HK1190160A - Carrier immunoglobulins and uses thereof - Google Patents

Description

Carrier immunoglobulins and uses thereof

This application claims the benefit of U.S. provisional application No. 61/385,460 filed on 9/22/2010, which is hereby incorporated by reference in its entirety.

This application contains the ASCII "txt" standard sequence listing submitted via EFS-WEB on day 9/22 2011 for use as a paper print required by sections 1.821(c) and 1.821(e) of Computer Readable Form (CRF) and 37c.f.r. and is hereby incorporated by reference in its entirety. The name of the "txt" file created on 9/22/2011 is: A-1536-WO-PCTSeqList092211_ ST25.txt, and has a size of 501 kb.

Throughout this application, various publications are referenced within parentheses or square brackets. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background

1. Field of the invention

The present invention relates to immunoglobulins to which one or more pharmacologically active chemical moieties may be conjugated to improve pharmacokinetic properties.

2. Detailed description of the related Art

By "carrier" moiety is meant a pharmacologically inactive molecule to which a pharmacologically active chemical moiety, such as a non-peptide organic moiety (i.e., a "small molecule") or a polypeptide agent (e.g., an immunoglobulin of the invention) may be covalently conjugated or fused. Effective carriers have been found for preventing or slowing in vivo degradation of the pharmacologically active moiety, or reducing renal clearance, due to proteolysis of the pharmacologically active chemical moiety or other in vivo activity-reducing chemical modification, increasing the in vivo half-life or other pharmacokinetic properties of the therapeutic agent, e.g., increasing the rate of absorption, reducing toxicity or immunogenicity, improving solubility and/or improving manufacturability or storage stability compared to the unconjugated form of the pharmacologically active moiety.

Examples of such carrier moieties that have been used in the pharmaceutical industry include polyethylene glycol (see, e.g., Burg et al, erythropoetin conjugates with polyethylene glycol, WO01/02017), immunoglobulin Fc domains (see, e.g., Feige et al, Modified peptides therapeutic agents, U.S. Pat. No. 6,660,843), human serum Albumin (see, e.g., Rosen et al, Albumin fusion proteins, U.S. Pat. Nos. 6,926,898 and US 2005/0054051; Bridon et al, Protection of endogenous therapeutic peptide fusion protein binding to antibodies, U.S. Pat. No. 6,887,470), thyroxine transporters (see, e.g., Walker et al, Use of recombinant peptides/proteins to thyroid polypeptides fusion proteins), thyroxine transporters (see, e.g., Wallace et al, nucleic acid conjugates with thyroxine antibody binding to the antigen conjugate of the cell), and so-called thyroid protein antibody combinations such as Fc antibody A6778/antibody conjugates (see, e.g., heterologous peptide conjugates), thyroid protein conjugates, thyroid polypeptides, and thyroid polypeptides, for example as described in Sullivan et al, Toxin Peptide Therapeutic Agents, PCT/US2007/022831 (published as WO 2008/088422). Pharmacologically active moieties have also been conjugated to peptides or small molecules with affinity for long half-life serum proteins. (see, e.g., Blaney et al, methods and compositions for creating the serum half-life of pharmacological active agents by binding transmethylatin-selective ligands, U.S. Pat. No. 5,714,142; Sato et al, serum binding moieties, U.S. Pat. No. 2003/0069395A 1; Jones et al, Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225).

Fischer et al describe a peptide-immunoglobulin-conjugate, wherein the immunoglobulin consists of two heavy chains or two heavy chains and two light chains, and wherein the immunoglobulin is not a functionalizable immunoglobulin (Fischer et al, A peptide-immunoglobulin conjugate, WO2007/045463A 1).

The present invention provides an immunoglobulin which achieves superior uniformity and recombinant expression efficiency, in vitro stability without aggregation, photodegradation resistance and oxidation resistance, no cross-reactivity with human antigens, and good pharmacokinetic properties.

Summary of The Invention

The present invention relates to immunoglobulins which are used as carrier moieties. These immunoglobulins, including antibodies and antibody fragments, have reliable expression and purification characteristics, produce stable and relatively homogeneous products, and have prominent Pharmacokinetic (PK) properties in rats and cynomolgus monkeys. The immunoglobulins of the present invention have not been detected as binding to human proteins, cells or tissues. These immunoglobulins may also be used for a number of purposes including, but not limited to, quality control or analytical criteria for antibody-based drugs and as controls for biologically relevant isotype-matched antibodies.

Certain embodiments of the invention include an isolated immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

the heavy chain variable region comprises SEQ ID NO: 323[ VH10] and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188[ VL4] or SEQ ID NO: 190[ VL5 ]; or

The heavy chain variable region comprises SEQ ID NO: 321[ VH9] and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188[ VL4] or SEQ ID NO: 190[ VL5 ]; or

The heavy chain variable region comprises SEQ ID NO: 325[ VH11] and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 182[ VL1], SEQ ID NO: 188[ VL4] or SEQ id no: 190[ VL5 ].

Examples include antibodies #16435, 16436, 16438, 16439, 16440, 16441, and 16444 disclosed in table 2C. In an aqueous solution incubated under physiological conditions, for example, as measured by a surface plasmon resonance binding assay, 30 micromolar concentrations of an immunoglobulin of the invention typically do not significantly bind 30 nanomolar concentrations of soluble human IL-17R (SEQ ID NO: 89), as described herein.

Other embodiments of the invention include isolated immunoglobulins comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region wherein:

The light chain variable region comprises SEQ ID NO: 196[ VL8] and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 335[ VH16], SEQ ID NO: 349[ VH23], SEQ id no: 351[ VH24], SEQ ID NO: 353[ VH25], SEQ ID NO: 355[ VH26] or SEQ ID NO: 359[ VH28 ]; or

The light chain variable region comprises SEQ ID NO: 204[ VL12] and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349[ VH23] or SEQ ID NO: 355[ VH26 ]; or

The light chain variable region comprises SEQ ID NO: 202[ VL11] and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349[ VH23 ]; or

The light chain variable region comprises SEQ ID NO: 192[ VL6] and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 357[ VH27], SEQ ID NO: 359[ VH28] or SEQ id no: 369[ VH33 ]; or

The light chain variable region comprises SEQ ID NO: 194[ VL7] and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 335[ VH16], SEQ ID NO: 349[ VH23] or SEQ id no: 351[ VH24 ].

Examples include antibodies #1961, 1962, 1963, 1964, 1965, 1966, 2323, 2324, 2330, 4241, 4341, 10182, 10183, 10184 and 10188 disclosed in table 2C. In aqueous solutions incubated under physiological conditions, for example, 10 micromolar of an immunoglobulin of the invention typically does not significantly bind 10 nanomolar soluble human TR2(SEQ ID NO: 82), as measured by a surface plasmon resonance binding assay, as described herein.

In some embodiments, the immunoglobulins of the present invention are used as carriers for pharmacologically active chemical moieties, such as small molecules, peptides and/or proteins, to improve their PK properties. Pharmacologically active moieties may be conjugated, i.e., covalently bound, to the immunoglobulins of the present invention by chemical conjugation reactions, or they may be fused to immunoglobulins by recombinant gene expression.

The invention also provides materials and methods for making such inventive immunoglobulins, including isolated nucleic acids encoding the inventive immunoglobulins, vectors, and isolated host cells. Isolated nucleic acids encoding any of the immunoglobulin heavy and/or light chain sequences and/or VH and/or VL sequences are also provided. In related embodiments, expression vectors comprising any of the above-described nucleic acids are provided. In yet another embodiment, a host cell comprising any of the above-described nucleic acids or expression vectors is provided.

The immunoglobulin of the invention can be used for manufacturing pharmaceutical compositions or medicaments. The pharmaceutical composition or medicament of the invention comprises an immunoglobulin conjugated to a pharmacologically active agent, and a pharmaceutically acceptable diluent, carrier or excipient.

Various methods are contemplated in the present invention. For example, a method is provided that involves culturing the above-described host cell comprising an expression vector of the invention such that the encoded immunoglobulin is expressed. Such methods may further comprise the step of recovering the immunoglobulin from the host cell culture. In a related embodiment, an isolated immunoglobulin prepared by the above method is provided.

The above summary is not intended to define every aspect of the invention, and other aspects are described in other sections, such as the detailed description of the embodiments. The entire document is intended to be recited as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combinations of features are not present together in the same sentence, or paragraph, or section of the document.

In addition to the above, the invention also includes, as a further aspect, all embodiments of the invention which are in any case narrower than the scope of the variants defined in the specific paragraph above. For example, certain aspects of the invention are described as genera, and it is understood that each member of the genus is an aspect of the invention individually. In addition, an aspect described as a genus or a member of a selected genus should be understood to include a combination of two or more members of that genus. While the applicant has invented the full scope of the invention described herein, it is not the intention of the applicant to claim subject matter described in the prior art work of others. Thus, if statutory prior art within the scope of a claim is brought to the attention of the applicant by a patent office or other entity or individual, then the applicant has the right to exercise amendment rights under applicable patent laws to redefine the subject matter of the claim to expressly exclude such statutory prior art or obvious variations of statutory prior art from the scope of the claim. Variations of the invention defined by such amended claims are also intended as aspects of the invention.

Brief Description of Drawings

Fig. 1A-N show schematic structures of some embodiments of compositions of the present invention comprising one or more units (wave curves) of a pharmacologically active toxin peptide analog fused to one or more domains of an immunoglobulin via an optional peptidyl linker moiety (such as, but not limited to, L5 or L10 described herein). These schematic diagrams show more typical IgG1, but they are intended to apply also to IgG2, IgG3 and IgG4, where IgG2 would have 4 disulfide bonds in the hinge and the disulfide bonds connecting the heavy and light chains have a different arrangement. FIG. 1A represents a monovalent heterodimeric Fc-toxin peptide analog fusion wherein the toxin peptide analog is fused to the C-terminus of one of the immunoglobulin Fc domain monomers. FIG. 1B represents a bivalent homodimeric Fc-toxin peptide analog fusion wherein the toxin peptide analog is fused to the C-terminus of two immunoglobulin Fc domain monomers. FIG. 1C represents a monovalent heterodimeric toxin peptide analog-Fc fusion wherein the toxin peptide analog is fused to the N-terminus of one of the immunoglobulin Fc domain monomers. FIG. 1D represents a bivalent homodimeric toxin peptide analog-Fc fusion, wherein the toxin peptide analog is fused to the N-terminus of two immunoglobulin Fc domain monomers. FIG. 1E represents a monovalent heterotrimeric Fc-toxin peptide analog/Ab comprising an immunoglobulin Heavy Chain (HC) + an immunoglobulin Light Chain (LC) + an immunoglobulin Fc monomer fused at its C-terminus to a toxoprotein peptide analog. FIG. 1F represents a monovalent Heterotetrameric (HT) antibody HC-toxin peptide analog fusion in which the toxin peptide analog is fused to the C-terminus of one of the HC monomers. FIG. 1G represents a bivalent HT antibody Ab HC-toxin peptide analog fusion with toxin peptide analogs at the C-termini of both HC monomers. FIG. 1H represents a monovalent HT toxin peptide analog-LC Ab, wherein the toxin peptide analog is fused to the N-terminus of one of the LC monomers. FIG. 1I represents a monovalent HT toxin peptide analog-HC Ab, wherein the toxin peptide analog is fused to the N-terminus of one of the HC monomers. Fig. 1J represents a monovalent HT Ab LC-toxin peptide analog fusion (i.e., LC-toxin peptide analog fusion + LC +2(HC)), in which the toxin peptide analog is fused to the C-terminus of one of the LC monomers. Fig. 1K represents a bivalent HT Ab LC-toxin peptide analog fusion (i.e., 2 (LC-toxin peptide analog fusion) +2(HC)), where the toxin peptide analog is fused to the C-terminus of the two LC monomers. Fig. 1L represents a trivalent HT Ab LC-toxin peptide analog/HC-toxin peptide analog (i.e., 2 (LC-toxin peptide analog fusion) + HC-toxin peptide analog fusion + HC), where the toxin peptide analog is fused to the C-terminus of one of the two LC monomers and the HC monomer. Figure 1M represents a bivalent antibody in which a toxin peptide analog moiety is inserted into the internal loop of the immunoglobulin Fc domain of each HC monomer. Figure 1N represents a monovalent antibody in which a toxin peptide analog moiety is inserted into the internal loop of an immunoglobulin Fc domain of one of the HC monomers. Upon expression of a deoxyribonucleic acid (DNA) construct encoding a single strand, dimers or trimers will form spontaneously in certain host cells. In other host cells, the cells may be placed under conditions conducive to the formation of dimers/trimers, or dimers/trimers may be formed in vitro. If more than one HC monomer, LC monomer or immunoglobulin Fc domain monomer is part of a single embodiment, the individual monomers may be the same or different from each other as desired.

