBS) containing 100U/mL penicillin, 100. mu.g/mL streptomycin, and 0.25. mu.g/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 25mM Hepes.

A cell line. The non-secreting mouse myeloma fusion partner 653 received the cell biology technology services (CBS) group at 5-14-97 in the product development group of Centocor. Cell lines were expanded in RPMI medium (JRH Biosciences) supplemented with 10% (v/v) FBS (cell Culture labs), 1mM sodium pyruvate, 0.1mM NEAA, 2mM L-glutamine (all from JRH Biosciences), and cryopreserved in 95% FBS and 5% DMSO (Sigma) before being stored in a gas phase liquid nitrogen freezer in CBS. Cell banks are sterile (Quality Control centre, Malvern) and mycoplasma free (Bionique Laboratories). Cells were maintained in log phase cultures until confluent. Cells were washed with PBS, counted, and cell viability was determined (> 95%) via trypan blue dye exclusion prior to fusion.

Human TNF α was produced by a recombinant cell line, designated C237A, in Molecular Biology by Centocor. In a medium supplemented with 5% (v/v) FBS (cell Culture labs), 2mM L-glutamine (both from JRH Biosciences) and 0.5: the cell lines were expanded in IMDM medium (JRHBOSciences) at g/mL mycophenolic acid and cryopreserved in 95% FBS and 5% DMSO (Sigma) and then stored in a vapor phase liquid nitrogen freezer in CBS (13). Cell banks are sterile (Quality Control centre, Malvern) and mycoplasma free (Bionique Laboratories).

And (4) fusing the cells. Cell fusion was performed using 653 murine myeloma cells and live murine splenocytes in a 1: 1 ratio. Briefly, splenocytes are pelleted with myeloma cells. The pellet was slowly resuspended in 1mL of a 50% (w/v) PEG/PBS solution (PEG molecular weight 1,450g/mol, Sigma) at 37 ℃ at 30 ℃. Fusion was terminated by slow addition of 10.5mL RPMI medium (without additives) (JRH) (37 ℃) over 1 minute. The fused cells were centrifuged at 750rpm for 5 minutes. The cells were then resuspended in HAT medium (RPMI/HEPES medium containing 10% fetal bovine serum (JRH), 1mM sodium pyruvate, 2mM L-glutamine, 10 μ g/mL gentamicin, 2.5% Origen culture supplement (Fisher), 50 μ M2-mercaptoethanol, 1% 653 conditioned reflex RPMI medium, 100 μ M hypoxanthine, 0.4 μ M aminopterin, and 16 μ M thymidine) and then plated at 200 μ L/well in five 96-well flat-bottom tissue culture plates. The panels were then placed in a 5% CO holding chamber₂And 95% air in a humidified 37 ℃ incubator for 7-10 days.

Human IgG anti-TNF α antibodies were detected in mouse sera. Solid phase EIA was used to screen mouse sera for human IgG antibodies specific for human TNF α. Briefly, plates were coated overnight with 1. mu.g/mL TNF α in PBS. After washing in 0.15M saline containing 0.02% (v/v) Tween 20, wells were blocked with 1% (w/v) BSA in PBS, 200. mu.L/well for 1 hour at room temperature. Immediately, the plate is used or frozen at-20 ℃ for use. Mouse serum was incubated at 50 μ L/well on human TNF α coated plates for 1 hour at room temperature in two-fold serial dilutions. Plates were washed and then probed with 50 μ L/well HRP labeled goat anti-human IgG diluted 1: 30,000 in 1% BSA-PBS, Fc specific (accurate) for 1 hour at room temperature. Plates were washed again and 100. mu.L/well of citrate-phosphate substrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H) was added at room temperature ₂O₂And 1mg/mL OPD) for 15 minutes. Stop solution (4N sulfuric acid) was then added at 25 μ L/well and the OD read at 490nm using an automatic plate spectrophotometer.

And (3) detecting the human whole immunoglobulin in the hybridoma supernatant. Because the GenPharm mouse is capable of producing both mouse and human immunoglobulin chains, two separate EIA assays were used to test growth positive hybridoma clones for the presence of human light and human heavy chains. Plates were coated as described above and undiluted hybridoma supernatant was incubated on the plates for one hour at 37 ℃. The plates were washed and probed with HRP-conjugated goat anti-human kappa (Southern Biotech) antibody diluted 1: 10,000 in 1% BSA-HBSS or HRP-conjugated goat anti-human IgG Fc specific antibody diluted 1: 30,000 in 1% BSA-HBSS at 37 ℃ for 1 hour. The plate was then incubated with the substrate solution as described above. Hybridoma clones that gave no positive signal in both anti-human κ and anti-human IgG Fc EIA formats were discarded.

Isoforms. Isotyping of antibodies was done using EIA in a format similar to the specific titer used to screen immune sera from mice. EIA plates were coated with goat anti-human IgG (H + L) 10: g/mL in sodium carbonate buffer overnight at 4E ℃ and blocked as described above. The clear supernatant from the 24-well culture was incubated on the plate for one hour at room temperature. Plates were washed and were diluted 1: 4000 with HRP-labeled goat anti-human IgG in 1% BSA-PBS ₁、IgG₂、IgG₃Or IgG₄(binding site) was probed for one hour at room temperature. The plate was washed again and incubated with substrate solution as described above.

Results and discussion. Fully human anti-human TNF α monoclonal antibodies were generated. A fusion named GenTNV was performed once from GenPharm mice immunized with recombinant human TNF α protein. 196 growth positive hybrids were selected by this fusion. Eight hybridoma cell lines were identified that secreted fully human IgG antibodies reactive with human TNF α. Each of these eight cell lines secreted immunoglobulin of the human IgG1 kappa isotype and were subcloned twice in their entirety by limiting dilution to obtain stable cell lines (> 90% homogeneous). Table 1 lists cell line names and corresponding C code designations. Each of the cell lines was frozen in 12 vial study cell banks stored in liquid nitrogen.

Parental cells collected from wells of 24-well culture dishes for each of the eight well cell lines were handed over to the Molecular Biology group for transfection and further characterization at 2-18-99.

Table 1: GenTNV cell line nomenclature

Conclusion。

GenTNV fusions were performed using splenocytes from hybrid mice containing human variable and constant region antibody transgenes immunized with recombinant human TNF α prepared at Centocor. Eight fully human TNF α -reactive IgG monoclonal antibodies of the IgG1 κ isotype were generated. Parental cell lines were transferred to the Molecular Biology panel for further characterization and development. One of these novel human antibodies may prove useful in anti-inflammation, with potential beneficial effects of reduced immunogenicity and allergic complications, compared to remicade.

Reference documents:

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).

Scalon et al, Cytokine 7: 759-770(1995).

Example 4: cell lines expressing human anti-TNF α antibodies were cloned and prepared.

And (6) summarizing. A panel of eight human monoclonal antibodies (mabs) with TNV nomenclature was found to bind immobilized human TNF α with significantly high affinity. Seven of the eight mabs were shown to effectively block binding of huTNF α to the recombinant TNF receptor. Sequence analysis of the DNA encoding the seven mabs confirmed that all mabs had human V-regions. The DNA sequences also revealed that the three pairs of mabs were identical to each other, so that the original set of eight groups of mabs contained only four different mabs, represented by TNV14, TNV15, TNV148, and TNV 196. Based on the analysis of the deduced amino acid sequences of the mabs and the results of the in vitro TNF α neutralization data, mabs TNV148 and TNV14 were selected for further study.

Because during the database search, no proline residue at position 75 (frame 3) of the TNV148 heavy chain was found at this position in other human antibodies of the same subgroup, site-directed DNA mutagenesis was performed to encode a serine residue at this position so that it fits into a known germline framework e sequence. The serine modified mAb was designated TNV 148B. The 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 heavy and light chain genes of another recently cloned human mAb (12B75), disclosed in U.S. patent application 60/236,827 entitled "IL-12 Antibodies, Compositions, Methods and Uses", filed on 10/7/2000, which is disclosed as WO 02/12500, incorporated herein by reference in its entirety.

P3X63Ag8.653(653) cells or Sp2/0-Ag14(Sp2/0) mouse myeloma cells were transfected with the corresponding heavy and light chain expression plasmids and cell lines producing high levels of recombinant TNV148B and TNV14(rTNV148B and rTNV14) mAbs were selected by two rounds of subcloning. Evaluation of growth curves and stability of mAb production over time indicated that 653-transfectant clones C466D and C466C stably produced about 125: g/ml rTNV148B mAb, whereas Sp2/0 transfectant 1.73-12-122(C467A) stably produced about 25: rTNV148B mAb in g/ml. Similar analysis showed that Sp 2/0-transfectant clone C476A produced 18: rTNV14 in g/ml.

Brief introduction of the drawing. Groups of eight mabs from GenPharm/Medarex mice immunized with human TNF α (HCo12/KCo5 genotype) previously shown to bind human TNF α and have fully human IgG1, the kappa isotype. Whether an exemplary mAb of the invention is likely to have TNF α neutralizing activity is determined using a simple binding assay by evaluating its ability to block TNF α binding to a recombinant TNF receptor. Based on these results, DNA sequence results and in vitro characterization of several mabs, TNV148 was selected as the mAb to be further characterized.

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

Materials and methods：

Reagents and cells. TRIZOL reagent was purchased from Gibco BRL. Proteinase K is available from Sigma Chemical Company. Reverse transcriptase was obtained from Life Sciences, Inc., and Taq DNA polymerase from Perkin Elmer Cetus or Gibco BRL. Restriction enzymes were purchased from New England Biolabs. The QIAquick PCR purification kit was from Qiagen. QuikChange site-directed mutagenesis kit was purchased from Stratagene. Wizard plasmid minipreps kit and RNase were from Promega. The optical plate is available 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 shown in table 2.

TABLE 2 oligonucleotides used for cloning, engineering or sequencing TNV mAb genes.

The amino acids encoded by oligonucleotides 5' 14s and HuH-J6 are shown above the sequence. The 'M' amino acid residue represents a translation initiation codon. The underlined sequences in the oligonucleotides 5' 14s and HuH-J6 mark BsiWI and BstBI restriction sites, respectively. The diagonal lines in HuH-J6 correspond to the exon/intron boundaries. Note that the oligonucleotides whose sequences correspond to the minus strand are written in the 3 '-5' direction.

A single frozen vial of 653 mouse myeloma cells was obtained. Vials were thawed on the day and expanded in IMDM, 5% FBS, 2mM glutamine (medium) in T-flasks. These cells were maintained in continuous culture until they were transfected with anti-TNF DNA described herein after 2 to 3 weeks. Some cultures were harvested 5 days after the thawing day, pelleted by centrifugation, and resuspended in 95% FBS, 5% DMSO, aliquoted into 30 vials, frozen, and stored for future use. Similarly, a single frozen vial of Sp2/0 mouse myeloma cells was obtained. The vials were thawed, fresh freezes were prepared as described above, and frozen vials were stored in CBC freezers AA and AB. These cells were thawed and used for all Sp2/0 transfections described herein.

Assays for inhibiting TNF binding to a receptor. Hybridoma cell supernatants containing TNV mAb for determination of mAb blockade¹²⁵The ability of I-labeled TNF α to bind to the recombinant TNF receptor fusion protein p55-sf2 (Scallon et al, (1995) Cytokine 7: 759-770). 50: 1 in PBS 0.5: g/ml of p55-sf2 was added to the optical plate to coat the wells during one hour of incubation at 37 ℃. Serial dilutions of eight TNV cell supernatants were prepared in 96-well round bottom plates using PBS/0.1% BSA as diluent. Cell supernatants containing anti-IL-18 mAb were included as negative controls, and the same anti-IL-18 supernatants spiked with cA2 (anti-TNF chimeric antibody, Remikade, U.S. Pat. No. 5,770,198, incorporated herein by reference in its entirety) were included as positive controls. Will be provided with ¹²⁵I-labeled TNF α (58: Ci/: g, D.Sheary) was added to the 100: 1 cell supernatant to give a final TNF α concentration of 5 ng/ml. The mixture was preincubated for one hour at room temperature. The coated optical sheets were washed to remove unbound p55-sf2, and 50: l¹²⁵The I-TNF α/cell supernatant mixture was transferred to the optical plate. After 2 hours at room temperature, the plates were washed three times with PBS-Tween. Adding 100: microscint-20 was l and cpm binding was determined using a TopCount gamma counter.

V gene amplification and DNA sequence analysis. Hybridoma cells were washed once in PBS, and TRIZOL reagent was then added for RNA preparation. Will be between 7 × 10⁶And 1.7X 10⁷The cells between individuals were resuspended in 1ml TRIZOL. After addition of 200. mu.l chloroform the tube was shaken vigorously. The samples were centrifuged at 4 ℃ for 10 minutes. The aqueous phase was transferred to a new microcentrifuge tube and an equal volume of isopropanol was added. The tube was shaken vigorously and allowed to incubate for 10 minutes at room temperature. The samples were then centrifuged at 4 ℃ for 10 minutes. The precipitate was washed once with 1ml 70% ethanol and dried briefly in a vacuum desiccator. The RNA pellet was resuspended in 40. mu.l of DEPC-treated water. The quality of the RNA preparation was determined by fractionating 0.5. mu.l in a 1% agarose gel. The RNA was stored in a-80 ℃ freezer until use.

To prepare the heavy and light chain cDNAs, a mixture was prepared containing 3. mu.l of RNA and 1. mu.g of either oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) (see Table 1) in a volume of 11.5. mu.l. The mixture was incubated in a water bath at 70 ℃ for 10 minutes and then cooled on ice for 10 minutes. Separate mixtures were prepared consisting of 2.5. mu.l 10 XTRT buffer, 10. mu.l 2.5mM dNTP, 1. mu.l reverse transcriptase (20 units) and 0.4. mu.l RNase inhibitor RNase (1 unit). 13.5. mu.l of this mixture was added to 11.5. mu.l of the cooled RNA/oligonucleotide mixture and the reaction was incubated at 42 ℃ for 40 minutes. The cDNA synthesis reaction was then stored in a-20 ℃ freezer until use.

Unpurified heavy and light chain cdnas were used as templates to PCR amplify the variable region coding sequences. Five oligonucleotide pairs (366/354, 367/354, 368/354, 369/354, and 370/354, table 1) were tested simultaneously for their ability to prime heavy chain DNA amplification. Two oligonucleotide pairs (362/208 and 363/208) were tested simultaneously for their ability to prime light chain DNA amplification. Using 2 units of PLATINUM^TMHigh fidelity (HIFI) Taq DNA polymerase was used for PCR in a total volume of 50. mu.l. Each reaction included 2. mu.l of cDNA reaction, 10pmole of each oligonucleotide, 0.2mM dNTP, 5. mu.l of 10 XHIFI buffer and 2mM magnesium sulfate. The thermocycler program was 95 ℃ for 5 minutes followed by 30 cycles (94 ℃ for 30 seconds, 62 ℃ for 30 seconds, 68 ℃ for 1.5 minutes). Then a final incubation at 68 ℃ for 10 minutes was performed.

To prepare PCR products for direct DNA sequencing, QIAquick was used^TMPCR purification kits purified them according to the manufacturer's protocol. The DNA was eluted from the spin column using 50. mu.l of sterile water, and then dried to a volume of 10. mu.l using a vacuum drier. Then 1. mu.l of the purified PCR product, 10. mu.M of the oligonucleotide primer, 4. mu.l of the BigDye Terminator were used^TMThe reaction mixture was prepared and a DNA sequencing reaction was set up with 14. mu.l of sterile water in a total volume of 20. mu.l. The heavy chain PCR product prepared with oligonucleotide pair 367/354 was sequenced with oligonucleotide primers 159 and 360. The light chain PCR product prepared with oligonucleotide pair 363/208 was sequenced with oligonucleotides 34 and 163. The thermal cycler program for sequencing was 25 cycles (96 ℃ for 30 ℃ C.)Seconds, 50 ℃ for 15 seconds, 60 ℃ for 4 minutes), followed by overnight at 4 ℃. The reaction products were separated by polyacrylamide gel and detected using an ABI377DNA sequencer.

Site-directed mutagenesis was performed to change the amino acids. Altering a single nucleotide in the TNV148 heavy chain variable region DNA sequence to replace Pro with a serine residue in TNV148 mAb⁷⁵. Complementary oligonucleotides 399 and 400 (Table 1) were designed and ordered to use QuikChange as described by the manufacturer^TMSite-directed mutagenesis methods make this change. The two oligonucleotides were first fractionated through a 15% polyacrylamide gel and the main band was purified. Mutagenesis reactions were prepared using 10ng or 50ng TNV148 heavy chain plasmid template (p1753), 5. mu.l 10 Xreaction buffer, 1. mu.l dNTP mix, 125ng primer 399, 125ng primer 400 and 1. mu.l Pfu DNA polymerase. Sterile water was added to bring the total volume to 50. mu.l. The reaction mixture was then incubated in a thermal cycler programmed to incubate at 95 ℃ for 30 seconds, and then cycle 14 times, with 95 ℃ being incubated continuously for 30 seconds, 55 ℃ for 1 minute, 64 ℃ for 1 minute, 68 ℃ for 7 minutes, followed by 30 ℃ for 2 minutes (1 cycle). These reactions were designed to incorporate mutagenic oligonucleotides into otherwise identical newly synthesized plasmids. To remove the original TNV148 plasmid, the samples were incubated at 37 ℃ for 1 hour after addition of 1. mu.l of DpnI endonuclease, which cleaves only the original methylated plasmid. One μ l of the reaction was then used to transform Epicurian Coli XL1-Blue supercompetent E.coli by standard heat shock methods and the transformed bacteria were identified after plating on LB-ampicillin agar plates. Using Wizard as described by the manufacturer ^TMThe kit prepared plasmid minipreps. From Wizard^TMAfter the column elution samples, with ethanol precipitation of plasmid DNA to further purification of plasmid DNA, and then heavy suspension in 20 u l sterile water. DNA sequence analysis was then performed to identify plasmid clones with the desired base changes and confirm that no other base changes were inadvertently introduced into the TNV148 coding sequence. Using the same parameters described in section 4.3, one μ l of plasmid was subjected to a cycle sequencing reaction prepared with 3 μ l of the BigDye mixture, 1 μ l of the pUC19 forward primer and 10 μ l of sterile water.

Construction of expression vector from 12B75 gene. Several recombinant DNA procedures were performed to prepare a new human IgG1 expression vector and a new human kappa expression vector from previously cloned genomic copies of the heavy and light chain genes encoding 12B75, respectively, as disclosed in U.S. patent application 60/236,827, entitled "IL-12 Antibodies, Compositions, Methods and Uses", filed on 7/10/2000, which is published as WO 02/12500, which is incorporated herein by reference in its entirety. The final vector is designed to allow simple one-step replacement of the existing variable region sequence with any appropriately designed PCR amplified variable region.

To modify the 12B75 heavy chain gene in plasmid p1560, a 6.85kb BamHI/HindIII fragment containing the promoter and variable region was transferred from p1560 to pUC19 to make p 1743. The smaller size of this plasmid compared to p1560 enables the use of QuikChange according to the manufacturer's protocol^TMMutagenesis (using oligonucleotides BsiWI-1 and BsiWI-2) introduced a unique BsiWI cloning site upstream of the translation initiation site. The resulting plasmid was designated p 1747. To introduce a BstBI site at the 3 'end of the variable region, 5' oligonucleotide primers with SalI and BstBI sites were designed. This primer was used with the pUC reverse primer to amplify a 2.75kb fragment from p 1747. This fragment was then cloned back into the naturally occurring SalI site in the 12B75 variable region and HindIII site, thereby introducing a unique BstB1 site. The resulting intermediate vector (designated p1750) can accept variable region fragments with BsiWI and BstBI termini. To prepare a heavy chain vector version in which the constant region was also derived from the 12B75 gene, the BamHI-HindIII insert in p1750 was transferred to pBR322 to have an EcoRI site downstream of the HindIII site. The resulting plasmid p1768 was then digested with HindIII and EcoRI and ligated to the 5.7kb HindIII-EcoRI fragment from p1744, obtained by cloning the large BamHI-BamHI fragment of p1560 into pBC. The resulting plasmid p1784 was then used as a vector for the TNV Ab cDNA fragment with BsiWI and BstBI termini. Additional work was done to prepare expression vectors p1788 and p1798, which included the IgG1 constant region from the 12B75 gene, and which differed from each other by how many of the 12B75 heavy chain J-C introns they contained.

To modify the 12B75 light chain gene in plasmid p1558, a 5.7kb SalI/AflII fragment containing the 12B75 promoter and variable region was transferred from p1558 to the XhoI/AflII site of plasmid L28. This new plasmid p1745 provides a smaller template for the mutagenesis step. QuikChange Using oligonucleotides (C340salI and C340sal2)^TMMutagenesis introduces a unique SalI restriction site at the 5' end of the variable region. The resulting intermediate vector, p1746, has unique SalI and AflII restriction sites allowing the cloning of variable region fragments. Any variable region fragment cloned into p1746 is preferably ligated to the 3' half of the light chain gene. To prepare restriction fragments from the 3' half of the 12B75 light chain gene that can 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, HindIII and NotI, and which contains ends that can be ligated to KpnI and SacI sites. This linker was cloned between KpnI and SacI sites of pBC to give plasmid p 1757. The 7.1kb fragment containing the 12B75 light chain constant region, generated by digestion of p1558 with AflII and then partial digestion with HindIII, was cloned between the AflII and HindIII sites of p1757 to give p 1762. The new plasmid contains unique sites for BsiWI and AflII, where BsiWI/AflII fragments containing promoter and variable regions can be transferred, binding both halves of the gene.

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 restriction enzymes BsiWI and BstBI and then cloned between the BsiWI and BstBI sites of plasmid L28 (L28 was used since an intermediate vector p1750 based on 12B75 was not prepared yet). DNA sequence analysis of the clone insert showed that the resulting construct was correct and no errors were introduced during PCR amplification. The assigned identification numbers for these L28 plasmid constructs (TNV14, TNV15, TNV148B and TNV196) are shown in table 3.

The BsiWI/BstBI insert of the TNV14, TNV148 and TNV148B heavy chains was transferred from the L28 vector to the newly prepared intermediate vector p 1750. The assigned identification numbers of these intermediate plasmids are shown in Table 2. This cloning step and subsequent steps were not completed for TNV15 and TNV 196. The variable regions were then transferred into two different human IgG1 expression vectors. Restriction enzymes EcoRI and HindIII were used to transfer the variable regions into the IgG1 vector p104 previously used by Centocor. The resulting expression plasmids encoding Gm (f +) isotype IgG1 were designated p1781(TNV14), p1782(TNV148) and p1783(TNV148B) (see table 2). The variable region was also cloned upstream of the IgG1 constant region from the 12B75(Genpharm) gene. Those expression plasmids encoding IgG1 of the G1m (z) allotype are also listed in Table 3.

TABLE 3 plasmid identification numbers for various heavy and light chain plasmids.

The L28 vector or the pBC vector represents the original Ab cDNA clone. The inserts in those plasmids were transferred to incomplete 12B 75-based vectors to prepare intermediate plasmids. An additional transfer step produces the final expression plasmid, which is introduced into the cells after linearization or used to purify the mAb gene insert prior to cell transfection. (ND) is not tested.

The light chain PCR product was digested with restriction enzymes SalI and SacII and then cloned between the SalI and SacII sites of plasmid pBC. The two different light chain forms (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 from p1748 and p1749 were then cloned into the intermediate vector p1746 between the SalI and AflII sites to make p1755 and p1756, respectively. The 5 'half of these light chain genes were then ligated to the 3' half of the genes by transferring the BsiWI/AflII fragments from p1755 and p1756 to the newly prepared construct p1762 to prepare the final expression plasmids p1775 and p1776, respectively (table 2).

Cell transfection, screening and subcloning. A total of 15 mouse myeloma cell transfections were performed with various TNV expression plasmids (see table 3 in the results and discussion section). These transfections differ by (1) whether the host cell is Sp2/0 or 653; (2) the heavy chain constant region is encoded by either the Centocor previous IgG1 vector or the 12B75 heavy chain constant region; (3) the mAb is TNV148B, TNV148, TNV14, or a novel HC/LC combination; (4) whether the DNA is a linearized plasmid or a purified Ab gene insert; and (5) the presence or absence of the complete J-C intron sequence in the heavy chain gene. In addition, several transfections were repeated to increase the likelihood of screening a large number of clones.

Sp2/0 cells and 653 cells were transfected with a mixture of heavy and light chain DNA (8 g-12: g each), respectively, by electroporation under standard conditions as described previously (Knight DM et al, (1993) Molecular Immunology 30: 1443-1453). For transfection numbers 1, 2, 3 and 16, the appropriate expression plasmids were linearized by digestion with restriction enzymes prior to transfection. For example, SalI and NotI restriction enzymes were used to linearize TNV148B heavy chain plasmid p1783 and light chain plasmid p1776, respectively. For the remaining transfections, the DNA insert containing only the mAb gene was 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 insert was then purified by agarose gel electrophoresis and qiax purification resin. Cells transfected with purified gene inserts were simultaneously transfected with 3-5: g of the PstI-linearized pSV2gpt plasmid (p13) as a source of selection marker. After electroporation, cells were seeded in IMDM, 15% FBS, 2mM glutamine in 96-well tissue culture dishes at 37 ℃ with 5% CO₂Incubation in an incubator. Two days later, 2 x MHX selection (1 x MHX ═ 0.5: g/ml mycophenolic acid, 2.5: g/ml hypoxanthine, 50: g/ml xanthine) with equal volumes of IMDM, 5% FBS, 2mM glutamine was added and the plates incubated for an additional 2 to 3 weeks while colonies formed.