Fig. 2A-B show that an embodiment of the antibody of the invention does not bind to IL 17R. Antibody 16429 was immobilized to a CM5 sensor chip and a 100% IL-17 binding signal without antibody binding in solution was established using 10nM of IL-17R in the absence of antibody. In FIG. 2A, 10nM, 100nM, and 1000nM indicator antibody samples were incubated with 10nMIL-17R to determine antibody binding in solution. In FIG. 2B, a 30,000nM antibody sample was incubated with 30nM IL-17R to determine antibody binding in solution. In FIGS. 2A-B, the reduced IL-17R binding signal after antibody incubation is indicative of binding of the antibody to IL-17R in solution.

FIG. 3 shows the relative preparation of GRO-alpha by human foreskin fibroblasts incubated with 5ng/ml IL-17 and 0.1. mu.M, 1. mu.M and 10. mu.M samples of the indicator antibody. The conditioned cell culture media was then assessed for GRO- α levels using a GRO- α sandwich ELISA.

Figure 4A shows antibodies (from top to bottom): 16435. 16436, 16439, 16440, 16441 and 16444, which were eluted from two size exclusion columns in series (TSK-GEL G3000SWXL, 5mM size, 7.8X300mM, Tosohbioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (pH6.8) flowing at 0.5 mL/min.

Fig. 4B shows the antibody shown in fig. 4A above (from top to bottom): 16435. 16436, 16439, 16440, 16441 and 16444.

FIG. 5 shows non-reducing analysis on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) performed at 220V using non-reducing loading buffer and staining with QuickBlue (Boston biologicals) for 2. mu.g of antibodies 16435, 16436, 16437, 16438, 16439, 16440, 16429, 16430, 16433, 16434, 16441 and 16444. Molecular weight standard NovexThe standards were pre-stained.

FIG. 6 shows a reduction analysis on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) at 220V using non-reducing loading buffer and staining with QuickBlue (Boston biologicals) for 2. mu.g of antibodies 16435, 16436, 16437, 16438, 16439, 16440, 16429, 16430, 16433, 16434, 16441 and 16444. Molecular weight standard NovexThe standards were pre-stained.

Fig. 7A-B show titers of antibodies 16435 and 16444, respectively. Expression libraries were created by transfecting CHO DHFR (-) host cells with the corresponding HC and LC expression plasmids. Small-scale (5-mL; FIG. 7A) expression assays were performed in CD6-D assay medium using a 6 day prior loading method, while large-scale (3-L; FIG. 7B) assays were performed using a 11 day fed batch method with peptone medium. Titer levels were measured using a protein AHPLC-based assay.

FIGS. 8A-B show chromatograms of antibodies 16435 (FIG. 8A) and 16444 (FIG. 8B) eluted from SP-HP Sepharose (sepharose) columns (GELife Sciences) at 7 ℃ using a gradient of 20 column volumes to 50% S-Buffer B (20mM acetic acid, 1M NaCl, pH5.0), where absorbance was measured at 300 nm.

FIGS. 9A-B show analysis of 16435 (FIG. 9A) and 16444 (FIG. 9B) antibodies on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) performed at 220V and stained with QuickBlue (Boston biologicals). Lanes labeled "NR" contain non-reducing sample buffer, while those labeled "red" contain reducing sample buffer.

Figure 10 shows antibodies: 16435 and 16444, using two size exclusion columns in series (TSK-GEL G3000SWXL, 5mM size, 7.8X300mM, Tosohbioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (pH6.8) flowing at 0.5 mL/min.

Figure 11 shows antibodies by DSC using MicroCal VP-DSC: analysis of IgG2 monoclonal antibody comparisons 16444 and 16435, where the samples were heated from 20 ℃ to 95 ℃ at a rate of 1 ℃ per minute. The protein concentration in 10mM sodium acetate, 9% sucrose (pH5.0) was 0.5 mg/ml.

FIGS. 12A-D show analysis of 16435 (FIGS. 12A-B) and 16444 (FIGS. 12C-D) antibodies by reduced (FIGS. 12A and 12C) and non-reduced (FIGS. 12B and 12D) CE-SDS, where absorbance was detected at 220 nm. Bare fused silica capillaries 50 μm × 30.2cm were used for separation analysis.

Figure 13 shows size exclusion analysis of antibodies 16435 and 16444 after coating with aluminum foil ("dark") or exposure to fluorescence ("light") for 3 days at room temperature, with elution from two tandem size exclusion columns (TSK-GEL G3000SWXL, 5mM particle size, 7.8x300mM, TosohBioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (ph6.8) flowing at 0.5 mL/min.

FIGS. 14A-D show HIC analysis of 16435 (FIGS. 14A and 14C) and 16444 (FIGS. 14B and 14D) antibodies after 3 days of coating with aluminum foil ("dark", FIGS. 14A-B) or exposure to fluorescence ("light", FIGS. 14C-D) at room temperature using two Dionex ProPacHIC-10 columns in tandem, mobile phase A being 1M ammonium sulfate, 20mM sodium acetate (pH5.0), and mobile phase B being 20mM sodium acetate, 5% acetonitrile (pH 5.0). The sample was eluted with a 0-100% linear gradient at 0.8 ml/min over 50 min and the absorbance was measured at 220 nm.

Figure 15 shows binding of antibody to TRAIL (huTR 2). Antibody 16449 was immobilized to a CM5 sensor chip and 100% TRAIL binding signal without antibody binding in solution was established using 1nM TRAIL in the absence of antibody. To determine antibody binding in solution, antibody samples of 7pM to 10nM were incubated with 1nM TRAIL. The decreased TRAIL binding signal after antibody incubation is indicative of binding of the antibody to TRAIL in solution.

Figure 16 shows that an embodiment of the antibody of the invention does not bind to TRAIL (huTR 2). Antibody 16449 was immobilized to a CM5 sensor chip and a 100% TRAIL binding signal without antibody binding in solution was established using 10nM TRAIL in the absence of antibody. To determine antibody binding in solution, antibody samples at 50 and 1000nM were incubated with 10nM trail. The decreased TRAIL binding signal after antibody incubation is indicative of binding of the antibody to TRAIL in solution.

Figure 17 shows that an embodiment of the antibody of the invention does not bind to TRAIL (huTR 2). Antibody 16449 was immobilized to a CM5 sensor chip and a 100% TRAIL binding signal without antibody binding in solution was established using 10nM TRAIL in the absence of antibody. To determine antibody binding in solution, 1000nM antibody samples were incubated with 10nM trail. The decreased TRAIL binding signal after antibody incubation is indicative of binding of the antibody to TRAIL in solution.

Figure 18 shows that the antibody embodiment of the invention listed on the y-axis did not bind to TRAIL (huTR 2). Antibody 16449 was immobilized to a CM5 sensor chip and a 100% TRAIL binding signal without antibody binding in solution was established using 10nM TRAIL in the absence of antibody. To determine antibody binding in solution, antibody samples of 1000 and 10000nM were incubated with 10nM TRAIL. The decreased TRAIL binding signal after antibody incubation is indicative of binding of the antibody to TRAIL in solution.

Figure 19 shows that the antibody embodiments of the invention listed on the x-axis did not bind to TRAIL (huTR 2). Antibody 16449 was immobilized to a CM5 sensor chip and a 100% TRAIL binding signal without antibody binding in solution was established using 10nM TRAIL in the absence of antibody. To determine antibody binding in solution, 50000nM antibody samples were incubated with 10nM TRAIL. The decreased TRAIL binding signal after antibody incubation is indicative of binding of the antibody to TRAIL in solution.

Figures 20A-B show that the antibody embodiments of the present invention listed on the x-axis do not bind to TRAIL (huTR 2). Antibody 16449 was immobilized to a CM5 sensor chip and a 100% TRAIL binding signal without antibody binding in solution was established using 10nM TRAIL in the absence of antibody. To determine antibody binding in solution, antibody samples of 1000, 10000 and 50000nM were incubated with 10nM TRAIL. The decreased TRAIL binding signal after antibody incubation is indicative of binding of the antibody to TRAIL in solution.

Figure 20C shows the results of in vitro cell-based TRAIL activity assays. Samples of antibodies 4241 and 4341 were compared to the positive control IgG1 anti-TR 2mAb 16449. The prepared antibody sample was added to the TRAIL-sensitive human ascites colorectal adenocarcinoma cell line Colo 205. Detection of TRAIL-mediated caspase-3 activation by measuring an increase in relative luminescence was taken as a positive marker of apoptosis. Unlike positive control antibody 16449, antibodies 4241 and 4341 do not activate caspase-3.

FIG. 21A shows 2. mu.g of antibody 1870[ aka16451]16449, 16450, 10185, 10184, 4341, 10183 and 10182 at 220Non-reducing analysis on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) performed at V using non-reducing loading buffer and staining with QuickBlue (Boston biologicals). The molecular weight standard was NovexSeeBlue prestained standard. Molecular weight standard NovexThe standards were pre-stained.

FIG. 21B shows 2. mu.g of antibody 1870[ aka16451]16449, 16450, 10185, 10184, 4341, 10183 and 10182 reduction analysis on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) at 220V using non-reducing loading buffer and staining with QuickBlue (Boston biologicals). Molecular weight standard NovexThe standards were pre-stained.

FIGS. 22A-F show size exclusion chromatography of 50 μ g of antibody 4241 (FIG. 22A), 4341 (FIG. 22B), 10182 (FIG. 22C), 10183 (FIG. 22D), 10184 (FIG. 22E) and 10185 (FIG. 22F), wherein the antibody is injected at 1 mL/min into 50mM NaH₂PO₄Absorbance was measured at 280nm on a Phenomenex BioSep SEC-3000 column (7.8x300mM) in 250mM NaCl (ph 6.9).

Figures 23A-B show titers of antibodies 4241 and 4341, respectively. Expression libraries were created by transfecting CHO DHFR (-) host cells with the corresponding HC and LC expression plasmids. Small-scale (5-mL; FIG. 23A) expression assays were performed in CD6-D assay medium using a 6 day prior loading method, while large-scale (3-L; FIG. 23B) assays were performed using a 11 day fed batch method with peptone medium. Titer levels were measured using a protein a HPLC based assay.

FIGS. 24A-B show reduction analysis on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) at 220V of samples processed with antibodies 4241 (FIG. 24A) and 4341 (FIG. 24B) using non-reducing loading buffer and stained with QuickBlue (Boston biologicals).

FIG. 25 shows the overlap of chromatograms of antibodies 4341 and 4241 on SP-HP Sepharose columns (GE Life sciences) with a gradient elution at 7 ℃ using 20 column volumes to 50% S-Buffer B (20mM acetic acid, 1M NaCl, pH5.0) and absorbance observed at 300 nm.

FIGS. 26A-B show analysis of 4241 (FIG. 26A) and 4341 (FIG. 26B) antibodies on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) at 220V, stained with QuickBlue (Boston biologicals). Lanes labeled "NR" contain non-reducing sample buffer, while those labeled "red" contain reducing sample buffer.

Fig. 27A-B show antibodies: full size (FIG. 27A) and scale-up (FIG. 27B) analyses of 4241 (upper panel) and 4341 (lower panel) using two size exclusion columns in series (TSK-GELG3000SWXL, 5mM size, 7.8X300mM, TosohBioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (pH6.8) flowing at 0.5 mL/min.

Figure 28 shows analysis of antibodies 4341 and 4241 by DSC using MicroCal VP-DSC, where the sample was heated from 20 ℃ to 95 ℃ at a rate of 1 ℃ per minute. The protein concentration in 10mM sodium acetate, 9% sucrose (pH5.0) was 0.5 mg/ml.

FIGS. 29A-D show analysis of 4241 (FIGS. 29A-B) and 4341 (FIGS. 29C-D) antibodies by reducing (FIGS. 29A and 29C) and non-reducing (FIGS. 29B and 29D) CE-SDS, where absorbance was detected at 220 nm. Bare fused silica capillaries 50 μm × 30.2cm were used for separation analysis.