Cell supernatants collected from wells with colonies were assayed for human IgG by ELISA as described. Briefly, cell supernatants at various dilutions were incubated in 96-well EIA plates coated with polyclonal goat anti-human IgG Fc fragment, and bound human IgG was then detected using alkaline phosphatase conjugated goat anti-human IgG (H + L) and appropriate color substrates. A standard curve was included on each EIA plate, which was used as a standard curve for the same purified mAb measured in cell supernatants to enable quantification of human IgG in the supernatants. Cells from those colonies that appear to produce the most human IgG are passaged into 24-well plates for additional production assays in spent culture, followed by identification of the parent clones with the highest yield.

The highest yielding parental clones were subcloned to identify higher yielding subclones and to generate more homogeneous cell lines. A96 well tissue culture plate was seeded at 1 XMHX with one cell per well or four cells per well of IMDM, 5% FBS, 2mM glutamine at 37 ℃ and 5% CO₂Incubate in incubator for 12 to 20 days until colonies are evident. Cell supernatants were collected from wells containing one colony per well and analyzed by ELISA as described above. Selected colonies were passaged to 24-well plates and the culture was allowed to deplete before the highest-yielding subclones were identified by quantifying human IgG levels in their supernatants. This process was repeated when the first round of subcloning was selected to undergo the second round of subcloning. The best secondary subclones were selected for cell line development.

Characterization of cell subcloning. The best second round of subcloning was selected and growth curves were performed to evaluate mAb production levels and cell growth characteristics. A T75 flask was replaced with a 1X 10 flask⁵Individual cells/ml were seeded in 30ml IMDM, 5% FBS, 2mM glutamine and 1X MHX (or serum free medium). Aliquots of 300. mu.l were removed at 24-hour intervals and viable cell density was determined. The analysis was continued until the number of viable cells was less than 1X 10⁵Individual cells/ml. The concentration of antibody present in the collected cell supernatant aliquot is determined. ELISA assays were performed using standard rTNV148B or rTNV14 JG 92399. 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) diluted 1: 1000.

To compare growth rates in the presence of different amounts of MHX selection, different growth curve analyses were also performed on the two cell lines. Cell lines C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2mM glutamine) and cultured for two more days. The two cell cultures were then divided into three cultures which contained no MHX, 0.2X MHX or 1X MHX (1X MHX ═ 0.5: g/ml mycophenolic acid, 2.5: g/ml hypoxanthine, 50: g/ml xanthine). One day later, 1X 10 of the culture was used ⁵An initial density of individual cells/ml was inoculated into a fresh T75 flask,and cells were counted at 24 hour intervals for one week. Aliquots for mAb production were not collected. The doubling time of these samples was calculated using the formula provided in SOP PD 32.025.

Additional studies were performed to evaluate the stability of mAb generation over time. Cultures were grown in 24-well plates with or without MHX-selected IMDM, 5% FBS, 2mM glutamine. When the culture becomes confluent, the culture breaks into fresh cultures and the old culture is allowed to disappear. At this point, an aliquot of the supernatant was removed and stored at 4 ℃. Aliquots were removed over a period of 55-78 days. At the end of this period, the supernatants were tested for the amount of antibody present in an anti-human IgG Fc ELISA as described above.

Results and discussion.

Inhibiting the binding of TNF to the recombinant receptor.

A simple binding assay was performed to determine whether the eight TNV mabs contained in the hybridoma cell supernatants could block the binding of TNF α to the receptor. The concentration of TNV mAb in the supernatant of the respective cells was first determined by standard ELISA analysis of human IgG. The recombinant p55 TNF receptor/IgG fusion protein p55-sf2 was then coated onto EIA plates and allowed to dry ¹²⁵I-labeled TNF α binds to the p55 receptor in the presence of varying amounts of TNV mAb. As shown in figure 1, all but one of the eight TNV mabs (TNV122) effectively blocked TNF α binding to the p55 receptor. In fact, TNV mAb appears to be more effective at inhibiting TNF α binding than the cA2 positive control mAb incorporated into the negative control hybridoma supernatant. These results are interpreted to indicate that TNV mabs are highly likely to block TNF α bioactivity in cell-based assays and in vivo, thus requiring additional analysis.

And (5) analyzing a DNA sequence.

The RNA was confirmed to encode human mAb.

As a first step to characterize the seven TNV mabs (TNV14, TNV15, TNV32, TNV86, TNV118, TNV148 and TNV196) that showed TNF α blocking activity in the receptor binding assay, total RNA was isolated from the seven hybridoma cell lines producing these mabs. Each RNA sample was then used to prepare human antibody heavy or light chain cDNA comprising the complete signal sequence, the complete variable region sequence and part of the constant region sequence of each mAb. These cDNA products were then amplified in a PCR reaction and the PCR-amplified DNA was directly sequenced without first cloning the fragments. The sequenced heavy chain cDNA was > 90% identical to one of the five human germline genes present in mouse DP-46 (FIG. 2). Similarly, the sequenced light chain cDNA was 100% or 98% identical to one of the human germline genes present in the mouse (fig. 3). These sequence results confirm that the RNA molecules transcribed into cDNA and sequenced encode human antibody heavy chains and human antibody light chains. It should be noted that because the variable region was PCR amplified using an oligonucleotide mapped to the 5' end of the signal sequence coding sequence, the first few amino acids of the signal sequence may not be the actual sequence of the original TNV translation product, but they do represent the actual sequence of the recombinant TNV mAb.

Unique neutralizing mabs.

Analysis of the cDNA sequences of the entire variable regions of both the heavy and light chains of each mAb showed that TNV32 was identical to TNV15, TNV118 was identical to TNV14, and TNV86 was identical to TNV 148. The results of the receptor binding assay are consistent with DNA sequence analysis, i.e. both TNV86 and TNV148 block TNF binding by about 4-fold over both TNV118 and TNV 14. Therefore, the follow-up work was only directed against four unique TNV mabs, TNV14, TNV15, TNV148 and TNV 196.

Correlation of four mAbs

The DNA sequence results show that the genes encoding the heavy chains of the four TNV mabs are highly homologous to each other and appear to be all derived from the same germline gene DP-46 (fig. 2). In addition, because each of the heavy chain CDR3 sequences are so similar and identical in length, and because they all use the J6 exon, they are apparently caused by a single VDJ gene rearrangement event, followed by somatic changes, making each mAb unique. DNA sequence analysis showed that there were only two different light chain genes in the four mabs (fig. 3). The light chain variable region coding sequences in TNV14 and TNV15 are identical to each other and to a representative germline sequence of the Vg/38K family of human kappa chains. TNV148 and TNV196 light chain coding sequences are identical to each other but differ from the germline sequences at two nucleotide positions (fig. 3).

The deduced amino acid sequences of the four mabs revealed the relevance of the actual mabs. The four mabs contained four different heavy chains (fig. 4), but only two different light chains (fig. 5). The differences between TNV mAb sequence and germline sequence were mainly limited to CDR domains, but the three mAb heavy chains also differed from germline sequences in the framework regions (fig. 4). TNV14 is identical, TNV15 differs by one amino acid, TNV148 differs by two amino acids, and TNV196 differs by three amino acids compared to the DP-46 germline-encoded Ab framework region.

cloning of cDNA, site-specific mutagenesis, and assembly of the final expression plasmid. cloning of cDNA. Based on the DNA sequence of the PCR amplified variable region, the new oligonucleotide is instructed to perform another round of PCR amplification with the aim of adapting the coding sequence to be cloned for cloning into the expression vector. In the case of the heavy chain, the product of this second round of PCR was digested with the restriction enzymes BsiWI and BstBI and cloned into the plasmid vector L28 (plasmid identification numbers are shown in table 2). In the case of the light chain, the second round PCR product 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 the previous sequences obtained from direct sequencing of the PCR products, revealing the most abundant nucleotides at each position in the potentially heterogeneous population of molecules.

Site-specific mutagenesis to alter TNV 148. Upon neutralization of TNF α bioactivity, the mabs TNV148 and TNV196 were consistently observed to be four-fold stronger than the next best mAb (TNV 14). However, as described above, TNV148 and TNV196 heavy chain framework sequences differ from germline framework sequences. Comparison of TNV148 heavy chain sequences with other human antibodies indicates that many other human mabs contain an Ile residue at position 28 in framework 1 (only mature sequences are counted), while the Pro residue at position 75 in framework 3 is an unusual amino acid at this position.

Similar comparisons of TNV196 heavy chains indicate that three amino acids different from the germline sequence in framework 3 may be rare in human mabs. These differences may render TNV148 and TNV196 immunogenic if administered to humans. Since TNV148 has only one amino acid residue of interest, which is considered to be insignificant for TNF α binding, site-specific mutagenesis techniques are used to alter individual nucleotides in the TNV148 heavy chain coding sequence (in plasmid p 1753) to encode germline Ser residues instead of Pro residues at position 75. The resulting plasmid was designated p1760 (see Table 2). The resulting gene and mAb were designated TNV148B to distinguish them from the original TNV148 gene and mAb (see fig. 5).

And (5) assembling the final expression plasmid. New antibody expression vectors were prepared based on the 12B75 heavy and light chain genes previously cloned as genomic fragments. Although different TNV expression plasmids were prepared (see table 2), in each case the 5' flanking sequence, promoter and intron enhancer were derived from the corresponding 12B75 gene. For the light chain expression plasmid, the entire J-C intron, constant region coding sequence, and 3' flanking sequences were also derived from the 12B75 light chain gene. For the heavy chain expression plasmids that resulted in the final producer cell line (p1781 and p1783, see below), the human IgG1 constant region coding sequence was derived from the expression vector previously used by Centocor (p 104). Importantly, the final producer cell lines reported here expressed different allotypes of TNV mAb (Gm (f +)), rather than the original hybridoma-derived TNV mAb (G1m (z)). This is because the 12B75 heavy chain gene from GenPharm mouse encodes an Arg residue at the C-terminus of the CH1 domain, while the Centocor IgG1 expression vector p104 encodes a Lys residue at this position. Other heavy chain expression plasmids (e.g., p1786 and p1788) were prepared in which the J-C intron, the entire constant region coding sequence, and the 3' flanking sequence were derived from the 12B75 heavy chain gene, but no cell line transfected with these genes was selected as the producer cell line. The vector was carefully designed to allow one-step cloning of future PCR-amplified V-regions, which would result in the final expression plasmid.

The PCR amplified variable region cDNA was transferred from the L28 or pBC vector to an intermediate stage, 12B 75-based vector, which provided a promoter region and part of the J-C intron (plasmid identification number see table 2). The restriction fragments containing the 5 'half of the antibody gene were then transferred from these intermediate stage vectors to the final expression vector, which provided the 3' half of the corresponding gene to form the final expression plasmid (plasmid identification numbers see table 2).

Cell transfection and subcloning. Expression plasmids were linearized by restriction digestion, or the antibody gene insert in each plasmid was 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, Ab gene specific characteristics defined by Ab, whether the gene was on a linearized whole plasmid or purified gene insert, and 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.

Highest yield rTNV148B cell line

The 653 parental lines (5-10: g/ml generated in spent 24-well cultures) from rTNV148B with the best yields were subcloned to screen the higher yielding cell lines and to generate a more uniform cell population. Two subclones of parental lines 2.320, 2.320-17, and 2.320-20 produced approximately 50: g/ml, 5-fold higher than its parent line. Clones of the second round subclone lines 2.320-17 and 2.320-20 were sub-leading

The identification numbers of the heavy and light chain plasmids encoding each mAb are shown. In the case of transfection with purified mAb gene inserts, plasmid p13(pSV2gpt) was included as the source of gpt selection marker. The heavy chain constant region is encoded by the same human IgG1 expression vector used to encode remicade ('old') or by a constant region included within the 12B75(GenPharm/Medarex) heavy chain gene ('new'). H1/L2 refers to a "novel" mAb consisting of the TNV14 heavy chain and the TNV148 light chain. Plasmids p1783 and p1801 differ only in how many J-C introns their heavy chain genes contain. The right side shows the transfection number, which defines the first number of the common name of the cell clone. Cell lines C466(A, B, C, D) and C467A, which produced rTNV148B, were derived from transfection nos. 2 and 1, respectively. Cell line C476A, which produced rTNV14, was derived from transfection No. 3.

Table 4 summary of cell transfections.

ELISA assay of the spent 24-well culture supernatants showed that these second round subclones all produced between 98g/ml and 124: g/ml, which is at least 2-fold greater than the first round of subcloning. These 653 cell lines were assigned the C code designation as shown in table 5.

The three yield-optimized Sp2/0 parental lines from rTNV148B transfected 1 were subcloned. Two rounds of subcloning of parental line 1.73 led to the identification of 25: g/ml clone. This Sp2/0 cell line was designated C467A (Table 5).

Highest yield rTNV14 cell line

The three yield-optimized Sp2/0 parental lines from rTNV14 transfected 3 were subcloned once. Subclone 3.27-1 was found to be the highest yield of the used 24 well culture, with a yield of 19: g/ml. This cell line was designated C476A (Table 5).

TABLE 5. summary of selected producer cell lines and their C-codes.

The first digit of the original clone name indicates transfection from the cell line. All C-encoding cell lines reported here were derived from transfection of complete plasmids of the heavy and light chains linearized with restriction enzymes.

Characterization of subcloned cell lines

To more carefully characterize cell line growth and determine mAb production levels on a larger scale, growth curve analysis was performed using T75 cultures. The results showed that each of the four C466 series cell lines reached between 1.0X 10⁶And 1.25X 10⁶Peak cell density between individual cells/ml, and maximum mAb accumulation levels between 110g/ml and 140: g/ml (FIG. 7). In contrast to this, the present invention is,the best yield of Sp2/0 subclone C467A reached 2.0X 10⁶Peak cell density of individual cells/ml and 25: maximal mAb accumulation levels in g/ml (figure 7). No growth curve analysis was performed on cell line C476A producing rTNV 14.

Additional growth curve analysis was performed to compare the growth rates selected for different concentrations of MHX. Recent observations indicate that C466 cells cultured in the absence of MHX grow faster than the same cells cultured in normal amounts of MHX (1X). Since cytotoxic concentrations of compounds such as mycophenolic acid tend to be measured on the order of magnitude, it is believed that the use of lower concentrations of MHX may result in significantly faster cell doubling times without sacrificing stability of mAb production. Cell lines C466A and C466B were cultured in: no MHX, 0.2X MHX, or 1X MHX. Viable cell counts were performed every 24 hours for 7 days. These results do reveal MHX concentration dependent cell growth rate (fig. 8). Cell line C466A showed a doubling time of 25.0 hours in 1X MHX, but only 20.7 hours without MHX. Similarly, cell line C466B showed a doubling time of 32.4 hours in 1X MHX, but only 22.9 hours without MHX. Importantly, the doubling time of both cell lines in 0.2X MHX was closer to that of the cell line in 1X MHX than observed in the absence of MHX (figure 8). This observation presents the possibility of enhancing the performance of cells in a bioreactor, where doubling time is an important parameter and can be achieved by using less MHX. However, although the stability test results (see below) indicate that cell line C466D was able to stably produce rTNV148B for at least 60 days even in the absence of MHX, the stability test also showed higher levels of mAb production when cells were cultured in the presence of MHX compared to the absence of MHX.

To evaluate mAb production from various cell lines over a period of about 60 days, stability tests were performed on cultures with or without MHX selection. Not all cell lines maintain high mAb yields. After only two weeks in culture, clone C466A produced about 45% less than at the beginning of the study. The yield of clone C466B also appeared to be significantly reduced. However, clones C466C and C466D maintained fairly stable yields, with C466D showing the highest absolute yield level (fig. 9).

Conclusion

From the first eight human mAb panel directed against human TNF α, TNV148B was selected as preferred based on several criteria including protein sequence and TNF neutralization potency, as well as TNV 14. The preparation production is more than 100: g/ml rTNV148B and 19: cell line of rTNV14 g/ml.

Example 5: arthritis mouse study with anti-TNF antibody and controls using single bolus injection

At approximately 4 weeks of age, Tg197 study mice were assigned to one of 9 treatment groups based on gender and body weight and treated with a single intraperitoneal bolus dose of either 1mg/kg or 10mg/kg of either dulcoside phosphate buffer (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148 or TNV 196).

As a result:when the weight was analyzed as a change compared to pre-dose, animals treated with 10mg/kg cA2 showed consistently higher weight gain throughout the study than animals treated with D-PBS. Body weight increased significantly at weeks 3 to 7. Animals treated with 10mg/kg TNV148 also achieved significant weight gain at study week 7. (see FIG. 10).

Fig. 11A to 11C show progression of disease severity based on the arthritis index. The arthritis index was lower in the 10mg/kg cA2 treated group than in the D-PBS control group starting at week 3 and continuing for the remainder of the study (week 7). Animals treated with 1mg/kg TNV14 and animals treated with 1mg/kg cA2 showed no significant reduction in AI after week 3 when compared to the D-PBS treated group. There was no significant difference between each of the 10mg/kg treatment groups when compared to the other groups at similar doses (10mg/kg cA2 compared to 10mg/kg TNV14, 148 and 196). When comparing the 1mg/kg treatment groups, 1mg/kg TNV148 showed AI at 3, 4 and 7 weeks significantly below 1mg/kg cA 2. At 3 and 4 weeks, 1mg/kg TNV148 was also significantly lower than the 1mg/kg TNV14 treated group. Although TNV196 still showed a significant reduction in AI at study week 6 (when compared to the D-PBS treated group), TNV148 was the only 1mg/kg treatment that remained significant at the end of the study.

Example 6: arthritis mouse study using anti-TNF antibodies and controls as multiple bolus doses

At approximately 4 weeks of age, Tg197 study mice were assigned to one of 8 treatment groups based on body weight and treated with either control preparation (D-PBS) or TNF antibody (TNV14, TNV148) at an intraperitoneal bolus dose of 3mg/kg (week 0). Injections were repeated for all animals at weeks 1, 2, 3 and 4. The test articles of groups 1-6 were evaluated for efficacy. Serum samples obtained from animals of groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3, and 4.

As a result:no significant difference was found when body weight was analyzed as a change from before dosing. Animals treated with 10mg/kg cA2 showed consistently higher weight gain throughout the study than animals treated with D-PBS. (see FIG. 12).

Fig. 13A to 13C show progression of disease severity based on the arthritis index. The arthritis index of the 10mg/kg cA2 treated group was significantly lower than that of the D-PBS control group starting at week 2 and continuing for the remainder of the study (week 5). Animals treated with 1mg/kg or 3mg/kg cA2 and animals treated with 3mg/kg TNV14 failed to achieve any significant reduction in AI at any time throughout the study when compared to the d-PBS treated group. Animals treated with 3mg/kg TNV148 showed a significant decrease when compared to the d-PBS treated group starting at week 3 and continuing up to week 5. At study weeks 4 and 5, 10mg/kg cA2 treated animals showed a significant reduction in AI when compared to lower doses (1mg/kg and 3mg/kg) of cA2, and also significantly lower at weeks 3 to 5 than TNV14 treated animals. Although there did not appear to be a significant difference between any of the 3mg/kg treatment groups, the AI of animals treated with 3mg/kg TNV14 was significantly higher than 10mg/kg at some time points, while the AI of animals treated with TNV148 was not significantly different from animals treated with 10mg/kg cA 2.

Example 7: arthritis mouse study using anti-TNF antibodies and controls as a single intraperitoneal bolus dose

At approximately 4 weeks of age, Tg197 study mice were assigned to one of 6 treatment groups based on gender and body weight and treated with a single intraperitoneal bolus dose of either 3mg/kg or 5mg/kg of antibody (cA2 or TNV 148). The study utilized D-PBS and a 10mg/kg cA2 control group.

When body weight was analyzed as the change from pre-dose, similar weight gain was obtained for all treatments. Animals treated with 3mg/kg or 5mg/kg TNV148 or 5mg/kg cA2 gained significant amounts of body weight early in the study (at weeks 2 and 3). Only animals treated with TNV148 maintained significant weight gain at the later time points. Animals treated with both 3mg/kg and 5mg/kg TNV148 showed significance at 7 weeks, and animals treated with 3mg/kg TNV148 were still significantly elevated at 8 weeks post injection. (see fig. 14).

Figure 15 shows progression of disease severity based on the arthritis index. All treatment groups showed some degree of protection at earlier time points, with 5mg/kg cA2 and 5mg/kg TNV148 showing significant reductions in AI at weeks 1 to 3, and all treatment groups showing significant reductions at week 2. At a later stage of the study, animals treated with 5mg/kg cA2 showed some degree of protection, with significant reductions at weeks 4, 6, and 7. The low dose of cA2 and TNV148 (3mg/kg) showed a significant reduction at week 6 and all treatment groups showed a significant reduction at week 7. At the end of the study (week 8), none of the treatment groups were able to maintain a significant reduction. There was no significant difference between any of the treatment groups (not including the saline control group) at any time point.

Example 8: use of anti-TNF antibodies and controls as a single belly between anti-TNF antibodies and modified anti-TNF antibodies Intramembranous bolus dose arthritis mouse study

The efficacy of a single intraperitoneal dose of TNV148 (from hybridoma cells) and rTNV148B (from transfected cells) was compared. At approximately 4 weeks of age, Tg197 study mice were assigned to one of 9 treatment groups based on gender and body weight and treated with a single intraperitoneal bolus dose of 1mg/kg of du' S phosphate buffer S PBS (D-PBS) or antibody (TNV148, rTNV 148B).

When the weight was analyzed as a change compared to pre-dose, animals treated with 10mg/kg cA2 showed consistently higher weight gain throughout the study than animals treated with D-PBS. Body weight increased significantly at week 1 and 3 to 8. Animals treated with 1mg/kg TNV148 also gained significant weight gain at study weeks 5, 6 and 8. (see FIG. 16).

Figure 17 shows progression of disease severity based on the arthritis index. The arthritis index was lower in the 10mg/kg cA2 treated group than in the D-PBS control group starting at week 4 and continuing for the remainder of the study (week 8). Both the TNV148 treatment group and the 1mg/kg cA2 treatment group showed a significant reduction in AI at week 4. Although the previous study (P-099-017) showed that TNV148 was slightly effective in reducing the arthritis index after a single intraperitoneal bolus of 1mg/kg, the present study showed that the AI was slightly higher in both versions of the TNV antibody treatment group. Although (except for week 6) the 1mg/kg cA2 treated group did not increase significantly when compared to the 10mg/kg cA2 group and the TNV148 treated group was significantly higher at weeks 7 and 8, there was no significant difference in AI between 1mg/kg cA2, 1mg/kg TNV148, and 1mg/kg TNV148B at any time point in the study.

Example 9: for preparing Process for the preparation of (golimumab)

Background to golimumab

Therapies using anti-TNF α agents have been successfully used to treat inflammatory arthritis, but early anti-TNF α agents have limitations in terms of safety, dosing regimen, cost, and/or immunogenicity. To address some of the limitations, fully human anti-TNF α mabs, termed(golimumab). Golimumab (also known as CNTO 148 and rTNV148B) is of the immunoglobulin G1(IgG1) heavy chain isotype (G1m [ z)]Allotype) and κFully human monoclonal antibodies of the light chain isotype. The golimumab has a sequence comprising SEQ ID NO: 36 and a light chain comprising SEQ ID NO: 37 (LC). The molecular weight of golimumab is in the range of 149,802 daltons to 151,064 daltons.

Golimumab forms a high affinity, stable complex with soluble and transmembrane bioactive forms of human tumor necrosis factor alpha (TNF α) with high affinity and specificity, which prevents TNF α from binding to its receptor and neutralizes TNF α bioactivity. No binding to other TNF α superfamily ligands was observed; specifically, golimumab does not bind or neutralize human lymphotoxin. TNF α is synthesized primarily by activated monocytes, macrophages and T cells as a transmembrane protein that self-associates to form a homotrimer of biological activity and is rapidly released from the cell surface by proteolysis. Binding of TNF α to the p55 or p75 TNF receptor results in the aggregation of receptor cytoplasmic domains and initiation of signaling. Tumor necrosis factor has been identified as a key sentinel cytokine produced in response to various stimuli and subsequently promotes the inflammatory response by activating caspase-dependent apoptosis pathways and the transcription factor Nuclear Factor (NF) -kb and activating protein-1 (AP-1). Tumor necrosis factor alpha also modulates the immune response through its role in the immune cell organization of the germinal center. Elevated expression of TNF α has been associated with chronic inflammatory diseases such AS Rheumatoid Arthritis (RA) and spondyloarthropathies such AS psoriatic arthritis (PsA) and Ankylosing Spondylitis (AS). TNF α is an important mediator of joint inflammation and structural damage characteristic of these diseases.

Clinical trials with golimumab

In a global, randomized, double-blind, placebo-controlled phase 3 study (study C0524T09) of Subcutaneous (SC) administration of golimumab in Ankylosing Spondylitis (AS) subjects, golimumab proved to be effective in improving signs and symptoms, physical function and health-related quality of life (HRQOL) in subjects affected by Ankylosing Spondylitis (AS). Furthermore, safety analysis showed that SC golimumab is generally well tolerated and shows a safety profile similar to that observed with other anti-TNF α agents.