FIG. 30 shows the analysis of 4241 (upper panel) and 4341 (lower panel) antibodies using ion exchange HPLC (SP-5PW, 10 μ M particles, 7.5mM IDx7.5cm, Tosohbioscience, 08541) with 20mM acetic acid (pH5.0) as buffer A, 20mM acetic acid, 1M NaCl (pH5.0) as buffer B, flowing at 1 mL/min with an 80 min linear gradient of 0-40% buffer B.

FIGS. 31A-B show HIC analysis of 4241 (FIG. 31A) and 4341 (FIG. 31B) antibodies before and after light exposure using two Dionex ProPac HIC-10 columns in tandem, mobile phase A being 1M ammonium sulfate, 20mM sodium acetate (pH5.0), and mobile phase B being 20mM sodium acetate, 5% acetonitrile (pH 5.0). The sample was eluted with a 0-100% linear gradient at 0.8 ml/min over 50 min and the absorbance was observed at 220 nm.

Fig. 32 shows representative pharmacokinetic profiles of 16435, 16444, 4241 and 4341 antibodies determined in adult Sprague-Dawley rats (8-12 weeks of age) by subcutaneous injection of 5mg/kg and blood collection from the tail vein at 0, 0.25, 1, 4, 24, 48, 72, 96, 168, 336, 504, 672, 840 and 1008 hours post-dose. Serum concentrations were then determined using an anti-human Fc based ELISA.

Figure 33 shows a representative pharmacokinetic profile of 16435 antibody determined in male cynomolgus monkeys using a single IV dose of 1mg/kg or 10 mg/kg. Serum samples were collected at 0.25, 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 360, 408, 456, 504, 552, 600, 648 and 672 hours before and after administration. The 16435 antibody level of the samples was determined using an anti-IgG sandwich ELISA.

Figure 34 shows a schematic structural view of one embodiment of the composition of the invention comprising a pharmacologically active toxin peptide analog of one unit fused to one immunoglobulin via an optional peptidyl linker moiety (wave curve).

FIG. 35 shows Coomassie Brilliant blue stained Tris-glycine 4-20% SDS-PAGE of the final monovalent 16435IgG2-L10-Shk [1-35, Q16K ] product. Products were isolated from 4 different expression pools. Lanes 1-10 were loaded as follows: novex Mark12 broad range protein standards (10. mu.l), 2. mu.g non-reduced library 1 product, 2. mu.g non-reduced library 2 product, 2. mu.g non-reduced library 3 product, 2. mu.g non-reduced library 4 product, Novex Mark12 broad range protein standards (10. mu.l), 2. mu.g reduced library 1 product, 2. mu.g reduced library 2 product, 2. mu.g reduced library 3 product, 2. mu.g reduced library 4 product.

FIGS. 36A-D show size exclusion chromatography analysis of 30 μ g of the final pool 1, 2, 3 and 4 of 3742 product, where the final pool was injected at a rate of 1 mL/min onto a Phenomenex BioSepSEC-3000 column (7.8X300mM) equilibrated with 50mM NaH2PO4, 250mM NaCl (pH6.9) and absorbance was measured at 280 nm.

FIGS. 37A-D show LC-MS analysis of reduced light chain from the final 3742 sample. The product was chromatographed through a Waters MassREP mini desalting column using a Waters ACQUITY UPLC system. The column was set to 80 ℃ and the protein was eluted using a linear gradient of increasing acetonitrile concentration in 0.1% formic acid. The column eluate was directed to a Waters LCT PremierESI-TOF mass spectrometer for mass analysis. The instrument was run in forward V mode. The capillary voltage was set to 3,200V and the cone voltage to 80V. Mass spectra were collected from 800 to 3000m/z using MaxEntl software supplied by the instrument manufacturer and deconvoluted.

FIGS. 38A-D show reduced heavy chain LC-MS analysis of the final 3742 samples. The product was chromatographed through a Waters MassREP mini desalting column using a Waters ACQUITY UPLC system. The column was set to 80 ℃ and the protein was eluted using a linear gradient of increasing acetonitrile concentration in 0.1% formic acid. The column eluate was directed to a Waters LCT PremierESI-TOF mass spectrometer for mass analysis. The instrument was run in forward V mode. The capillary voltage was set to 3,200V and the cone voltage to 80V. Mass spectra were collected from 800 to 3000m/z using MaxEntl software supplied by the instrument manufacturer and deconvoluted.

FIG. 39A shows non-reducing analysis on 1.0mm Tris-glycine 4-20% SDS-PAGE (Novex) at 220V using non-reducing loading buffer and staining with QuickBlue (Boston biologicals) of conditioned media for antibody fusions 10162, 10163 and 10164 and conditioned media from mock transfections. Molecular weight standards are indicated in kDa.

FIG. 39B shows Coomassie Brilliant blue stained Tris-glycine 4-20% SDS-PAGE of the final 10162, 10163 and 10164 products. In lanes 1 and 5, Novex Mark12 standard was loaded. For lanes 2-4 (non-reduced) and 6-8 (reduced), 2. mu.g of product was loaded.

FIGS. 40A-C show size exclusion of 50 μ g of fusion antibodies 10162 (FIG. 40A), 10163 (FIG. 40B) and 10164 (FIG. 40C)Chromatography, in which the antibody was injected at a rate of 1 mL/min into 50mM NaH₂PO₄Absorbance was measured at 280nm on a phenomenex biosep SEC-3000 column (7.8x300mM) in 250mM NaCl (ph 6.9).

FIGS. 41A-C show reduced light chain LC-MS analysis of final 4341-ShK (1-35, Q16K) (FIG. 41A), 4341-FGF21 (FIG. 41B) and 16435-FGF21 (FIG. 41C) samples. The product was chromatographed through a Waters MassREP mini desalting column using a Waters ACQUITY UPLC system. The column was set to 80 ℃ and the protein was eluted using a linear gradient of increasing acetonitrile concentration in 0.1% formic acid. The column eluate was directed to a Waters LCTPremier ESI-TOF mass spectrometer for mass analysis. The instrument was run in forward V mode. The capillary voltage was set to 3,200V and the cone voltage to 80V. Mass spectra were collected from 800 to 3000m/z and deconvoluted using MaxEnt1 software supplied by the instrument manufacturer.

FIGS. 42A-C show reduced heavy chain LC-MS analysis of final 4341-ShK (1-35, Q16K) (FIG. 42A), 4341-FGF21 (FIG. 42B) and 16435-FGF21 (FIG. 42C) samples. The product was chromatographed through a Waters MassREP mini desalting column using a Waters ACQUITY UPLC system. The column was set to 80 ℃ and the protein was eluted using a linear gradient of increasing acetonitrile concentration in 0.1% formic acid. The column eluate was directed to a Waters LCTPremier ESI-TOF mass spectrometer for mass analysis. The instrument was run in forward V mode. The capillary voltage was set to 3,200V and the cone voltage to 80V. Mass spectra were collected from 800 to 3000m/z and deconvoluted using MaxEnt1 software supplied by the instrument manufacturer.

FIG. 43 shows representative PK profiles for antibodies 16435 and 4341 (both at 5mg/kg doses) in SD rats.

Figure 44 shows a representative PK profile for consecutive doses (5mg/kg) of antibody 16435 or 4341 in cynomolgus monkeys.

Detailed description of the embodiments

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definition of

Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes a population of a plurality of cells.

"polypeptide" and "protein" are used interchangeably herein and include molecular chains of two or more amino acids covalently linked by peptide bonds. These terms do not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. These terms include post-translational modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation, and the like. Furthermore, protein fragments, analogs, mutant or variant proteins, fusion proteins, and the like are also included within the meaning of the polypeptides. These terms also include molecules that contain one or more amino acid analogs or non-canonical or non-natural amino acids that can be expressed recombinantly using known protein engineering techniques. In addition, fusion proteins can be derivatized by well-known organic chemistry techniques as described herein.

Reference to the term "isolated protein" means that the subject protein (1) is free of at least some other proteins that normally occur naturally with it; (2) substantially free of other proteins from the same source (e.g., from the same species); (3) recombinant expression from a cell of a heterologous species or species; (4) has been isolated from at least about 50% of polynucleotides, lipids, carbohydrates or other substances with which it is naturally associated; (5) operatively associated (by covalent or non-covalent interactions) with a polypeptide with which it is not naturally associated; and/or (6) not naturally occurring. An "isolated protein" typically constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA of synthetic origin or any combination thereof may encode such an isolated protein. Preferably, the isolated protein is substantially free of proteins or polypeptides or other contaminants present in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

"variants" of a polypeptide (e.g., an immunoglobulin or antibody) comprise amino acid sequences in which one or more amino acid residues are inserted into, deleted from, and/or substituted into the amino acid sequence associated with another polypeptide sequence. Variants include fusion proteins.

The term "fusion protein" means that the protein comprises polypeptide components derived from more than one parent protein or polypeptide. Fusion proteins are typically expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with a nucleotide sequence encoding a polypeptide sequence from a different protein, and optionally separated therefrom by a linker. The fusion gene can then be expressed as a single protein by the recombinant host cell.

"secreted" proteins refer to those proteins that can be directed to the ER, secretory vesicles, or extracellular space due to the secretion of a signal peptide sequence, as well as those proteins that are released into the extracellular space without necessarily including a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein may undergo extracellular processing, thereby producing a "mature" protein. Release into the extracellular space can occur by a number of mechanisms, including extracellular secretion and proteolytic cleavage. In some other embodiments of the compositions of the invention, the toxin peptide analogs can be synthesized as secreted proteins by the host cell, which can then be further purified from the extracellular space and/or culture medium.

"soluble" as used herein in reference to a protein produced in a host cell by recombinant DNA techniques, is a protein that is present in an aqueous solution; if the protein comprises a twin arginine signal amino acid sequence, the soluble protein is transported to the periplasmic space in a gram-negative bacterial host or secreted into the culture medium by a eukaryotic host cell capable of secretion or by a bacterial host with the appropriate gene (e.g., the kil gene). Thus, a soluble protein is a protein that is not present in inclusion bodies within the host cell. Alternatively, depending on the context, a soluble protein is a protein that is not found integrated in the cell membrane; in contrast, an insoluble protein is a protein that exists in a host cell in a denatured form within cytoplasmic granules (referred to as inclusion bodies), or again, depending on the context, an insoluble protein is a protein that exists in cell membranes, including but not limited to the cytoplasmic membrane, the mitochondrial membrane, the chloroplast membrane, the endoplasmic reticulum membrane, and the like.

"soluble human IL-17R" is a polypeptide having the amino acid sequence (huIL-17R-FpH) as follows: LRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLPKPIPDGDPNHQSKNPLVPDCEHARMKVTTPCMSSGSLWDPNTTVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWEPRSGSSDYKDDDDKGSSHHHHHH// SEQ ID NO: 89.

"soluble human TR 2" is a fusion polypeptide in monomeric or dimeric form (huTR2 long-huFc (IgG1) having the following amino acid sequence:

MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALTTQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPASPCSLSGVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISPTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO：82。

by "under physiological conditions" with respect to incubation buffer and immunoglobulin or other binding assay reagent is meant incubation under conditions of temperature, pH and ionic strength that allow biochemical reactions such as non-covalent binding reactions to occur. The temperature is typically room or ambient temperature up to about 37 ℃ and at ph 6.5-7.5.

The term "recombinant" refers to a material (e.g., a nucleic acid or polypeptide) that has been altered, either artificially or synthetically (i.e., not naturally) by human intervention. The alteration may be made to a material that is within its natural environment or state, or to a material that is removed from its natural environment or state. For example, a "recombinant nucleic acid" is a nucleic acid that is prepared by recombining a nucleic acid, e.g., during cloning, DNA shuffling, or other well-known molecular biological procedures. Examples of such Molecular biology procedures can be found in Maniatis et al, Molecular cloning.A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. (1982). A "recombinant DNA molecule" is composed of DNA segments joined together by these molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule that is expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and/or expresses a recombinant nucleic acid.

The term "polynucleotide" or "nucleic acid" includes single-and double-stranded nucleotide polymers containing two or more nucleotide residues. The nucleotide residues that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives; ribose modifications, such as 2 ', 3' -dideoxyribose; and internucleotide linkage modifications, such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoramidate.

The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotide residues. In some embodiments, the oligonucleotide is 10 to 60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides may be single-stranded or double-stranded, e.g., for use in the construction of mutant genes. The oligonucleotide may be a sense or antisense oligonucleotide. The oligonucleotide may comprise a label for a detection assay, including a radioactive label, a fluorescent label, a hapten or an antigenic label. Oligonucleotides may be used, for example, as PCR primers, cloning primers, or hybridization probes.

"polynucleotide sequence" or "nucleotide sequence" or "nucleic acid sequence" are used interchangeably herein and, depending on the context, are the primary sequence of nucleotide residues or a string representing the primary sequence of nucleotide residues in a polynucleotide (including oligonucleotides, DNA, and RNA, nucleic acids). A given nucleic acid or complementary polynucleotide sequence can be determined from any specified polynucleotide sequence. Including DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and represents either the sense or antisense strand. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left direction of the double stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5 'to 3' addition of nascent RNA transcripts is called the direction of transcription; a sequence region from 5 'on the DNA strand having the same sequence as the RNA transcript to the 5' end of the RNA transcript is referred to as an "upstream sequence"; the sequence region from 3 'on the DNA strand having the same sequence as the RNA transcript to the 3' end of the RNA transcript is referred to as the "downstream sequence".

As used herein, an "isolated nucleic acid molecule" or "isolated nucleic acid sequence" is a nucleic acid molecule that is (1) identified and isolated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source, or (2) cloned, amplified, labeled, or otherwise distinguished from background nucleic acid such that the nucleic acid sequence of interest can be determined. An isolated nucleic acid molecule is distinguished from its naturally occurring form or state. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses an immunoglobulin (e.g., an antibody), where, for example, the nucleic acid molecule is located at a chromosomal location that is different from that of the native cell.

As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along the mRNA chain and also determines the order of amino acids along the polypeptide (protein) chain. Thus the DNA sequence encodes an RNA sequence and encodes an amino acid sequence.

The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Genes typically contain coding sequences and/or regulatory sequences required for expression of these coding sequences. The term "gene" applies to a particular genomic or recombinant sequence, as well as to the cDNA or mRNA encoded by that sequence. A "fusion gene" comprises a coding region that encodes a toxin peptide analog. Genes also include, for example, non-expressed nucleic acid fragments that form recognition sequences for other proteins. The non-expressed regulatory sequences include transcriptional control elements to which regulatory proteins, such as transcription factors, bind, thereby causing transcription of adjacent or nearby sequences.

By "expression of a gene" or "expression of a nucleic acid" is meant transcription of DNA into RNA (optionally including modification of RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent post-translational modification of the polypeptide), or both transcription and translation, as the context dictates.

As used herein, the term "coding region" or "coding sequence" when used in reference to a structural gene refers to a nucleotide sequence that encodes the amino acids found in a nascent polypeptide as a result of translation of an mRNA molecule. In eukaryotes, the coding region is bounded on the 5 'side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by one of the three triplets (i.e., TAA, TAG, TGA) of the designated stop codon.

The term "control sequence" or "control signal" refers to a polynucleotide sequence that can affect the expression and processing of a coding sequence linked thereto in a particular host cell. The nature of such control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosome binding site, and a transcription termination sequence. Control sequences for eukaryotes may include promoters, transcription enhancer sequences or elements, including one or more transcription factor recognition sites, polyadenylation sites, and transcription termination sequences. The control sequences may include leader sequences and/or fusion partner sequences. Promoters and enhancers consist of short DNA arrays that specifically interact with cellular proteins involved in transcription (Maniatis et al, Science 236: 1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, as well as viruses (similar control elements, i.e., promoters, are also present in prokaryotes). The choice of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range, while others are effective in a limited subset of cell types (for review see Voss et al, Trends biochem. Sci., 11: 287(1986) and Maniatis et al, Science 236: 1237 (1987)).

The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein-encoding information into a host cell.

The term "expression vector" or "expression construct" as used herein refers to a recombinant DNA molecule comprising a desired coding sequence and appropriate nucleic acid control sequences required for expression of the operably linked coding sequence in a particular host cell. Expression vectors can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing of coding regions operably linked to the introns. Nucleic acid sequences required for expression in prokaryotes include promoters, optional operator sequences, ribosome binding sites and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. If desired, a secretion signal peptide sequence may also optionally be encoded by the expression vector, operably linked to the coding sequence of interest, so that the recombinant host cell can secrete the expressed polypeptide, to facilitate easier isolation of the polypeptide of interest from the cell. Such techniques are well known in the art. (e.g., Goodey, Andrew R.; et al, Peptide and DNA sequences, U.S. Pat. No. 5,302,697; Weiner et al, Compositions and methods for Protein analysis, U.S. Pat. No. 6,022,952 and U.S. Pat. No. 6,335,178; Uemura et al, Protein expression vector and analysis therof, U.S. Pat. No. 7,029,909; Ruben et al, 27 man secreted proteins, U.S. Pat. No. 2003/0104400A 1).

The terms "operable combination", "operable order" and "operably linked" as used herein refer to the joining of nucleic acid sequences in such a way as to produce a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule. The term also refers to the linkage of amino acid sequences in such a way that a functional protein is produced. For example, a control sequence in a vector "operably linked" to a protein coding sequence is linked to the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

The term "host cell" means a cell that has been transformed or is capable of being transformed with a nucleic acid and thereby expressing a gene of interest. The term includes progeny of a parent cell, whether or not the progeny is morphologically or genetically identical to the original parent cell, so long as the gene of interest is present. Any of a number of useful and well known host cells can be used in the practice of the present invention. The choice of a particular host depends on a variety of factors recognized in the art. Such factors include, for example, compatibility with the chosen expression vector, toxicity of the peptide encoded by the DNA molecule, conversion rate, ease of peptide recovery, expression characteristics, biosafety and cost. The balance of these factors must be achieved with the following recognition: not all hosts may be equally efficient for expression of a particular DNA sequence. Within these general guidelines, microbial host cells useful in the culture medium include bacteria (e.g., E.coli: (E.coli)) Escherichia colisp.), yeast (e.g., Saccharomyces (Zymobacter)), (yeastSaccharomycessD.)) and other fungal cells, insect cells, plant cells,Mammalian (including human) cells, e.g., CHO cells and HEK-293 cells. Modification can also be performed at the DNA level. The DNA sequence encoding the peptide may be altered to codons more compatible with the host cell of choice. For theEscherichia coliOptimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which can facilitate DNA processing in a selected host cell. The transformed host is then cultured and purified. The host cell may be cultured under conventional fermentation conditions such that the desired compound is expressed. These fermentation conditions are well known in the art.

The term "transfection" means that a cell takes up foreign or exogenous DNA, and when exogenous DNA has been introduced into the cell membrane, the cell has been "transfected". Various transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology 52: 456; sambrook et al, 2001, Molecular Cloning: a Laboratory Manual, supra; davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; chu et al, 1981, Gene 13: 197. these techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

The term "transformation" refers to an alteration of the genetic characteristics of a cell, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed when it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombine with the DNA of the cell by physically integrating into the chromosome of the cell, or may remain transiently as an episomal element (episomal element) without being replicated, or may replicate independently as a plasmid. When transforming DNA is replicated with cell division, the cell is considered to have been "stably transformed".

By "physiologically acceptable salt" of a composition of matter, for example a salt of an immunoglobulin (e.g., an antibody), is meant any salt or salts that are known or later discovered to be pharmaceutically acceptable. Some non-limiting examples of pharmaceutically acceptable salts are: an acetate salt; a trifluoroacetate salt; hydrohalic acid salts, such as hydrochloride and hydrobromide salts; a sulfate salt; a citrate salt; a maleate salt; a tartrate salt; a glycolate; a gluconate salt; a succinate salt; a mesylate salt; a benzenesulfonate salt; gallate (gallic acid is also known as 3, 4, 5 trihydroxybenzoic acid) such as Pentagalloylglucose (PGG) and epigallocatechin gallate (EGCG) salts; salts of cholesterol sulfate; pamoate salts; tannates and oxalates.

A "domain" or "region" of a protein (used interchangeably herein) is any portion of the entire protein, up to and including the entire protein, but generally including less than the entire protein. Domains may, but need not, fold independently of the remainder of the protein chain and/or be associated with a particular biological, biochemical or structural function or location (e.g., a ligand binding domain, or a cytoplasmic, transmembrane or extracellular domain).

"treatment" is an intervention intended to prevent the development of a disorder or to modify the pathology of a disorder. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Persons in need of treatment include those already with the disorder as well as those who are to be prevented from the disorder. "treatment" includes any sign of successful improvement of an injury, lesion or condition, including any objective or subjective parameter, such as reduction of symptoms; (iii) alleviating; attenuating or making the injury, lesion, or condition more tolerable to the patient; slowing the rate of deterioration or decline; making the final point of deterioration less debilitating; improving the physical or mental health of the patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including physical examination, patient self-reporting, neuropsychiatric examination; and/or the results of psychiatric evaluations.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate symptoms and/or underlying causes, prevent the occurrence of symptoms and/or underlying causes thereof, and/or ameliorate or repair the damage caused by or associated with migraine. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state (e.g., transplant rejection or GVHD, inflammation, multiple sclerosis, cancer, diabetes, neuropathy, pain) or one or more symptoms, particularly a state or one or more symptoms associated with a disease state, or to prevent, hinder, delay or reverse the progression of a disease state or any other adverse symptoms associated with a disease in any way (i.e., to provide "therapeutic efficacy"). A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have a desired prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of, or reducing the likelihood of the onset (or recurrence) of, migraine symptoms, or multiple sclerosis symptoms. The full therapeutic or prophylactic effect does not necessarily occur as a result of administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

"mammal" for therapeutic purposes means any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, rats, mice, monkeys, etc. The mammal is preferably a human.

The term "naturally occurring" as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells and the like refers to materials that occur in nature.

The term "antibody", or interchangeably "Ab", is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human, humanized or chimeric antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab ', F (Ab') 2, Fv, single chain antibodies, diabodies) that bind to an antigen, including the Complementarity Determining Regions (CDRs) of the foregoing, so long as they exhibit the desired biological activity. Multimers or aggregates of intact molecules and/or fragments, including chemically derivatized antibodies, are contemplated. Antibodies of any isotype or subtype are contemplated, including IgG, IgM, IgD, IgA and IgE, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, or any allotype. Different isoforms have different effector functions; for example, IgG1 and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity.

The term "antigen binding protein" (ABP) includes antibodies or antibody fragments as defined above, as well as recombinant peptides or other compounds comprising sequences derived from CDRs having the desired antigen binding properties such that they specifically bind to a target antigen of interest.

In general, an antigen binding protein, such as an antibody or antibody fragment, "specifically binds" an antigen of interest (e.g., IL-17R or TR2) when the antigen has a binding affinity for the antigen that is significantly higher than its affinity for other unrelated proteins under similar binding assay conditions and is therefore capable of recognizing the antigen. When dissociation constant (K)_D) Is less than or equal to 10^-8M, an antigen binding protein is generally said to "specifically bind" to its target antigen. When K is_DIs less than or equal to 5 multiplied by 10^-9M, the antibody specifically binds to the antigen with "high affinity", and when K_DIs less than or equal to 5 multiplied by 10^-10M, the antibody specifically binds to the antigen with "very high affinity". In one embodiment, the antibody will be between about 10^-8M and 10^-10K between M_DBinds to an antigen of interest, and in another embodiment, the antibody will be at ≦ 5 × 10^-9K of_DAnd (4) carrying out combination.