Given the consistent safety and efficacy of SC golimumab, it is expected that IV golimumab will also demonstrate acceptable safety consistent with other anti-TNF α agents in rheumatic diseases (such AS RA, PsA, and AS). Intravenous golimumab has been specifically studied in phase 3 studies (CNTO148ART3001), which form the basis for the approval of treatment for RA. The CNTO148ART3001 study is a randomized, double-blind, placebo-controlled, multicenter, two-arm study of efficacy and safety of IV administration of golimumab 2mg/kg infusion over a period of 30 minutes ± 10 minutes at week 0, week 4 and every 8 weeks thereafter (q8w) in subjects with active RA despite concurrent Methotrexate (MTX) treatment. Despite MTX, subjects with active RA were randomly assigned to receive placebo infusions or IV administration of 2mg/kg of golimumab at week 0, 4 and every 8 weeks to week 24. Starting at week 24, all subjects were treated with IV golimumab at week 100. IV golimumab has been shown to provide substantial benefits in improving RA signs and symptoms, physical function and health-related quality of life, as well as inhibiting the progression of structural damage. Golimumab (CNTO148ART3001) administered intravenously in the treatment of RA showed robust efficacy and acceptable safety with low incidence of infusion reactions.

Recently, two phase 3 studies were designed to evaluate the efficacy and safety of Intravenous (IV) golimumab in treating subjects with active Ankylosing Spondylitis (AS) and active psoriatic arthritis (PsA). The subject's IV route of administration is being evaluated because currently available IV anti-TNF α agents have limitations in immunogenicity, infusion response, and longer infusion times (60 to 120 minutes) compared to 30 minutes ± 10 minutes infusion of IV golimumab. Patients may also prefer a maintenance dose schedule of IV golimumab rather than more frequent administration compared to SC doses.

Overview of the manufacturing Process

Simponi (golimumab) was manufactured in a 9-stage process involving continuous perfusion of cell cultures followed by purification. An overview of the manufacturing process is provided in fig. 18.

As used herein, the terms "culture", "culturing", "cultured" and "cell culture" refer to a population of cells suspended in a culture medium under conditions suitable for survival and/or growth of the population of cells. As will be clear to one of ordinary skill in the art from the context, these terms as used herein also refer to a combination comprising a cell population and a medium in which the cell population is suspended. Cell cultures include cells grown, for example, by batch, fed-batch, or perfusion cell culture methods, and the like. In certain embodiments, the cell culture is a mammalian cell culture.

Cell lines useful in the present invention include mammalian cell lines including, but not limited to, chinese hamster ovary cells (CHO cells), human embryonic kidney cells (HEK cells), baby hamster kidney cells (BHK cells), mouse myeloma cells (e.g., NS0 cells and Sp2/0 cells), and human retinal cells (e.g., per. c6 cells).

As used herein, the term "chemically-defined medium" or "chemically-defined media" refers to a synthetic growth medium in which the identity and concentration of all components are known. Chemically-defined media do not contain bacteria, yeast, animal or plant extracts, animal serum or plasma, but they may or may not include individual plant or animal-derived components (e.g., proteins, polypeptides, etc.). Chemically defined media may contain inorganic salts such as phosphates, sulfates, etc. necessary to support growth. The carbon source is defined and is typically a sugar such as glucose, lactose, galactose, etc., or other compounds such as glycerol, lactate, acetate, etc. Although certain chemically-defined media also use phosphate as a buffer, other buffers, such as citrate, triethanolamine, etc., may also be used. As used herein, a chemically-defined medium comprises no more than a micromolar amount of any metal ion. Examples of commercially available chemically-defined media include, but are not limited to, the CD hybridoma culture medium from ThermoFisher and the CD hybridoma AGT ^TMCulture medium, various Dulbecco's Modified Eagle's (DME) medium (Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham's nutrient mix (Sigma-Aldrich Co; SAFC Biosciences, Inc), combinations thereof, and the like. Methods of preparing chemically defined media are known in the art, for example, in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. patent application publications 2008/0009040 and 2007/0212770.

As used herein, the term "bioreactor" refers to any vessel that can be used for the growth of cell cultures. The bioreactor can be of any size as long as it can be used for cultured cells. In certain embodiments, such cells are mammalian cells. Typically, the bioreactor will be at least 1 liter and may be 10 liters, 100 liters, 250 liters, 500 liters, 1,000 liters, 2,500 liters, 5,000 liters, 8,000 liters, 10,000 liters, 12,000 liters or greater, or any volume therebetween. Internal conditions of the bioreactor, including but not limited to pH and temperature, are optionally controlled during the culture period. The bioreactor may be constructed of any material suitable for maintaining a mammalian cell culture suspended in a culture medium under the culture conditions of the present invention, including glass, plastic, or metal. As used herein, the term "production bioreactor" refers to a final bioreactor for producing a polypeptide or glycoprotein of interest. The volume of the production bioreactor is typically at least 500 liters, and can be 1,000 liters, 2,500 liters, 5,000 liters, 8,000 liters, 10,000 liters, 12,000 liters or more, or any volume therebetween. One of ordinary skill in the art will recognize and will be able to select a suitable bioreactor for practicing the present invention.

For large scale production of golimumab expressed in Sp2/0 cells, preculture, cell expansion and cell production were carried out in stages 1 and 2. In phase 1, preculture was started from a single working cell bank vial of transfected Sp2/0 cells expressing HC and LC sequences of golimumab, and the cells were expanded in culture flasks, disposable culture bags, and 50L perfusion seed bioreactors equipped with internal spin filters or 200L perfusion seed bioreactors equipped with alternating tangential flow hollow fiber filter (ATF) cell retention systems. Cells were cultured until the cell density and volume required to inoculate a 500L or 1000L preparation bioreactor was obtained. In stage 2, cell cultures were continuously perfused in 500L or 1000L preparation bioreactors using the ATF system. Cell culture permeate (harvest) was collected from the ATF system while cells were returned to the bioreactor and culture was replenished with fresh media. The biomass removed from the bioreactor may be combined with the harvest withdrawn from the ATF system, and then may be clarified to produce a combined harvest for further processing.

In stages 3 to 8, purification of golimumab from cell culture harvest is performed by a combination of affinity and ion exchange chromatography steps and steps to inactivate or remove potential viral contaminants (solvent/detergent treatment and virus removal filtration). In stage 3, protein a affinity chromatography is used to clarify and purify the harvest and/or the combined harvest. The resulting Direct Product Capture (DPC) eluate was frozen until further processing. The DPC eluates were filtered and combined in stage 4 after thawing and subsequently treated in stage 5 with tri-n-butyl phosphate (TNBP) and polysorbate 80(PS 80) to inactivate any lipid enveloped viruses that may be present.

In stage 6, the TNBP and PS80 reagents and impurities were removed from the golimumab product using cation exchange chromatography. The golimumab product was further purified in stage 7 using anion exchange chromatography to remove DNA, viruses and impurities that may be present. In stage 8, the purified golimumab product was diluted and filtered through a virus-retaining filter.

The final preparation of golimumab is performed in phase 9. The ultrafiltration step concentrates the golimumab product, and the diafiltration step adds formulation excipients and removes in-process buffer salts. PS80 was added and the intermediate was filtered into polycarbonate containers for frozen storage as formulation entity (FB) to be used for Drug Substance (DS) and Drug Product (DP).

As used herein, the terms "drug substance" (abbreviated "DS") and "drug product" (abbreviated "DP'") refer to compositions for use as commercial medicaments, e.g., for clinical trials or as commercially available medicaments. DS is an active ingredient intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease or to affect the structure or any function of the human body. DP (also known as pharmaceutical products, drugs, medicaments or medicaments) is a drug used for the diagnosis, cure, mitigation, treatment or prevention of diseases or for influencing the structure or any function of the human body. The formulation entity (FB) generated during the manufacturing process is the Drug Substance (DS). DP is DS that has been prepared as a pharmaceutical product for sale and/or administration to a patient.

Description of cell culture Using Sp2/0 cells in Large Scale manufacturing Process

Stage 1

Preculture and amplification

The first stage of Simponi (golimumab) preparation was to prime pre-culture from Working Cell Bank (WCB) vials of transfected Sp2/0 cells expressing HC and LC sequences of golimumab and subsequently expand the cell culture in flasks, disposable bags and 50L or 200L seed bioreactors. Cells were cultured until the cell density and volume required to inoculate a 500L or 1000L preparation bioreactor was obtained. A flow chart for stage 1 is provided in fig. 19, which shows the pre-incubation and amplification steps with in-process control and process monitoring tests.

Manufacturing process

Frozen flasks from WCB were thawed and diluted to 0.2-0.4X 10 with chemically defined medium (CD-A medium) supplemented with 6mM L-glutamine, 0.5mg/L mycophenolic acid, 2.5mg/L hypoxanthine and 50mg/L xanthine⁶Seeding density of Viable Cells (VC)/mL. The activity of the culture when thawed must be > 50%. At temperature and CO₂Controlled humidity CO₂The incubator maintained initial passage in the flask. Incubate the culture for 2-3 days until 0.6X 10 is obtained⁶Minimum cell density of VC/mL.

Expansion is achieved by sequential expansion of the culture in flasks and disposable bags. By diluting with CD-A mediumRelease at 0.2-0.4X 10⁶Cell density of VC/mL began each passage. Passages were incubated for 2 days to 3 days at each amplification step until 0.6X 10 was obtained⁶Minimum cell density of VC/mL. Once in disposable culture bag at a ratio of 0.8 × 10 or more⁶VC/mL and > 80% culture activity achieved sufficient culture volume and cultures were inoculated into 50L or 200L seed bioreactors.

Each pre-culture was sub-sampled for Viable Cell Density (VCD), culture viability and microscopy. The pre-culture was sampled for bioburden prior to inoculation of the 50L or 200L seed bioreactor. The preculture can be maintained for up to 30 days after thawing. If microbial contamination is detected or the maximum duration is exceeded, the pre-incubation is terminated. The spare preculture may be retained when inoculating the seed bioreactor or may be thawed starting with a new WCB vial. The ready preculture was expanded as described above and subjected to the same in-process control and operating parameters as the primary culture. The pre-culture can be maintained as ready as needed and used to inoculate a 50L or 200L seed bioreactor.

When the preculture meets the inoculation criteria, the contents of the disposable bag are transferred to a 50L or 200L seed bioreactor to achieve ≥ 0.3X 10⁶Inoculation density of VC/mL. The 50L or 200L seed bioreactor was fed with CD-a culture medium and operated in perfusion mode at full working volume. The pH, temperature and dissolved oxygen concentration of the culture were controlled to support cell growth. Expanding 50L or 200L seed bioreactor cultures until a 2.0X 10 or more is obtained at 80% or more culture activity⁶Cell density of VC/mL. Samples were taken from 50L or 200L seed bioreactor cultures throughout the VCD, culture viability and microscopy. Prior to inoculation of 500L or 1000L preparation bioreactors, 50L or 200L seed bioreactors were sampled for bioburden. If the VCD of the 50L or 200L seed bioreactor reaches more than or equal to 2.0X 10⁶VC/mL and 500L or 1000L preparation bioreactor is not ready for inoculation, then culture can be continued in perfusion mode until the longest culture of 6 days after inoculation of 50L seed bioreactor and 7 days after inoculation of 200L seed bioreactorThe duration of time. If microbial contamination is detected or the maximum duration is exceeded, the 50L or 200L seed bioreactor operation is terminated.

Stage 2

Bioreactor preparation

The second stage in the manufacturing process is the perfusion of the cell culture in a 500L or 1000L preparation bioreactor. Cell culture permeate (harvest) was collected from the preparation bioreactor while retaining cells via an Alternating Tangential Flow (ATF) hollow fiber cell retention device and the culture was replenished with fresh media. FIG. 20 provides a flow diagram depicting a 500-L or 1000-L preparative bioreactor process.

Manufacturing process

Inoculation of the 500L or 1000L preparation bioreactor is performed by transferring the contents of a 50L or 200L seed bioreactor into a 500L or 1000L preparation bioreactor containing a chemically defined medium supplemented with 6mM L glutamine, 0.5mg/L mycophenolic acid, 2.5mg/L hypoxanthine and 50mg/L xanthine (CD-A medium). The volume transferred must be sufficient to produce ≧ 0.3X 10⁶Seeding density of Viable Cells (VC)/mL. The culture is maintained at a temperature of 34.0-38.0 ℃, a pH of 6.80-7.40 and a dissolved oxygen concentration of 10% -80%. Sampling of Viable Cell Density (VCD), culture activity, bioburden and immunoglobulin g (igg) concentration was performed throughout the 500L or 1000L preparation.

After inoculation, the media feed rate to the culture is increased according to a predetermined schedule until the maximum feed rate is reached. The maximum feed rate was controlled between 0.80 reactor volume/day and 1.50 reactor volume/day. When the full working volume of the bioreactor is reached, perfusion is initiated using the ATF system to separate the cells from the permeate. The permeate was continuously withdrawn through the ATF filter while the cell culture was circulated between the ATF system and the bioreactor. The ATF permeate was collected in a bioprocess vessel (BPC).

When VCD reaches more than or equal to 8.5 multiplied by 10⁶VC/mL, but not later than 50 inoculationsOn day 15 after 0L or 1000L bioreactor preparation, the bioreactor media feed was switched from CD-A to chemically defined media (CD-B media) supplemented with 6mM L glutamine, 0.5mg/L mycophenolic acid, 2.5mg/L hypoxanthine, 50mg/L xanthine and 10mM sodium acetate. Controlling the viable cell density in the bioreactor to at least 12.0 x 10 by means of a variable biomass removal stream from the culture⁶Target of VC/mL.

The biomass removed from the bioreactor can be discarded or combined with ATF permeate and clarified by filtration.

The ATF permeate was designated as the harvest stream. Ethylenediaminetetraacetic acid (EDTA) was added to the harvest stream to a concentration of 5mM-20 mM. After disconnecting from the bioreactor, the harvest was stored in a bioprocess container (BPC) in an environment of 2 ℃ to 8 ℃ for a period of up to 21 days. Each harvested BPC was sampled for IgG concentration, endotoxin and bioburden prior to direct product capture (stage 3).

Perfusion cell culture operations in 500L or 1000L production bioreactors lasted up to 60 days after inoculation. On the last day of operation of a 500L or 1000L preparation bioreactor, cultures were sampled for mycoplasma and exotic virus testing. Bioreactor IgG concentrations were monitored and only information was reported.

Description of the Small Scale preparation of Golomikon expressed in CHO cells

Generation of CHO cells expressing golimumab

CHO cell lines were originally generated from the ovaries of adult chinese hamsters by t.t.puck. CHO-K1(CCL-61) is a subclone of a parental CHO cell line lacking the proline synthesis gene. CHO-K1 was also deposited at the European Collection of cell cultures CHO-K1(ECACC 85051005). Celltech Biologics (now Lonza Biologics) established a Master Cell Bank (MCB) of CHO-K1, 024M, for adaptation of CHO-K1 to suspension and serum-free media. The adapted cell line was designated CHOK1 SV. The CHOK1SV cell line was usedFurther adaptation was performed in protein-free medium to produce cellular MCB designated 269-M. Cells derived from 269-M MCB were transfected as described below to generate a CHO cell line expressing golimumab.

Using cell culture plates and shake flasks at 37 ℃ and 5% CO ₂The humidified incubator of (1) generates, expands and maintains cell lines. The conventional inoculation density in shake flasks was 3X 10⁵Viable cells/mL (vc/mL). All shake flask cultures were maintained at 130 revolutions per minute (rpm), with an orbit of 25mm, a number of wells of 96(DW, Thermo Scientific, Waltham, MA, Cat. No. 278743), a culture at 800rpm, and an orbit of 3 mm.

Culture medium identified as MACH-1, an in-house developed chemically-defined medium for CHO cell culture, was used to generate Golomicron-expressing CHO clones. The basal medium used for routine passaging of the CHO host cell line was MACH-1 supplemented with 6mM L-glutamine (Invitrogen, Carlsbad, CA, Cat. No. 25030-. Unless otherwise indicated, CHO cells transfected with the Glutamine Synthetase (GS) gene were grown in MACH-1+ MSX (MACH-1 supplemented with 25. mu. M L-methionine sulfoximine (MSX, Sigma, St. Louis, MO, Cat. No. M5379-1G) to inhibit glutamine synthetase function). For the high dose fed-batch shake flask and bioreactor experiments, cells were cultured in MACH-1+ F8 (MACH-1 supplemented with 8g/kg F8 (supplement with proprietary growth enhancers) to further support cell growth and antibody production). Proprietary feed media was used for shake flask and bioreactor experiments.

The DNA encoding the gene of interest was cloned into a Glutamine Synthetase (GS) dual gene expression plasmid (Lonza Biologics). The expression of the Heavy (HC) and Light (LC) chain genes is driven by the human cytomegalovirus (hCMV-MIE) promoter alone. The GS gene selection marker driven by the simian virus SV40 promoter allows selection of transfected cells in the presence of MSX in glutamine-free medium.

Prior to each transfection, 1 aliquot of plasmid DNA containing both HC and LC coding regions of golimumab was linearized by restriction enzyme digestion. Using BTX ECM 830 cell fusion Instrument (Harvard Apparatus, Ho)lliston, MA) linearized 15. mu.g aliquots of DNA were transfected into 1X 10⁷In an aliquot of individual cells. Cells were electroporated 3 times at 250 volts in 4mm gap cuvettes with 15 millisecond pulse lengths and 5 second pulse intervals. The transfected cells were transferred to MACH-1+ L-glutamine in shake flasks and incubated for 1 day. The transfectants were centrifuged and then resuspended in MACH-1+25uM MSX for selection and transferred to shake flasks for 6 days of incubation.

Following chemical selection, cells were seeded in single cell suspensions in custom glutamine-free Methocult medium (method cell, StemCell Technologies, inc., Vancouver, BC, catalog No. 03899) containing 2.5% (w/v) methylcellulose in Dartbucker Modified Eagle Medium (DMEM) basal medium. The working solution also contained 30% (v/v) gamma-irradiated dialyzed fetal bovine serum (dFBS. IR, Hyclone, Logan, UT, cat # SH30079.03), 1 XGS supplement (SAFC, St. Louis, MO, cat # 58672-100M), 1.5mg animal component-free protein G Alexa Fluor 488 conjugate (protein G, Invitrogen, Carlsbad, CA, cat # C47010), 25. mu.M MSX, Darber's modified eagle's medium with F12 (DMEM/F12, Gibco/Invitrogen, Carlsbad, CA, cat # 21331-020) and cell suspension.

Protein G recognizes human monoclonal antibodies and binds to IgG secreted by the cells. Protein G was conjugated to a fluorescent-labeled Alexa Fluor 488 such that the cell colonies secreting the most antibody would show the highest level of fluorescence. After 12 to 18 days of incubation, colonies with the highest fluorescence levels were picked into 100 μ L of phenol red containing MACH-1+ MSX in 96-well plates using a ClonePix FL colony picker (Molecular Devices, Sunnyvale, Calif.) and incubated for 5-7 days without shaking. After 5-7 days, cells from 96-well plates were expanded by addition to 50-100 μ L of phenol red-containing MACH-1+ MSX in 96DW plates (Thermo Scientific, Waltham, MA, Cat. 278743) and shaken at 800rpm with 3mm orbits. 96DW plates were fed and titrated 7 days after 96DW inoculation via Octet (ForteBio, Menlo Park, Calif.). The first 10 cultures corresponding to the highest batch 96DW overgrowth titer were expanded into shake flasks in MACH-1+ MSX and frozen cell banks were created with cells suspended in MACH-1+ MSX medium containing 10% DMSO.

Cell culture for small-scale production

As in the large-scale preparation of golimumab expressed in Sp2/0 cells, preculture, cell expansion and cell preparation were carried out in stages 1 and 2 for small-scale preparation of golimumab expressed in chinese hamster ovary cells (CHO cells). In phase 1, preculture was started from a vial of a single cell bank of transfected CHO cells expressing golimumab HC and LC sequences, and the cells were then expanded in culture flasks. Cells were cultured until the cell density and volume required to inoculate a 10L preparation bioreactor were obtained. In phase 2, the cell culture was run in fed-batch mode in a 10L preparation bioreactor. The cultures were fed with concentrated glucose-based and amino acid-based feeds as needed for the duration of the 15 day bioreactor run. Upon completion of the production bioreactor run, the cell culture harvest is clarified to remove biomass and filtered for further processing.

Purification of small-scale preparations

The purification steps for small-scale preparation of golimumab are the same as for large-scale manufacturing process, except that the small-scale preparation omits the stage 8 virus filtration step. Briefly, for small-scale preparation, the purification of golimumab from cell culture harvest was performed in stages 3 to 7 by a combination of affinity and ion exchange chromatography steps and steps to inactivate or remove potential viral contaminants (solvent/detergent treatment and virus removal). In stage 3, protein a affinity chromatography is used to clarify and purify the harvest and/or the combined harvest. The resulting Direct Product Capture (DPC) eluate was frozen until further processing. The DPC eluates were filtered and combined in stage 4 after thawing and subsequently treated in stage 5 with tri-n-butyl phosphate (TNBP) and polysorbate 80(PS 80) to inactivate any lipid enveloped viruses that may be present.

In stage 6, the TNBP and PS80 reagents and impurities were removed from the golimumab product using cation exchange chromatography. The golimumab product was further purified in stage 7 using anion exchange chromatography to remove DNA, viruses and impurities that may be present. As mentioned above, the 8 th stage filtration through the virus retention filter was omitted from the CHO-derived golimumab product purification process.

The final preparation of golimumab is carried out in stage 9 (referred to as large scale stage). The ultrafiltration step concentrates the golimumab product, and the diafiltration step adds formulation excipients and removes in-process buffer salts. PS 80 was added and the bulk intermediate was filtered into polycarbonate containers for bulk storage as formulated.

Method

Method for determining Viable Cell Density (VCD) and% activity

Total CELL/mi, viable CELL/ml (VCD), and% activity are typically determined using a Beckman Coulter Vi-CELL-XR CELL activity analyzer using protocols, software, and reagents provided by the manufacturer. Alternatively, a CEDEX automated cell counting system is also used. However, it should also be noted that other methods for determining VCD and% activity are well known to those skilled in the art, for example using a hemocytometer and trypan blue exclusion.

Determination of biological Activity (potency)

The measurement of biological activity (potency) of golimumab was performed with an in vitro assay based on the ability of golimumab to protect WEHI 164 cells (mouse BALB/c fibrosarcoma cells, available from Walter and Eliza Hall Institute, Melbourne, Australia) from TNF α -induced cytotoxicity. Each assay plate contained 500ng/mL (6 replicates) of a 100 μ L serial dilution of Simponi test preparation and Simponi reference standard. TNF-. alpha.was then added and the plates were incubated. After neutralization and incubation, WEHI 164 cells were added to the microtiter plate, followed by another incubation step. Thereafter, metabolic substrates (which are indicators of living cells) are added and the converted substrates are measured spectrophotometrically.

Fitting test System Using 4-parameter logic analysisArticle and reference standard neutralization curves. 50% Effective Dose (ED) by comparing Simponi reference standard and Simponi test preparation₅₀) The performance is calculated.

During the execution of the bioactive procedure, the following system suitability acceptance criteria were applied to produce valid results:

reference standard substance:

each neutralization curve must be a sigmoidal curve with a lower plateau within 40% of the mean OD value of the cell + TNF-alpha control of the 3 assay plates, a higher plateau within 25% of the mean OD value of the cell-only control of the 3 assay plates, and linear sections between the plateaus.

The slope of each curve must be ≥ 0.7 and ≤ 3.5.

R of each curve²The value must be ≧ 0.97.

All repetitive ED₅₀The value must be ≥ 2ng/mL and ≤ 20 ng/mL.

Mean ED₅₀The RSD of the value (n-6) must be 20% or less.

TNF- α cytotoxicity curves:

the TNF-. alpha.cytotoxicity curves must show sigmoidal curves with a lower plateau, an upper plateau and a linear section between the plateaus.

For each fitted curve, the slope must be ≦ 2.0.

R of TNF-. alpha.cytotoxicity Curve²The value must be ≧ 0.97.

The OD at a TNF-. alpha.concentration of 1.68ng/mL should fall between 0.1 and 0.4.

Comparison:

the OD range per plate (difference between the mean OD values of cell + TNF control and cell only control) must be ≧ 0.68.

For each plate, the mean OD value of the cell-only control (n 6) must be ≧ 0.75. The RSD of the cell-only control must be ≦ 20%.

The mean OD value of the cell + TNF-alpha control (n 6) for each plate must be ≦ 0.50. And the RSD of the TNF-alpha control must be 20% or less.

Test article:

The slope of each curve must be ≥ 0.7 and ≤ 3.5.

R of each curve²The value must be ≧ 0.97.

Mean ED₅₀The RSD of the ratio (n-6) must be 25% or less.

The average slope ratio between the test article and reference standard curves is ≥ 0.8 and ≤ 1.2, which ensures that the slope values of the test article and reference standard curves are comparable (have a difference of no more than 20%).

The mean upper asymptote value of the neutralization curve of the test article differs by no more than 10% from the mean upper asymptote value of the reference standard (the difference in mean upper asymptote value is ≦ δ 10%).

The mean lower asymptote value of the neutralization curve of the test article differs from the mean lower asymptote value of the reference standard by no more than 15% (the difference in mean lower asymptote value is ≦ δ 15%).