By "antigen-binding region" or "antigen-binding site" is meant the portion of a protein that specifically binds a particular antigen, such as IL-17R or TR 2. For example, the portion of an antigen binding protein that comprises amino acid residues that interact with an antigen and confer upon the antigen binding protein its specificity and affinity for the antigen is referred to as an "antigen binding region". The antigen binding region typically includes one or more "complementary binding regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions ("FRs"). The 'CDR' is helpfulAmino acid sequences with antigen binding specificity and affinity. The "framework" region can help maintain the proper conformation of the CDRs to facilitate binding between the antigen binding region and the antigen. In traditional antibodies, CDRs are embedded within frameworks in the heavy and light chain variable regions, where they constitute the regions responsible for antigen binding and recognition. The variable region of an immunoglobulin antigen binding protein comprises at least three heavy or light chain CDRs within the framework regions (designated framework regions 1-4, FR1, FR2, FR3 and FR4, Kabat et al, 1991, supra; see also Chothia and Lesk, 1987, supra), see also supra (Kabat et al, 1991, Sequencesof Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD; see also Chothia and Lesk, 1987, J.mol.biol.). 196: 901-; chothia et al, 1989, Nature342：877-883)。

An "isolated" immunoglobulin, e.g., an antibody or antibody fragment, is an immunoglobulin that has been recognized and isolated from one or more components of its natural environment or culture medium in which it is secreted by the producing cells. The "contaminant" component of its natural environment or culture medium is a material that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will (1) be purified to greater than 95% by weight, and most preferably greater than 99% by weight of the antibody, or (2) be purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions, optionally using staining, such as coomassie blue or silver staining. An isolated naturally occurring antibody includes an antibody in situ within a recombinant cell, as at least one component of the antibody's natural environment will not be present. However, isolated antibodies are typically prepared by at least one purification step.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Compared to polyclonal antibody systems that typically include different antibodies directed against different epitopes Agents, monoclonal antibodies that are antigen binding proteins, are highly specific binders, directed against a single antigenic site or epitope. Non-limiting examples of monoclonal antibodies include murine, rabbit, rat, chicken, chimeric, humanized or human antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments that bind antigen (including Fab, Fab ', F (ab')₂Fv, single chain antibody, diabody), macroantibody (maxibody), nanobody, and recombinant peptides comprising the foregoing CDRs, as long as they exhibit the desired biological activity, or variants or derivatives thereof.

The modifier "monoclonal" indicates the identity of the antibody obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the invention may be produced by first monoclonal antibodies produced by a method described by Kohler et al, Nature, 256: 495[1975], or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be used, for example, in Clackson et al, Nature, 352: 624-: 581-597(1991) from phage antibody libraries.

A "multispecific" binding agent or antigen-binding protein or antibody targets more than one antigen or epitope.

A "bispecific", "dual specificity" or "bifunctional" binding agent or antigen binding protein or antibody is a hybrid having two distinct antigen binding sites. Dual antigen binding proteins, antigen binding proteins and antibodies belong to the class of multi-antigen binding proteins, antigen binding proteins or multi-specific antibodies and can be produced by a variety of methods including, but not limited to, hybridoma fusion or ligation of Fab' fragments. See, e.g., Songsivilai and Lachmann, l990, clin. exp. immunol.79: 315- > 321; kostelny et al, 1992, J.Immunol.148: 1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes, which may be located on the same or different protein targets.

The term "immunoglobulin" encompasses a complete antibody comprising two dimerized Heavy Chains (HC) and each heavy chain covalently linked to a Light Chain (LC); a single, non-dimerized immunoglobulin heavy chain and a covalently linked light chain (HC + LC); or chimeric immunoglobulins (light chain + heavy chain) -Fc heterotrimers (so-called "half-antibodies"). An "immunoglobulin" is a protein, but not necessarily an antigen binding protein.

In an "antibody", each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain of about 220 amino acids (about 25kDa) and a "heavy" chain of about 440 amino acids (about 50-70 kDa). The amino-terminal portion of each chain includes a "variable" ("V") region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. The variable regions differ between different antibodies. The constant regions are identical between different antibodies. In the case of an antibody that is an antigen binding protein, there are three hypervariable subregions within the variable region of each heavy or light chain that help determine the specificity of the antibody for antigen. However, within the scope of the present invention, embodiments of immunoglobulins, such as antibodies, need not be antigen binding proteins, or need not be known to specifically bind antigens. Variable domain residues between hypervariable regions are called framework residues and are generally to some extent homologous between different antibodies. Immunoglobulins can be assigned to different classes based on the constant domain amino acid sequence of their heavy chains. Human light chains are classified into kappa (. kappa.) and lambda (. lamda.) light chains. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain further comprises a "D" region of about more than 10 amino acids. See generally fundamentals immunology chapter 7 (Paul, w. editor, 2 nd edition, Raven Press, n.y. (1989)). Within the scope of the present invention, "antibody" also encompasses recombinantly produced antibodies as well as antibodies that are glycosylated or lack glycosylation.

The term "light chain" or "immunoglobulin light chain"includes full-length light chains and fragments thereof having variable region sequences sufficient to confer binding specificity. Full-length light chains comprise a variable region V_LAnd a constant region C_L. The variable region of the light chain is located at the amino terminus of the polypeptide. Light chains include kappa and lambda chains.

The term "heavy chain" or "immunoglobulin heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity. The full-length heavy chain comprises a variable region V_HAnd three constant regions C_H1、C_H2 and C_H3。V_HThe domain is located at the amino terminus of the polypeptide, and C_HThe domain is at the carboxy terminus, where C_H3 is closest to the carboxy terminus of the polypeptide. Heavy chains are divided into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In various embodiments of the invention, the heavy chain may be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. Several of these can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. Different IgG isotypes may have different effector functions (mediated by the Fc region), such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (fcyr) on the surface of immune effector cells, such as natural killer cells and macrophages, leading to phagocytosis or lysis of target cells. In CDC, antibodies kill target cells by triggering the complement cascade at the cell surface.

An "Fc region", or "Fc domain" or "immunoglobulin Fc domain" as used interchangeably herein, comprises two heavy chain fragments which, in a complete antibody, constitute the C of the antibody_H1 and C_H2 fields. The two heavy chain fragments are linked by two or more disulfide bonds and by C_HThe 3 domains are held together by hydrophobic interactions.

The term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) Is responsible for the epitope that increases the serum half-life of the IgG molecule in vivo.

An "allotype" is a variant form of an antibody sequence, usually in the constant region, which may be immunogenic in humans and encoded by a specific allele. Allotypes have been identified for five of the human IGHC genes, namely IGHG1, IGHG2, IGHG3, IGHA2 and IGHE genes, and are designated G1m, G2m, G3m, A2m and Em allotypes, respectively. At least 18 Gm allotypes are known: nG1m (1), nG1m (2), G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, G1, c5, u, V, G5). There are two A2m allotypes, A2m (1) and A2m (2).

For a detailed description of the structure and production of antibodies, see Roth, d.b. and Craig, n.l., Cell, 94: 411-414(1998), which is incorporated herein by reference in its entirety. Briefly, the process of generating DNA encoding the heavy and light chain immunoglobulin sequences occurs first in developing B cells. V, D, J and the constant (C) gene segment are typically present relatively close together on a single chromosome prior to rearrangement and ligation of the various immunoglobulin gene segments. During B cell differentiation, one of each of the appropriate family members of V, D, J (or in the case of light chain genes only V and J) recombines to form functionally rearranged variable regions of the heavy and light chain immunoglobulin genes. This process of gene fragment rearrangement appears to be sequential. First, a heavy chain D-to-J junction is performed, followed by a heavy chain V-to-DJ junction and a light chain V-to-J junction. In addition to the rearrangement of V, D and J segments, further diversification was created in the main repertoire of immunoglobulin heavy and light chains by variable recombination at the location where the V and J segments join in the light chain and the D and J segments join in the heavy chain. This change in the light chain typically occurs within the last codon of the V gene segment and the first codon of the J segment. Similar ligation does not occur exactly at D and J on the heavy chain chromosome _HAnd may extend up to 10 nucleotides between fragments. In addition, multiple nucleotides may be presentTo insert D and J_HAnd V_HAnd D gene segments, these nucleotides are not encoded by genomic DNA. The addition of these nucleotides is referred to as N-region diversification. The net effect of this rearrangement in the variable region gene segments and the variable recombination that can occur during this ligation is the generation of a master antibody repertoire.

The term "hypervariable" region refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from the complementarity determining regions or CDRs [ i.e., residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable domain and 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al, Sequences of Proteins of immunological Interest, 5 th edition, Public Health Service, national institutes of Health, Bethesda, Md. (1991) ]. Even a single CDR can recognize and bind antigen, but has a lower affinity than the entire antigen binding site that contains all CDRs.

Substitutions of residues from the hypervariable "loop" are defined by Chothia et al j.mol.biol.196: 901-917(1987) is described as residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable domain and 26-32(H1), 53-55(H2) and 96-101(H3) in the heavy chain variable domain.

"framework" or "FR" residues are those residues of the variable region, but not the hypervariable region.

An "antibody fragment" comprises a portion of an intact full-length antibody, preferably the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a bivalent antibody; linear antibodies (Zapata et al, Protein Eng., 8 (10): 1057-1062 (1995)); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments (referred to as "Fab" fragments, each having a single antigen-binding site) and a residual "Fc" fragment comprising the constant region. The Fab fragment contains all the variable domains as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). The Fc fragment displays carbohydrates and is responsible for many antibody effector functions (e.g., binding complement and cellular receptors) that distinguish one type of antibody from another.

Pepsin treatment to give F (ab')₂A fragment having two "single chain Fv" or "scFv" antibody fragments comprising the VH and VL domains of an antibody, wherein the domains are present in a single polypeptide chain. Fab fragments differ from Fab' fragments in that they contain several additional residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the structure required for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol.113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994).

The "Fab fragment" consists of one light chain and one heavy chain C_H1 and variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

"Fab' fragment" comprising one light chain and one heavy chain comprising V_HDomain and C_H1 part of the Domain and C_H1 and C_H2 domain such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments, thereby forming F (ab')₂A molecule.

“F(ab′)₂Fragment "comprises two light chains and two comprise C_H1 and C_H2 domain such that an interchain disulfide bond is formed between the two heavy chains. Thus F (ab')₂The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH VL dimer. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with lower affinity than the entire binding site.

A "single chain antibody" is an Fv molecule in which the variable region of the heavy chain and the variable region of the light chain have been joined by a flexible linker to form a single polypeptide chain that forms the antigen binding region. Single chain antibodies are discussed in detail in International patent application publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference in their entirety.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains (wherein the domains are present in a single polypeptide chain), and optionally comprising V_HAnd V_LA polypeptide linker between the domains which enables the Fv to form the structure required for antigen binding (Bird et al, Science 242: 423-. Fragment "Fd" consists of V_HAnd C_H1 domain.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and form two antigen binding sites. Bivalent antibodies are described, for example, in EP404,097; WO 93/11161; and Hollinger et al, proc.natl.acad.sci.usa, 90: 6444- > 6448(1993) is described more fully.

A "domain antibody" is an immunologically functional immunoglobulin fragment that comprises only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more V_HThe region is covalently linked to a peptide linker to form a bivalent domain antibody. Two V of bivalent domain antibody_HThe regions may target the same or different antigens.

The term "competition" as used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means that competition between the antigen binding proteins is determined by an assay in which the antigen binding protein under test (e.g., an antibody or immunologically functionalized fragment thereof) prevents or inhibits specific binding of a reference antigen binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., IL-17R or fragment thereof, or TR2 or fragment thereof). Various types of competitive binding assays can be used, for example: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-; direct labeling of RIA using an I-125 labeled solid phase (see, e.g., Morel et al, 1988, mol. Immunol.25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al, 1990, Virology 176: 546-552); direct-labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol.32: 77-82); and surface plasmon resonance ( (ii) a E.g., Fischer et al, Apeptide-immunoglobulin-conjugate, WO2007/045463a1, example 10, which is incorporated herein by reference in its entirety) or KinExA. Typically, such assays involve the use of purified antigen bound to a solid surface or cells carrying either of these, unlabeled test immunoglobulin or antigen binding protein, and labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of a test antigen binding protein. The test immunoglobulin or antigen binding protein is typically present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include binding proteins with reference antigensAntigen binding proteins that bind the same epitope as well as antigen binding proteins that bind a proximal epitope sufficiently close to the epitope bound by the reference antigen binding protein to cause steric hindrance. Further details regarding methods for determining competitive binding are provided in the examples herein. Generally, when a competing antigen binding protein is present in excess, it will inhibit specific binding of a reference antigen binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

When an immunoglobulin (e.g., an antibody or antibody fragment) "does not significantly bind" to an antigen, this means that an excess of a particular immunoglobulin does not compete with a reference antigen binding protein (e.g., with a positive control antibody) so as not to inhibit its binding to the target antigen by > 39%, or > 30%, or > 20%, or > 10%. With respect to specific binding to soluble human IL-17R, the positive control antibody is antibody 16429, as described herein. With respect to specific binding to soluble human TR2, the positive control antibody is antibody 16449, as described herein.