Method for determining oligosaccharide composition

Determination of oligosaccharide composition by HPLC

The N-linked oligosaccharide composition of golimumab was determined by a normal phase anion exchange HPLC method with fluorescence detection using an Agilent 1100/1200 series HPLC system with Chemstation/Chemstore software. To quantify the relative amount of glycans, the N-linked oligosaccharides were first cleaved from the reduced and denatured test preparation using N-glycanase (PNGase F). The released glycans were labeled with anthranilic acid, purified by filtration using a 0.45- μm nylon filter, and analyzed by normal phase anion exchange HPLC with fluorescence detection. The HPLC chromatogram serves as a profile that can be used to identify and quantify the relative amount of N-linked oligosaccharides present in the sample. Glycans were identified by co-elution with oligosaccharide standards and retention times based on widely characterized historical results. A representative HPLC chromatogram of the golimumab reference standard is shown in fig. 21.

The amount of each glycan was quantified by peak area integration and expressed as a percentage of the total glycan peak area (% peak area). Results for G0F, G1F, G2F, total neutral species, and total charged glycans can be reported. The other neutral species were the sum of all integrated peaks between 17 minutes and 35 minutes, excluding the peaks corresponding to G0F, G1F, and G2F. The total neutral glycans are the sum of G0F, G1F, G2F, and other neutral species. The total charged glycans are the sum of all mono-sialylated glycan peaks eluting between 42 minutes to 55 minutes and all bi-sialylated glycan peaks eluting between 78 minutes to 90 minutes.

A mixture of oligosaccharide standards (G0F, G2F, G2F + N-acetylneuraminic acid (NANA) and G2F +2NANA) was analyzed in parallel as a positive control for labeling reactions, as a standard for peak identification and as a measure of system suitability. Reconstituted oligosaccharides G0F (catalog No. GKC-004301), G2F (catalog No. GKC-024301), SA1F (catalog No. GKC-124301), and SA2F (catalog No. GKC-224301) or equivalents available from Prozyme were used as reference standards. For system suitability purposes, a method blank negative control and a pre-labeled G0F standard were also run. During the performance of the oligosaccharide mapping procedure, the following system suitability and assay (test article) acceptance criteria were applied to produce valid results:

System applicability criteria:

the resolution (USP) between the G0F peak and the G2F peak in the oligosaccharide standard must be ≧ 3.0.

The theoretical plate number (cut line method) of the G0F peak in the oligosaccharide standard must be not less than 5000.

The total glycan peak area of the golimumab reference standard must be > 1.5 times the main glycan peak area of the pre-labeled G0F.

Re-injecting the reference standard at a smaller injection volume if any reference standard glycan peak is out of scale.

The retention time of the G0F peak in the golimumab reference standard must be within 0.4 minutes of the retention time of G0F in the oligosaccharide standard.

Assay acceptance criteria:

the method blank must have no detectable peaks co-eluting with the oligosaccharide peaks specified in golimumab.

The total glycan peak area of each test preparation must be ≧ 1.5 times the main glycan peak area of the pre-labeled G0F standard.

Re-injecting the sample with a smaller injection volume along with a normal volume of pre-labeled G0F, oligosaccharide standard, method blank, and reference standard if any sample glycan peak is out of scale.

The retention time of the G0F peak in each test article must be within 0.4 minutes of the retention time of the G0F peak in the oligosaccharide standard.

If the assay fails to meet any acceptance criteria, the assay is invalid.

Determination of oligosaccharide composition by IRMA

IdeS-RMA (IRMA) method allows in-use(IgG degrading enzyme of Streptococcus pyogenes (IdeS), purchased from Genovis AB (SKU: A0-FR1-050)) the major glycoforms were distinguished by Reduced Mass Analysis (RMA) after enzymatic treatment of immunoglobulin G (IgG). See also, for example, U.S. patent 7,666,582. Reduced Mass Analysis (RMA) involves disulfide bond reduction of an antibody, followed by complete mass analysis of the heavy chain of the antibody and its attached glycan moieties. Some antibodies show a large degree of heterogeneity due to the presence of N-terminal modifications (such as pyroglutamate formation and carboxylation). Thus, disulfide reduction and heavy chain mass measurement can produce complex deconvolution peak patterns. Thus, in some applications, proteolytic generation of antibody fragments is more desirable than generation of light and heavy chains using reducing agents such as Dithiothreitol (DTT). Traditionally, papain and pepsin were used to generate antibody fragments, all of which are laborious. Cleavage of IgG with pepsin requires extensive optimization and it is lowAt an acidic pH. Papain requires an activator, and both F (ab') 2 and Fab can be obtained depending on the reaction conditions, thus generating a heterogeneous library of fragments. These disadvantages can be overcome by using novel enzymes To avoid. The cracking process is very fast, simple and importantly does not require optimization. It is performed at neutral pH, thereby generating the precise F (ab') 2 and Fc fragments. No further degradation or over-digestion normally associated with other proteolytic enzymes such as pepsin or papain was observed. Importantly, becauseOnly the C-terminus of the disulfide bridge in the heavy chain is cleaved, so no reduction step is required, and the intact F (ab') 2 and two residual Fc fragments are obtained.

Definition of

H: hexose (mannose, glucose and galactose)

Man 5: mannose 5

N: n-acetylhexosamine (N-acetylglucosamine and N-acetylgalactosamine)

F: fucose sugar

S: sialic acid (N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA))

G0: asialo-galactose-free afucosylated biantennary oligosaccharides

G0F: asialo-galactose-free fucosylated biantennary oligosaccharides

G1: asialo-monogalactosylated-afucosylated biantennary oligosaccharides

G1F: asialo-monogalactosylated-fucosylated biantennary oligosaccharides

G2: asialo-digalactosylated-afucosylated biantennary oligosaccharides

G2F: asialo-digalactosylated-fucosylated biantennary oligosaccharides

GlcNAc: N-acetyl-D-glucosamine

Lys: lysine

Lys-Lys: truncated heavy chain (absence of C-terminal lysine residue)

- + Lys: heavy chain containing C-terminal lysine

Ppm: parts per million

Device

Thermo Scientific Q active (Plus) Mass Spectroscopy

Agilent1200 HPLC System

Applied Biosystems POROS R2/102.1 mm D100 mm L column

Thermo Scientific QOxctive tube software

Thermo Scientific protein deconvolution software

An analytical balance capable of weighing 0.01mg

Vortex mixer, of any suitable type

Water bath or heating block, of any suitable type

Calibrated thermometers-10 ℃ to 110 ℃, any suitable type

Calibrated pipettes

Microcentrifuge of any suitable type

Protocol

IdeS digestion of samples

Samples (equal to 50. mu.g IgG).

Add 1. mu.l (50 units) of IdeS enzyme to 50. mu.g IgG, briefly vortex, centrifuge rapidly, and incubate at 37 ℃ for 30 minutes (stock enzyme 5000 units/100. mu.l, 1 unit of enzyme completely digests 1. mu.g IgG in 30 minutes at 37 ℃)

The samples were quickly centrifuged and transferred to LC-MS vials, and the sample vials were loaded into an Agilent1200 autosampler

LC-MS method

Preparation of solutions

Mobile phase a (0.1% Formic Acid (FA) in ultrapure water) -999 mL of ultrapure water was added to a 1L HPLC mobile phase bottle, 1mL of FA was added and stirred. The solution can be stored at room temperature for 2 months.

Mobile phase B (0.1% FA, 99.9% acetonitrile) -999 mL acetonitrile was added to a 1L HPLC mobile phase vial, 1mL FA was added and stirred. The solution can be stored at room temperature for 2 months.

LC method

Column: applied Biosystems POROS R2/102.1 mm D × 100mm L

Column temperature: 60 deg.C

Autosampler temperature: 4 deg.C

·300μL/min

Sample volume: 5 μ L

Mobile phase a: ultrapure water solution of 0.1% FA

Mobile phase B: 0.1% FA in acetonitrile

Table 6: LC gradiometer

Time (minutes)	Mobile phase B%
		0.0	10
6.0	30
		11.9	42
12.0	95
		15.9	95
16.0	10
		21.0	10

MS method

Scanning parameters are as follows:

scan type: full MS

Scan range: 700 to 3500m/z

Fragmentation: in-source CID 35.0eV

Resolution: 17500

Polarity: is just

Locking quality: open, m/z 445.12002

AGC target: 3e6

Maximum sample injection time: 250

HESI source:

sheath flow rate: 32

Flow rate of auxiliary gas: 7

Purge gas flow rate: 0

Spray voltage (| kV |): 4.20

Capillary temperature (c): 280

S-lens RF level: 55.0

Heater temperature (c): 80

Data analysis

Analysis based on deconvolution mass spectrometry recorded the relative content of each detected glycan species. Figure 22 shows a representative deconvolution mass spectrum of IRMA analysis of golimumab produced in Sp2/0 cells. The main structures determined by IRMA analysis include, for example, Man 5(H5N2), G0(H3N4), G0F (H3N4F1), G1F-GlcNAc (H4N3F1), H5N3, G1(H4N4), H5N3F1, G1F (H4N4F1), G2(H5N4), G2F (H5N4F1), G1FS (H4N4F1S1), H6N4F1, G2FS (H5N4F1S1), H7N4F1, H6N4F1S1, G2FS2(H5N4F1S 2). The percentage of each of these structures was monitored. The measured peak intensities represent the percentage of each structure (as a percentage of the total number of assignments) after normalization. Glycans observed with masses outside the 100ppm mass deviation threshold are not included in the calculations, (e.g., (G1F-GlcNAc-Lys, (' H5N3-Lys, (' G1-Lys), ' H5N3F1-Lys, and ' G2-Lys as indicated, these are indicated by an asterisk (' x '). moreover, Man5-Lys is not always detected in the spectra because it has very low intensity, but it will be considered when present and included in the calculations.as detected on both isoforms of the Fc fragment with and without terminal lysines, the percentages of glycans are calculated, e.g., g. G0F percentage is (% G0F-Lys +% G0F + Lys) (' 1) (' 36F-GlcNAc + Lys) (' 1+ H3 + 1H 3+ 3H 3+ Lys) G2+ Lys, G2FS-Lys, H6N4F1S 1-Lys, G2FS2-Lys, H6N4F1-Lys, and H7N4F 1-Lys. Most of these structures are low abundance, cannot be resolved from neighboring peaks with higher intensity, or are less detectable than the method.

Note that: differences between HPLC and IRMA methods (see, e.g., table 7 below) may result from co-elution of multiple species in HPLC and IRMA underestimating some sialylated species, as some intensities are very close to the detectability of IRMA methods.

Table 7: comparison of IRMA and HPLC glycan abundance of representative Golomikan antibody samples generated in Sp2/0 cells

Capillary isoelectric focusing

Capillary isoelectric focusing (cIEF) separates proteins based on total charge or isoelectric point (pI). The method is used to monitor the distribution of charge-based isoforms in golimumab. Unlike gel-based IEF programs, cIEF provides a quantitative measure of the charged species present. In addition, the cIEF showed increased resolution, sensitivity and reproducibility compared to gel-based methods. The cIEF program separated 4 to 6 charge-based isoforms of Simponi (golimumab) at near baseline resolution, whereas IEF gel analysis separated only 4 to 5 species at partial resolution. Representative cIEF electropherograms of golimumab expressed in Sp2/0 cells are shown in fig. 23, with four major peaks labeled C, 1, 2, and 3 and minor peaks labeled B. Also shown are graphs representing the general relationship between the cIEF peak and reduced negative charge/degree of sialylation.

The cIEF determination is performed on a commercially available imaging cIEF analyzer equipped with an autosampler capable of maintaining a sample temperature of 10.5 ℃ or less in an ambient environment of 30 ℃ or less, such as an Alcott autosampler (GP Instruments, Inc.). The assay employs an inner wall coated silica capillary tube without an outer wall polyimide coating to allow for full column detection. In addition, a defined mixture of anolyte of dilute phosphoric acid and methylcellulose, catholyte of sodium hydroxide and methylcellulose, and wide range (pH 3-10) and narrow range (pH 8-10.5) ampholytes were used. The assay pre-treats both the test article and the Reference Standard (RS) with carboxypeptidase B (cpb) that removes the heavy chain C-terminal lysine and eliminates ambiguity due to the presence of multiple C-terminal variants.

Prior to each analysis, the autosampler temperature set point was set to 4 ℃, the autosampler was pre-chilled for at least 30 minutes, and the ambient room temperature of the laboratory was maintained at ≦ 30 ℃. The pre-treated test article and RS, sample vial, vial insert, reagents used in the assay (including purified water), mother liquor containing N, N' -Tetramethylethylenediamine (TEMED), which optimizes focusing within the capillary, ampholytes, pI 7.6 and 9.5 markers for internal standards, and Methylcellulose (MC) were kept on ice for at least 30 minutes prior to starting sample preparation. Samples were prepared on ice, and the time of addition of the mother liquor was recorded and exposure to TEMED was controlled. The assay must be completed within 180 minutes after the addition. The system suitability control was run once and the test article and RS run twice in the order of the following table (table 8):

Table 8: sample run sequence

Sample name	Sample vial location	Number of samples taken
			System applicability	1	1
Blank space	2	1
			CPB control	3	1
CPB-processed RS	4	2
			CPB treated sample 1	5	2
CPB-processed RS	6	2

After the sample was injected into the capillary by the syringe pump, an electric field (3kV) was applied to the capillary for 8 minutes, a pH gradient was formed, and the charge-based isoforms of golimumab were separated according to isoelectric point (pI). The protein isoforms in the capillaries were detected by imaging the entire capillary at 280nm and the data presented in the form of an electropherogram as a function of pI value and a 280. pI values were assigned by comparison to internal pI standards (pI 7.6 and 9.5) using instrument software, and peak areas were determined from the electropherograms using standard data acquisition software. The average pI and average peak area percentage for all duplicate entries ≧ LOQ peaks, the Δ pI value compared to the reference standard, and the sum of the area percentages of peaks C, 1, 2, and 3 are reported.

Characterization of golimumab cIEF isoforms

The reference cIEF spectrum of golimumab produced on Sp2/0 cells contained four major and minor peaks B labeled C, 1, 2, and 3 as shown in the representative electropherogram (fig. 23). For golimumab, the main source of variation in the cIEF spectrum is deamidation and isomerization of heavy chain asparagine 43(HC Asn 43). The basic peak 3 in cIEF represents the non-deamidated HC Asn43 as well as deamidated heavy chain isoaspartic acid 43(HC isoasp 43), while the more acidic peak represents deamidated HC Asp43 and the degree of sialylation. The predicted identities of the different cIEF peaks are listed in table 9. The cIEF assay was used as a general monitor of charge heterogeneity of golimumab, and a different assay was used as the primary assay to monitor deamidation.

^aTable 9: predicted identity of the peaks observed in cIEF electropherograms of FB

Introduction to manufacturing control strategy

During large-scale commercial production, manufacturing control strategies were developed to maintain consistent DS and DP characteristics of therapeutic proteins (e.g., therapeutic antibodies such as golimumab) with respect to oligosaccharide profile, biological activity (potency), and/or other characteristics of the Drug Substance (DS) and Drug Product (DP) (e.g., see the properties identified in tables 10 and 11). For example, during the 9 th stage of the manufacturing process, the golimumab glycosylation is monitored as a control during the formulation entity (FB), with appropriate upper and lower limit specifications for the average total neutral oligosaccharide%, total charged oligosaccharide% and individual neutral oligosaccharide species G0F, G1F and G2F%.

Table 10: representative comparison of selected Golomikon characteristics expressed in Sp2/0 cells and CHO cells

As shown in table 10, there were differences in some selected characteristics of golimumab produced in Sp2/0 cells and CHO cells. However, it has been previously determined that differences in deamidation profiles (e.g., the level of HC Asn43 deamidation) reflected in the results of both cIEF and peptide mapping are primarily due to differences in processing (e.g., purification and/or storage) of proteins. Similarly, differences in biological activity have been shown to be caused by differences in processing (i.e., large scale commercial production versus small scale production), and not thought to be caused by differences in host cells used to express the protein. Furthermore, the biological activity of golimumab expressed in CHO cells and purified on a small scale is still within specification.

Oligosaccharide profile of golimumab

Golimumab is N-glycosylated at a single site on each heavy chain, i.e. at asparagine 306. These N-linked oligosaccharide structures may be any one of a group of biantennary oligosaccharide structures linked to the protein via a primary amine of an asparagine residue, but on golimumab they consist mainly of biantennary core-fucosylated species, with galactose and sialic acid heterogeneity. Individual oligosaccharide classes include "G0F" (asialo, galactose free core-fucosylated biantennary glycans), "G1F" (asialo, galactose free core-fucose)Fucosylated biantennary glycans) and "G2F" (asialo, digalactose core-fucosylated biantennary glycans). During stage 9 of manufacture, golimumab glycosylation was monitored as an in-process control, with appropriate instructions for the total neutral oligosaccharides, total charged oligosaccharides and individual neutral oligosaccharide species G0F, G1F and G2F. A schematic overview of indium for some of the major N-linked oligosaccharide species in golimumab IgG is shown in figure 24. The role of some enzymes in the glycosylation maturation process is also shown, including some divalent cations (e.g., Mn) ²⁺And Cu²⁺) In these enzymatic processes.

Control of oligosaccharide profile

Control of the oligosaccharide profile of therapeutic antibodies is critical, as changes in the oligosaccharide profile of recombinant monoclonal antibodies can significantly affect antibody biological function. For example, biological studies have shown that the distribution of different glycoforms over the Fc region can significantly affect antibody efficacy, stability and effector function (J.Biosci.Bioeng.2014, Vol.117, No. 5, p.639-. In particular, afucosylation (J.mol.biol.368: 767-. In addition, it has been shown that high mannose levels adversely affect efficacy by increasing antibody clearance (glycobiology.2011, 21 (7): 949-. As changes in the oligosaccharide profile have these biological consequences, regulatory agencies need to control antibody glycosylation patterns to ensure compliance with batch release specifications for consistent, safe and effective products.

Effect of oligosaccharide Profile-expression in different cells

Two commonly used host cell lines for recombinant expression of antibodies are Chinese Hamster Ovary (CHO) cells and mouse myeloma cells (e.g., Sp2/0 cells). CHO cells express recombinant antibodies that may be nearly free of sialylglycans, and fucosylation rates of glycans can be as high as 99%. In contrast, mouse myeloma cells express recombinant antibodies that can contain up to 50% sialic acid and typically have less fucose. These differences may have a significant effect on antibody activity in vivo, for example, it has been shown that such differences may affect the structure of the Fc portion of the molecule, thereby altering antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (see, e.g., US patent No. 8975040). For example, a decrease in ADCC activity has been noted with increasing sialylated (charged) Fc glycans (Scalon et al, Mol Immunol, 2007, Vol. 44, p. 1524-1534).

The effect on ADCC activity may be particularly important for anti-TNF antibodies, as it has been shown that for some approved indications, the key mode of action (MOA) of anti-TNF antibodies may be the destruction of TNF-alpha producing cells by ADCC, e.g. for the treatment of Inflammatory Bowel Disease (IBD), such as Crohn's Disease (CD) and/or Ulcerative Colitis (UC). It has also been shown that Flixabi (CHO-derived Biomimic Remicade (infliximab) has a higher average ADCC activity in the NK92-CD16a cell line infliximab is produced in Sp2/0 cells and its oligosaccharide profile has a higher percentage of charged glycans than Flixabi produced in CHO cells (Lee et al, MAbs, 8.2017, vol. 6, p. 968-977.) it may therefore be advantageous to reduce sialylation, thereby reducing the percentage of charged glycans associated with anti-TNF antibodies.

In addition, antibodies produced in CHO and Sp2/0 cells may have significant differences in the level of two glycan epitopes, galactose- α -1, 3-galactose (α -gal) and sialylated N-glycan Neu5Gc- α -2-6-galactose (Neu5 Gc). For example, it has been shown that CHO cells can express antibodies with undetectable levels or only trace levels of α -Gal and Neu5Gc, whereas Sp2/0 cells can express much higher levels of both glycan structures (Yu et al, Sci rep.2016, 1/29/7: 20029). In contrast, humans have genetic defects in the gene for biosynthesis of α -gal, and the gene responsible for the production of Neu5Gc has been irreversibly mutated in all humans. Thus, α -Gal and Neu5Gc are not produced in humans. Furthermore, the presence of these non-human glycan epitopes on therapeutic antibodies may cause adverse immune responses in certain populations due to the high levels of pre-existing anti- α -Gal and Neu5Gc antibodies. For example, anti- α -gal IgE-mediated allergic reactions to cetuximab have been reported (Chung, C.H. et al, N Engl J Med.2008, 3.13 days; 358 (11): 1109-17), and the presence of circulating anti-Neu 5Gc antibodies has been reported to promote cetuximab clearance (Ghaderi et al, Nat Biotechnol.2010, 8 months, Vol.28, 8, p.863-867).

Oligose of golimumab expressed in Sp2/0 cells and CHO cells

Compiled HPLC data from a number of commercial production runs of Golomikon monoclonal antibody show that the DS or DP produced in Sp2/0 cells comprises an anti-TNF antibody comprising ≥ 82.0% to ≤ 94.4% of the total neutral oligosaccharide species, ≥ 5.6% to ≤ 18.0% of the total charged oligosaccharide species and ≥ 25.6% to ≤ 42.2% of the individual neutral oligosaccharide species G0F, ≥ 31.2% to ≤ 43.6% of G1F and ≥ 5.6% to ≤ 14.2% of G2F. As shown in table 11 and fig. 25, the oligosaccharide profile of golimumab expressed in CHO cells was significantly different from that of golimumab expressed in Sp2/0 cells. The oligosaccharide spectrum of golimumab produced in CHO cells was biased towards very low levels of charged glycans and higher levels of neutral glycans, mainly G0F, compared to golimumab produced in Sp2/0 cells. The oligosaccharide profile of golimumab produced in CHO cells contained > 99.0% of total neutral oligosaccharide species, < 1.0% of total charged oligosaccharide species and > 60.0% of individual neutral oligosaccharide species G0F, < 20.0% of G1F and < 5.0% of G2F. Furthermore, for golimumab produced in CHO cells no di-sialylated glycan species were detected by IRMA or HPLC, based on HPLC analysis mono-sialylated glycan species were at very low levels and could not be detected by IRMA analysis.

Table 11: total neutral oligosaccharide species, Total charged oligosaccharide species of Golomikon produced in Sp2/0 cells and CHO cells Representative results of IRMA and HPLC analysis of oligosaccharide species and other selected oligosaccharide species

Table 12: IRMA analysis of the Individual oligosaccharide species of Golomikne antibody produced in Sp2/0 cells and CHO cells Representative results of (1)

Conclusion

Thus, as described above, manufacturing control strategies were developed to maintain a therapeutic protein with consistent DS and DP characteristics in terms of oligosaccharide profile and/or other characteristics of the Drug Substance (DS) or Drug Product (DP) (e.g., DS and/or DP comprising the therapeutic antibody Ultezumab). In particular, control of the oligosaccharide profile of therapeutic antibodies is critical, as changes in the oligosaccharide profile can significantly affect antibody biological function. The control point for the oligosaccharide profile of a therapeutic antibody is the cell host selected for expression of the therapeutic antibody. As shown herein, golimumab expressed in Sp2/0 cells comprises an anti-TNF antibody having a sequence comprising SEQ ID NO: 36 and a Heavy Chain (HC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the oligosaccharide profile of the anti-TNF antibody comprises ≥ 82.0% to ≤ 94.4% of total neutral oligosaccharide species, ≥ 5.6% to ≤ 18.0% of total charged oligosaccharide species and ≥ 25.6% to ≤ 42.2% of the individual neutral oligosaccharide species G0F, ≥ 31.2% to ≤ 43.6% of G1F and ≥ 5.6% to ≤ 14.2% of G2F.

In contrast, golimumab expressed in CHO cells comprises a mammalian anti-TNF antibody having a heavy chain comprising SEQ ID NO: 36 and a Heavy Chain (HC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the oligosaccharide profile of the anti-TNF antibody comprises > 99.0% total neutral oligosaccharide species, < 1.0% total charged oligosaccharide species and > 60.0% single neutral oligosaccharide species G0F, < 20.0% G1F and < 5.0% G2F. Furthermore, for golimumab produced in CHO cells no di-sialylated glycan species were detected by IRMA or HPLC, based on HPLC analysis mono-sialylated glycan species were at very low levels and could not be detected by IRMA analysis.

These changes in the oligosaccharide profile of golimumab produced in CHO cells may provide multiple benefits in different therapeutic indications and different patient populations. For example, reduced sialylation (charged) Fc glycans on anti-TNF antibodies may increase ADCC activity in vivo, resulting in enhanced efficacy in treating certain diseases (e.g., Inflammatory Bowel Disease (IBD), such as Crohn's Disease (CD) and/or Ulcerative Colitis (UC)). In addition, in general, reduction of sialylated species and reduction of anti-TNF antibody-specific Neu5Gc produced in CHO cells may provide benefits by reducing adverse immunogenic reactions when administered to humans. For example, a reduced level of Neu5Gc may reduce clearance such that anti-TNF antibodies produced in CHO cells will have a longer half-life than anti-TNF antibodies expressed in Sp2/0 cells, particularly for a patient population with a higher level of anti-Neu 5Gc antibody.

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Methods of manufacture of compositions for the production of anti-TNF antibodies

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