Antibody antigen interaction may be by interaction with M^-1s^-1(k_a) Binding rate constant in units, or in s^-1(k_d) The dissociation rate constant in units, or the dissociation equilibrium constant in units of m (kd). The association rate constant, dissociation rate constant, or dissociation equilibrium constant can be determined using kinetic analysis techniques such as surface plasmon resonance ((ii) a E.g., Fischer et al, a peptide-immunoglobulin-conjugate, WO2007/045463a1, example 10, which is incorporated herein by reference in its entirety) or KinExA and is readily determined using the general procedures outlined by the manufacturer or other methods known in the art. By passing Or power derived from KinExAThe study data may be analyzed by the methods described by the manufacturer.

"measured by a surface plasmon resonance binding assay" with respect to determining whether a test immunoglobulin "does not significantly bind" means that the binding activity of the immunoglobulin is assessed based on surface plasmon resonance as measured in a solution equilibrium binding assay as described herein. Reference antigen binding proteins (e.g., antibody 16429 of human IL-17R or antibody 16449 of human TR2) were immobilized to the protein according to the manufacturer's instructions (BIACore, Inc., Piscataway, NJ)The 2000 research level sensor chip CM5 surface. Carboxyl groups on the sensor chip surface were activated by injecting 60 μ L of a mixture containing 0.2M N-ethyl-N' - (dimethylaminopropyl) carbodiimide (EDC) and 0.05M N-hydroxysuccinimide (NHS). The reference antigen binding protein was diluted with 10mM sodium acetate (pH4.0) and injected at 30. mu.L/min onto the activated chip surface for 6 min. Excess reactive groups on the surface were deactivated by injection of 60 μ L of 1M ethanolamine. The final immobilization level is typically about 6600 Resonance Units (RU). In the absence of soluble antigen binding protein (e.g., antibody), soluble target antigen (e.g., 10nM soluble human IL-17R or 30nM soluble human TR2) was used to establish 100% binding signal to an immobilized reference antigen binding protein (e.g., positive control antibody). The decreased target antigen binding signal after incubation of the test immunoglobulin is indicative of its level of binding to the target antigen in solution.

The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent, such as an antigen binding protein (including, for example, an antibody or immunologically functional fragment thereof), and that is otherwise capable of being used in an animal to produce an antibody that is capable of binding to the antigen. An antigen may have one or more epitopes capable of interacting with different antigen binding proteins, such as antibodies.

The term "epitope" is the portion of a molecule that is bound by an antigen binding protein (e.g., an antibody). The term includes any determinant capable of specifically binding an antigen binding protein such as an antibody or T cell receptor. Epitopes can be contiguous or non-contiguous (e.g., in a single chain polypeptide, amino acid residues that are not contiguous with each other in the polypeptide sequence but are bound by the antigen binding protein in the case of a molecule). In certain embodiments, epitopes may be mimetic in that they comprise a three-dimensional structure similar to the epitope used to produce the antigen binding protein and also comprise none or only some of the amino acid residues present in the epitope used to produce the antigen binding protein. More generally, epitopes are located on proteins, but in some cases may be located on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups, sugar side chains, phosphoryl groups, or sulfonyl groups of molecules such as amino acids, and can have specific three-dimensional structural characteristics and/or specific charge characteristics. Generally, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing the sequences. "percent identity" means the percentage of residues that are identical between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the compared molecules. For these calculations, the nulls in the alignment (if any) must be processed (address) by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in comparative Molecular Biology, (Lesk, a.m. editors), 1988, new york: oxford University Press; biocomputing information and genoproject, (Smith, d.w. ed), 1993, New York: academic Press; computer Analysis of Sequence Data, part I, (Griffin, a.m. and Griffin, h.g. editions), 1994, New Jersey: humana Press; von Heinje, g., 1987, Sequence Analysis in Molecular Biology, New York: academic Press; sequence Analysis Primer, (Gribskov, m. and Devereux, j. eds.), 1991, New York: M.Stockton Press; and Carillo et al, 1988, SIAM J. applied Math.48: 1073, respectively. For example, sequence identity can be determined by standard methods commonly used to compare the similarity of amino acid positions of two polypeptides. Two polypeptide or two polynucleotide sequences are aligned for best match to their corresponding residues (along the full length of one or both sequences or along a predetermined portion of one or both sequences) using a computer program such as BLAST or FASTA. The program provides default open and gap penalties, and a scoring matrix such as PAM250[ standard scoring matrix; see Dayhoff et al, Atlas of Protein Sequence and Structure, Vol.5, supplement 3(1978) for use in conjunction with computer programs. For example, the percent identity is then calculated as: the total number of identical matches is multiplied by 100 and then divided by the length of the longer sequence over the span of matches and the sum of the number of gaps introduced into the longer sequence to align the two sequences. In calculating percent identity, the sequences being compared are aligned in a manner that gives the greatest match between the sequences.

The GCG package is a computer program that can be used to determine percent identity and includes GAP (Devereux et al, 1984, Nucl. acid Res.12: 387; genetics computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to compare two polypeptides or two polynucleotides for which a percent sequence identity is to be determined. The sequences are aligned to best match their corresponding amino acids or nucleotides ("matching span", as determined by an algorithm). Gap opening penalties (which are calculated as 3x the average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and gap extension penalties (which are typically 1/10 for gap opening penalties) as well as comparison matrices such as PAM250 or BLOSUM62 are used in conjunction with the algorithm. In certain embodiments, standard comparison matrices (PAM250 comparison matrices see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure 5: 345-; BLOSUM62 comparison matrices see Henikoff et al, 1992, Proc. Natl. Acad. Sci. U.S. A.89: 10915-) -10919) are also used by the algorithm.

Recommended parameters for determining percent identity of a polypeptide or nucleotide sequence using the GAP program include the following:

The algorithm is as follows: needleman et al, 1970, J.mol.biol.48: 443-;

comparing the matrixes: BLOSUM62 from Henikoff et al, 1992 (supra);

gap penalties: 12 (but no penalty for end gaps)

Gap length penalty: 4

Similarity threshold: 0

Certain alignment schemes for aligning two amino acid sequences can result in only a small region of the two sequences matching, and this small alignment region can have very high sequence identity, even if there is no significant relationship between the two full-length sequences. Thus, the alignment method of choice (GAP program) can be adjusted if one wishes to obtain an alignment of at least 50 contiguous amino acids across the target polypeptide.

The term "modification" when used in connection with the immunoglobulins of the present invention (including antibodies and antibody fragments) includes, but is not limited to, one or more amino acid changes (including substitutions, insertions, or deletions); chemical modification; covalent modification by conjugation to a therapeutic or diagnostic agent; labels (e.g., using radionuclides or various enzymes); covalent polymer attachments such as pegylation (derivatization with polyethylene glycol) and insertion or substitution of unnatural amino acids by chemical synthesis. The modified immunoglobulins of the present invention will retain the binding (non-binding) properties of the unmodified molecules of the present invention.

The term "derivative" when used in connection with the immunoglobulins of the present invention (including antibodies and antibody fragments) refers to immunoglobulins that are covalently modified by conjugation to a therapeutic or diagnostic agent, a label (e.g., using a radionuclide or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol), and insertion or substitution of chemically synthesized unnatural amino acids. The derivatives of the invention will retain the binding properties of the non-derivatized molecules of the invention.

Embodiments of immunoglobulins

In full-length immunoglobulin light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain further comprises a "D" region of about more than ten amino acids. See, e.g., Fundamental Immunology, 2 nd edition, chapter 7 (Paul, w. editor) 1989, New York: raven Press (incorporated by reference in its entirety herein for all purposes). The variable region of each light/heavy chain pair typically forms an antigen binding site.

An example of a constant domain of the Heavy Chain (HC) of human IgG2 has the amino acid sequence:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKFYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO：86。

it is known in the art that constant region sequences of other IgG isotypes can be used to prepare recombinant forms of the immunoglobulin of the invention having an IgG1, IgG2, IgG3 or IgG4 immunoglobulin isotype as desired. Generally, human IgG2 can be used for targets that do not require effector function, while human IgG1 can be used in situations where such effector function is required (e.g., antibody-dependent cellular cytotoxicity (ADCC)). Human IgG3 has a relatively short half-life, whereas human IgG4 forms an antibody "half-molecule". There are four known allotypes of human IgG 1. The preferred allotype is referred to as the "hIgG 1 z" and also as the "KEEM" allotype. Human IgG1 allotypes "hIgG 1 za" (KDEL), "hIgG 1 f" (REEM), and "hIgG 1 fa" are also useful; all appear to have ADCC effector function.

The constant domain of the Heavy Chain (HC) of human hIgG1z has the amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISPTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO：87。

the constant domain of the Heavy Chain (HC) of human hIgG1za has the amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFRPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ I DNO：88。

the constant domain of the Heavy Chain (HC) of human hIgG1f has the amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO：127。

the constant domain of the Heavy Chain (HC) of human hIgG1fa has the amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO：90。

an example of a human immunoglobulin Light Chain (LC) constant region sequence is as follows (designated as "CL-1")

GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS//SEQ ID NO：91。

CL-1 is used to increase the pI of the antibody and is convenient. There are three other human immunoglobulin light chain constant regions, designated "CL-2", "CL-3" and "CL-7", which may also be used within the scope of the present invention. CL-2 and CL-3 are more common in the human population.

The CL-2 human Light Chain (LC) constant domain has the amino acid sequence:

Gqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthegstvektvaptecs//SEQ ID NO：92。

CL-3 human LC constant domain has the amino acid sequence:

gqpkaapsvtlfppsseelqankatlvelisdftpgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshksysjqvthegstvektvaptecs//SEQID NO：93。

CL-7 human LC constant domain has the amino acid sequence:

Gqpkaapsvtlfppsseelqankatlvclvsdfypgavtvawkadgspvkvgvettkpskqsnnkyaassylsltpeqwkshrsyscrvthegstvektvapaecs//SEQ ID NO：94。

the human LC κ constant region has the amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC//ESQIDNO：129。

the variable regions of immunoglobulin chains are generally shown to have the same overall structure, comprising relatively conserved Framework Regions (FRs) joined by three hypervariable regions, more commonly referred to as "complementarity determining regions" or CDRs. The CDRs from both chains of each heavy/light chain pair mentioned above are typically aligned by a framework region to form a specific epitope or region on a target (e.g., human IL-17R or human TR2) Domain-specific binding structure, however within the scope of the invention, the initial CDR sequences have been intentionally modified so as not to significantly bind to human IL-17R or TR2 targets. From N-terminus to C-terminus, the naturally occurring light and heavy chain variable regions both generally conform to these elements in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. Numbering systems have been designed to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is described in Kabat Sequences of Proteins of immunological interest (1987 and 1991, NIH, Bethesda, Md.), or Chothia&Lesk，1987，J.Mol.Biol.196：901-; chothia et al, 1989, Nature342: 878-883.

Some specific examples of full-length light and heavy chains of the provided antibodies and their corresponding amino acid sequences are summarized in tables 1A and 1B below. Table 1A shows exemplary light chain sequences. Table 1B shows exemplary heavy chain sequences, some of which comprise constant region human IgG2(SEQ ID NO: 86) and some of which comprise constant region human IgG1f (SEQ ID NO: 127). However, encompassed within the invention are immunoglobulins (e.g., IgG4 and IgG2, CL2 and CL1) that vary in sequence in the constant or framework regions of those listed in table 1A and/or table 1B. Additionally, Signal Peptide (SP) sequences including all sequences in table 1A and table 1B, e.g., VK-1SP signal peptide:

MDMRVPAQLLGLLLLWLRGARC(SEQ ID NO：103).

MEAPAQLLFLLLLWLPDTTG(SEQ ID NO：104).

MEWTWRVLFLVAAATGAHS(SEQ ID NO：105).

METPAQLLFLLLLWLPDTTG(SEQ ID NO：106).

MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 107), but any other suitable signal peptide sequence may be used within the scope of the invention. Another example of a useful signal peptide sequence is VH21SP MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 95). Other exemplary signal peptide sequences are shown in tables 1A-B.

Table 1a immunoglobulin light chain sequences. The signal peptide sequence is indicated by underlining.

Table 1b immunoglobulin heavy chain sequences. The signal peptide sequence is indicated by underlining.

Some useful embodiments of an isolated immunoglobulin comprising an antibody or antibody fragment comprise:

(a) comprises the amino acid sequence of SEQ ID NO: 113, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; and a polypeptide comprising SEQ ID NO: 110, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; or

(b) Comprises the amino acid sequence of SEQ ID NO: 125, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 122, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; or

(c) Comprises the amino acid sequence of SEQ ID NO: 101, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 98, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; or

(d) Comprises the amino acid sequence of SEQ ID NO: 119, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 116, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both.

In some cases, such antibodies comprise at least one heavy chain and one light chain, while in other cases, the variant forms comprise two identical light chains and two identical heavy chains. The following are within the scope of the invention: for example, one or more heavy chains and/or one or more light chains may lack one, two, three, four, or five amino acid residues from the N-terminus or C-terminus or both, relative to any of the heavy and light chains shown in table 1A and table 1B, due to post-translational modifications. For example, CHO cells typically cleave off the C-terminal lysine. As described herein, certain embodiments of conjugates comprising a chemical moiety having one or more pharmacological activities, such as a pharmacologically active polypeptide, can include heteromultimers, e.g., monovalent heterodimers, heterotrimers, or heterotetramers, as schematically shown in fig. 1F-1N (see also table 2D).

Variable domains of immunoglobulins, such as antibodies

The various heavy and light chain variable regions provided herein are shown in tables 2A-B. Each of these variable regions may be attached to the heavy and light chain constant regions described above to form the complete antibody heavy and light chains, respectively. In addition, each of the heavy and light chain sequences so formed may be combined to form a complete antibody structure. It is to be understood that the heavy and light chain variable regions provided herein may also be attached to other constant domains having sequences different from the exemplary sequences listed above.

Also provided are immunoglobulins (including antibodies or antibody fragments) comprising or including at least one V selected from the group consisting of those shown in table 2A below_L2、V_L3、V_L4 and V_L5 and at least one V selected from the group consisting of V as shown in table 2B below_H2、V_H3、V_H4、V_H5、V_H6、V_H7、V_H8、V_H9、V_H10 and V_H11, and immunologically functional fragments, derivatives, mutants and variants of these light and heavy chain variable regions. Examples of these embodiments can be found in tables 2C and 2D below.

Also provided are immunoglobulins (including antibodies or antibody fragments) comprising or including at least one V selected from the group consisting of those shown in table 2A below_L7、V_L8、V_L9、V_L10、V_L11、V_L12、V_L13、V_L14、V_L15 and V_L16 and at least one V selected from the group consisting of those shown in table 2B below _H13、V_H14、V_H15、V_H16、V_H17、V_H18、V_H19、V_H20、V_H21、V_H22、V_H23、V_H24、V_H25、V_H26、V_H27、V_H28、V_H29、V_H30、V_H31、V_H32、V_H33、V_H34、V_H35 and V_H36, and immunologically functional fragments, derivatives, mutants and variants of these light and heavy chain variable regions. Examples of these embodiments can be found in tables 2C and 2D below.

Exemplary embodiments of the immunoglobulin of the invention include those wherein:

the heavy chain variable region comprises SEQ ID NO: 323[ VH10 ]; and the light chain variable region comprises SEQ ID NO: 188[ VL4 ]; or

The light chain variable region comprises SEQ ID NO: 196[ VL8 ]; and the heavy chain variable region comprises SEQ ID NO: 353[ VH25 ]; or

The light chain variable region comprises SEQ ID NO: 202[ VL11 ]; and the heavy chain variable region comprises SEQ ID NO: 349[ VH23 ]; or

The heavy chain variable region comprises SEQ ID NO: 325[ VH11 ]; and the light chain variable region comprises SEQ ID NO: 190[ VL5 ].

Immunoglobulins of this type may generally be represented by the formula "V_Hx/V_Ly, "where" x "corresponds to the number of heavy chain variable regions contained in the immunoglobulin and" y "corresponds to the number of light chain variable regions contained in the immunoglobulin (generally, x and y are each 1 or 2).

TABLE 2A. exemplary V_LAnd (3) a chain. Optional N-terminal signal sequences are not shown, but may be present at V_LIn any "description" of (1).

TABLE 2B exemplary V_HAnd (3) a chain. Optional N-terminal signal sequences are not shown, but may be present at V_HIn any "description" of (1).

Table 2c. contains the indications V as disclosed in tables 2A and 2B above_LAnd V_H(or multimers thereof). Antibodies 16429 and 16449, also listed herein, are positive control antibodies to human IL-17R and TR2, respectively.

Table 2d. contains the indications V as disclosed in tables 2A and 2B above_LAnd V_H(or multimers thereof) and fusion partners as described in more detail in examples 5-6 herein.

Antibody #	VL	VH	Fusion partner
				3742	VL1	VH1	ShK(1-35，Q16K)
10162	VL4	VH10	FGF21
				10163	VL11	VH23	FGF21
10164	VL11	VH23	ShK(1-35，Q16K)

In some embodiments, immunoglobulins (including antibodies and antibody fragments) can be used as therapeutic molecules, either alone or in combination with other therapeutic agents, to achieve a desired effect. In such embodiments, the immunoglobulin (including antibodies and antibody fragments) of the invention further comprises from 1 to 24, from 1 to 16, from 1 to 8, or from 1 to 4 pharmacologically active chemical moieties, whether small molecules or polypeptides, conjugated thereto. Pharmacologically active small molecule or polypeptide chemical moieties may be conjugated at or through the N-terminal or C-terminal residues of immunoglobulin monomers (e.g., LC or HC monomers), chemical reactions are known in the art and are further described herein. Alternatively, the invention contemplates conjugating a pharmacologically active chemical moiety or moieties at or through a functional group on one or more side chains of one or more amino acid residues within the backbone of the immunoglobulin of the invention. Useful methods and internal conjugation sites (e.g., specific cysteine residues) within immunoglobulin chains are known in the art (e.g., Gegg et al, Modified Fc Molecules, published in WO2007/022070 and US20070269369, which are incorporated herein by reference in their entirety).

In other embodiments of the invention, wherein the pharmacologically active chemical moiety is a polypeptide, recombinant fusion proteins may be produced wherein the pharmacologically active polypeptide is inserted into the primary amino acid sequence of an immunoglobulin heavy chain within the internal loop of an Fc domain of an immunoglobulin heavy chain, rather than the N-terminus and/or C-terminus, as further described in the examples herein and in the art (e.g., Gegg et al, U.S. patent No. 7,442,778, U.S. patent No. 7,655,765, U.S. patent No. 7,655,764, U.S. patent No. 7,662,931, U.S. patent No. 7,645,861, published U.S. patent application US2009/0281286, and US2009/0286964, each of which is incorporated herein by reference in its entirety).

By "conjugated" is meant that at least two chemical moieties are covalently linked, or bound to each other directly or optionally through a peptide-based or non-peptide-based linker moiety that is itself covalently linked to both chemical moieties. For example, covalent attachment may be through an amino acid residue of a peptide or protein, including through an alpha amino group, an alpha carboxyl group, or through a side chain. The method by which covalent attachment is achieved is not critical, for example, whether "conjugation" is by chemical synthesis or by recombinant expression of a fused (i.e., conjugated) partner in a fusion protein.

As noted above, some embodiments of the compositions of the present invention relate to at least one pharmacologically active polypeptide moiety conjugated to a pharmacologically inactive immunoglobulin of the present invention, e.g., thereby constituting a recombinant fusion protein in which the pharmacologically active polypeptide moiety is conjugated to the pharmacologically inactive immunoglobulin of the present invention. The term "pharmacological activity" means that the substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level, pain perception) or disease state (e.g., cancer, autoimmune disorder, chronic pain), excluding the simple immunogenicity, if any, of the substance. Conversely, the term "pharmacologically inactive" means that the agent can be determined to have no activity affecting a medical parameter or disease state, not including the simple immunogenicity (if any) of the agent. Thus, pharmacologically active peptides or proteins include agonist or mimetic peptides and antagonist peptides as defined below. The present invention encompasses the use of any pharmacologically active protein having an amino acid sequence ranging from about 5 to about 80 amino acid residues in length and suitable for recombinant expression. In some useful embodiments of the invention, pharmacologically active proteins are modified in one or more ways relative to the native sequence of interest, including amino acid additions or insertions, amino acid deletions, peptide truncations, amino acid substitutions, or chemical derivatization of amino acid residues (accomplished by known chemical techniques), so long as the desired biological activity is maintained.

The terms "-mimetic peptide", "peptidomimetic" and "-agonist peptide" refer to a peptide or protein having a biological activity comparable to a naturally occurring protein of interest, such as, but not limited to, a toxin peptide molecule, e.g., an ShK or OSK1 toxin peptide, or a peptide analog thereof. These terms also include peptides that indirectly mimic the activity of a naturally occurring peptide molecule, for example, by potentiating the effect of the naturally occurring peptide molecule.

The terms "-antagonist peptide", "peptide antagonist" and "inhibitor peptide" refer to peptides that block or somehow interfere with the biological activity of a receptor of interest, or have biological activity comparable to known antagonists or inhibitors of a receptor of interest (such as, but not limited to, ion channels or G-protein coupled receptors (GPCRs)).

Examples of pharmacologically active proteins useful in the present invention include, but are not limited to, toxin peptides (e.g., OSK1 or OSK1 peptide analogs; ShK or ShK peptide analogs), IL-6 binding peptides, CGRP peptide antagonists, bradykinin B1 receptor peptide antagonists, parathyroid hormone (PTH) agonist peptides, parathyroid hormone (PTH) antagonist peptides, ang-1 binding peptides, ang-2 binding peptides, myostatin binding peptides, erythropoietin-mimetic (EPO-mimetic) peptides, FGF21 peptides, thrombopoietin-mimetic (TPO-mimetic) peptides (e.g., AMP2 or AMP5), Nerve Growth Factor (NGF) binding peptides, B cell activating factor (BAFF) binding peptides, and glucagon-like peptide (GLP) -1 or peptide mimetics thereof or GLP-2 or peptide mimetics thereof.

Glucagon-like peptide 1(GLP-1) and related peptide glucagon are produced by the differential processing of pro-glucagon and have opposite biological activities. The preproglucagon itself is produced in the alpha cells of the pancreas and in enteroendocrine L cells, which are mainly located in the distal small intestine and colon. In the pancreas, glucagon is selectively cleaved from the glucagon origin. In contrast, in the intestine, pro-glucagon is processed to form GLP-1 and glucagon-like peptides2(GLP-2) which correspond to amino acid residues 78-107 and 126-158 of pro-glucagon, respectively (see e.g. Irwin and Wong, 1995, mol.9: 267-277 and Bell et al, 1983, Nature304: 368-371). By convention, the amino acid numbering of GLP-1 is based on GLP-1(1-37) formed from cleavage from preproglucagon. The biologically active form results from further processing of the peptide which, in one numbering convention, results in GLP-1(7-37) -OH and GLP-1(7-36) -NH₂. GLP-1(7-37) -OH (or reduced to GLP-1(7-37)) and GLP-1(7-36) -NH₂Both had the same activity. For convenience, the term "GLP-1" is used to refer to both forms. In this numbering convention, the first amino acid of these processed peptides is His 7. However, another art-recognized numbering convention assumes the His starting with the numbering of the processed peptide as position 1 instead of position 7. Thus, in this numbering scheme GLP-1(1-31) is identical to GLP-1(7-37) and GLP-1(1-30) is identical to GLP-1 (7-36). Examples of GLP-1 mimetic polypeptide sequences include:

HGEGTFTSDQSSYLEGQAAKEFIAWLVKGRG//(SEQ ID NO：290)；

HGEGTFISDQSSYLEGQAAKEFIAWLQKGRG//(SEQ ID NO：291)；

HGEGTFTSDVSSYQEGQAAKEFIAWLVKGRG//(SEQ ID NO：292)；

HGEGTFTSDVSSYLEGQAAKEFIAQLVKGRG//(SEQ ID NO：293)；

HGEGTFTSDVSSYLEGQAAKEFIAQLQKGRG//(SEQ ID NO：294)；

HGEGTFTSDVSSYLEGQAAKEFIAWLQKGRG//(SEQ ID NO：295)；

HNETTFTSDVSSYLEGQAAKEFIAWLVKGRG//(SEQ ID NO：296)

HGEGTFTSDVSSYLENQTAKEFIAWLVKGRG//(SEQ ID NO：297)；

HGEGTFTSDVSSYLEGNATKEFIAWLVKGRG//(SEQ ID NO：298)；

HGEGTFTSDVSSYLEGQAAKEFIAWLVNGTG//(SEQ ID NO：299)；

HGEGTFTSDVSSYLEGQAAKEFIAWLVKNRT//(SEQ ID NO：300)；

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRNGT//(SEQ ID NO：301)；

HGFGTFTSDVSSYLEGQAAKEFIAWLVKGRGGTGNGT//(SEQ ID NO：302)；

And

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGNGT//(SEQ ID NO：303)。

human GLP-2 and GLP-2-mimetic analogs are also known in the art. (see, e.g., Prasad et al, Gluconlike peptide-2analogue enzyme research and reproduction, J.Peditator. Surg.2000.2 months; 35 (2): 357-59 (2000); Yusta et al, Glucone-like peptide-2receptor activity enzymes bad and glycerol synthase kinase-3in a protein kinase A-dependent manager and depression adaptation of luminescence inhibition of phosphorylation enzyme 3-kinase, J.l.Chem.277 (28): 24896-906 (2002)).

"toxin peptides" include peptides and polypeptides having the same amino acid sequence as a naturally occurring pharmacologically active peptide or polypeptide that can be isolated from the venom, and also include modified peptide analogs of such naturally occurring molecules. (see, e.g., Kalman et al, ShK-Dap22, a potentKv1.3-specific immunological rendering polypeptide, J.biol.chem.273 (49): 32697-707 (1998); Kem et al, U.S. Pat. No. 6,077,680; Mouhat et al, OsK1 derivatives, WO2006/002850A 2; Chandy et al, Analogs of SHKtoxin and the hair use in selective inhibition of Kv1.3 potassium channels, WO 2006/042151; Sullivan et al, Toxin Peptide therapeutics, WO2006/116156A2, all of which are incorporated herein by reference in their entirety). Snakes, scorpions, spiders, bees, snails, and sea anemones are some examples of organisms that produce venom that can act as a small biological activity that potently and selectively targets ion channels and receptors A rich source of toxin peptides or "toxins". An example of a toxin peptide is OSK1 (also known as OsK1), a scorpion from Strongylocentrotus israeli (R.israeli: (R) (R))Orthochirus scrobiculosus) Toxin peptide separated from scorpion venom. (e.g., Mouhat et al, K + channel types targeted by synthetic OSK1, a toxinfromOrthochirus scrobiculosusscorpion venom, biochem.j.385: 95-104 (2005); mouhat et al, pharmaceutical profiling of orthophorax chocerculosus toxin1analog with a trimmed N-terminal domain, Molec. Pharmacol.69: 354-62 (2006); mouhat et al, OsK1derivatives, WO2006/002850A 2). Another example is from sea anemone Caribbean sea anemone (A)Stichodactvla helianthus) The venom of (3) ShK. (e.g., Tudor et al, analysis of fluorescent solutions properties of the porous-channel brick ShK toxin, Eur. J. biochem.251 (1-2): 133-41 (1998); Pennington et al, Role of diagnostic in the structure and porous channel blocking activity of Shktoxin, biochem.38 (44): 14549-58 (1999); Kem et al, K diagnostic sites and methods of use, U.S. Pat. No. 6,077,680; Lebrun et al, neuropathies orientation in U.S. Pat. No. 6,689,749; Beeton et al, Targeting vector media tissue selection in cell tissue, U.S. Pat. No. 3,1364; tissue analysis of cell tissue of culture of cell, K diagnostic reagent of culture of 3.81, 1364).

Toxin peptides are generally between about 20 and about 80 amino acids in length, contain 2 to 5 disulfide bonds and form a very compact structure. Toxin peptides (e.g., venom from scorpions, sea anemones, and cardiospira) have been isolated and characterized for their effect on ion channels. These peptides appear to have evolved from a relatively small number of structural frameworks that are particularly well suited to address the key issues of potency and stability. For example, most scorpion and conotoxin peptides contain 10 to 40 amino acids and up to 5 disulfide bonds, forming an extremely compact and constrained structure (microbial protein) that is generally resistant to proteolysis. Conotoxin and scorpion toxin peptides can be divided into multiple superfamilies based on their disulfide linkage and peptide folding. The solution structure of many of these has been determined by NMR spectroscopy, showing its compact structure and verifying the conservation of its family folding. (e.g., Tudor et al, analysis of molecular and Solution properties of the pore-channel brick Ktoxin, Eur. J. biochem.251 (1-2): 133-41 (1998); Pennington et al, Role of tissue in the structure and pore channel block activity of ShK toxin, biochem.38 (44): 14549-58 (1999); Jaravine et al, thread-dimensional structure of tissue OSK1 from tissue culture tissue vector, 2006.36 (2004-1223-32); Cno-porous et al, Solution of tissue gradient, 32, tissue gradient, 11-32. 11. from tissue culture tissue vector, 11. 14. 11. from tissue culture tissue vector, 11. 12. 11. from tissue culture tissue strain J. (32. 11. 12. 11. from tissue culture tissue strain, 11. 12. 11. from tissue culture tissue strain, 2. 11. from tissue culture tissue strain, 2. 12. from tissue culture tissue strain, 2. 12. 11. origin tissue strain, 2. 12. from tissue culture tissue strain, 2. 12. origin tissue strain, 2. 12. origin strain, 2. 3. 11. from tissue strain, 2. 12. origin strain, 2. 11. 3. 11. from tissue strain, 2. 11. origin strain, 2. 3. 11. origin strain, 2. 11. origin strain. Examples of pharmacologically active toxin peptides for which the practice of the present invention is useful include, but are not limited to, ShK, OSK1, charybdotoxin (ChTx), kokuchiotoxin 1(kaliotoxin1) KTX1), or motonectin (maurotoxin), or toxin peptide analogs of any of these that are modified from the native sequence at one or more amino acid residues. Other examples are known in the art or may be found in Sullivan et al, WO06116156A2 or U.S. patent application No. 11/406,454 (titled: Toxin Peptide Therapeutic Agents, published as US 2007/0071764); mouhat et al, OsK1derivatives, WO2006/002850A 2; sullivan et al, U.S. patent application No. 11/978,076 (entitled: Conjunated Toxin Peptide therapeutics Agents, filed 10.25.2007 and published as US20090291885 at 26.11.2009), Sullivan et al, WO 2008/088422; lebrun et al, U.S. patent No. 6,689,749 and Sullivan et al, Selective and patent Peptide inhibitor kv1.3, U.S. provisional application No. 61/210,594 (filed 3/20/2009), each of which is incorporated by reference in its entirety.

The term "peptide analog" refers to a peptide having a sequence that differs from a naturally occurring peptide sequence by substitution, internal addition, or internal deletion of at least one amino acid residue and/or amino or carboxyl terminal truncation or addition of at least one amino acid. "internal deletion" refers to the absence of an amino acid at a position other than the N-terminus or C-terminus of a naturally occurring sequence. Likewise, "internal addition" refers to the occurrence of an amino acid at a position other than the N-terminus or C-terminus of a naturally occurring sequence. A "toxin peptide analog," such as, but not limited to, an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog, comprises modifications (e.g., amino acid residue substitutions, internal additions or insertions, internal deletions, and/or amino or carboxyl terminal truncations or additions as previously described) to the native toxin peptide sequence of interest.

"CGRP peptide antagonists" are those which preferentially bind CGRP₁Peptides of receptors such as, but not limited to, CGRP peptide analogs, and to antagonize, block, reduce, hinder or inhibit full-length native human alpha CGRP or beta CGRP-to-CGRP under conditions of physiological temperature, pH and ionic strength ₁An activated peptide of a receptor. CGRP peptide antagonists include full and partial antagonists. Such antagonist activity can be detected by known in vitro methods or in vivo functional assays. (see, e.g., Smith et al, Modifications to the N-terminal but not the C-terminal of calcein gene-related peptide (8-37) product anti-inflammatory sensitivity, J.Med.chem., 46: 2427-2435 (2003)). Examples of useful CGRP peptide antagonists are disclosed in Gegg et al, CGRP peptide antagonists and conjugates, WO2007/048026A2 and U.S. Ser. No. 11/584,177 (filed on 2006, 10/19, published as US2008/0020978A 1), the entire contents of which are incorporated herein by reference.

The terms "parathyroid hormone (PTH) agonist" and "PTH agonist" refer to molecules that bind to the PTH-1 or PTH-2 receptor and increase or decrease one or more PTH activity-determining parameters, as do full-length native human parathyroid hormone. Examples of useful PTH agonist peptides are disclosed in Table 1 of U.S. Pat. No. 6,756,480 entitled "Modulators of receptors for specific peptides and specific peptides-related proteins," which is incorporated herein by reference in its entirety. An exemplary PTH activity assay is disclosed in example 1 of U.S. patent No. 6,756,480.

The term "parathyroid hormone (PTH) antagonist" refers to a molecule that binds to the PTH-1 or PTH-2 receptor and blocks or prevents the normal effects of full-length native human parathyroid hormone on those parameters. Examples of useful PTH antagonist peptides are disclosed in table 2 of U.S. patent No. 6,756,480, which is incorporated herein by reference in its entirety. An exemplary PTH activity assay is disclosed in example 2 of U.S. patent No. 6,756,480.

The terms "bradykinin B1receptor antagonist peptide" and "bradykinin B1receptor peptide antagonist" mean peptides having antagonist activity against the human bradykinin B1receptor (hB 1). Such as Ng, et al, Antagonist of the bradykin B1 receiver, US2005/0215470A1 (published as US patent No. 7,605,120, 9/29, 2005); useful bradykinin B1receptor antagonist peptides are identified or obtained as described in U.S. patent nos. 5,834,431 or 5,849,863. An exemplary B1receptor activity assay is disclosed in examples 6 to 8 of US2005/0215470a 1.

The terms "Thrombopoietin (TPO) -mimetic peptide" and "TPO-mimetic peptide" mean a peptide which can be, for example, CwirlaEtc. of，(1997)，Science276: 1696-9, U.S. patent nos. 5,869,451 and 5,932,946 (which are incorporated by reference in their entirety); U.S. patent application No. 2003/0176352 (published 9/18/2003, which is incorporated by reference in its entirety); WO03/031589 (published on 17/4/2003); peptides identified or obtained as described in WO00/24770 (published 5/4/2000); and any of the peptides presented in Table 5 of published application US2006/0140934 (US Ser. No. 11/234,731, filed 2005, 9/23, entitled Modified Fc Molecules, the entire contents of which are incorporated herein by reference). One of ordinary skill in the art recognizes that each of these references enables the use of different peptides according to the disclosed procedures The library was selected for peptides different from those actually disclosed therein.

The terms "EPO-mimetic peptide" and "erythropoietin-mimetic peptide" mean a peptide that can be expressed, for example, as Wright et al (1996), Science 273: 458-63 and Naranda et al (1999), proc.natl.acad.sci.usa96: 7569-74, both of which are incorporated herein by reference in their entirety. Useful EPO-mimetics include the EPO-mimetics listed in Table 5 of published U.S. patent application US2007/0269369A1 and U.S. patent No. 6,660,843, both of which are hereby incorporated by reference in their entirety.

The term "ang-2 binding peptide" includes peptides that can be identified as disclosed in U.S. patent application No. 2003/0229023 (published 12/11/2003); WO03/057134 (published on 7/17/2003); peptides identified or obtained as described in U.S.2003/0236193 (published 12/25/2003) (each of which is incorporated herein by reference in its entirety); and any of the peptides presented in Table 6 of published application US2006/0140934 (US Ser. No. 11/234,731, filed 2005, 23.9, titled Modified Fc Molecules, which is incorporated herein by reference in its entirety). One of ordinary skill in the art recognizes that each of these references enables the use of different peptide libraries to select different peptides than are actually disclosed therein, following the disclosed procedures.

The terms "Nerve Growth Factor (NGF) binding peptide" and "NGF binding peptide" include peptides that can be identified or obtained as described in WO04/026329 (published 4/1/2004) as well as any of the peptides identified in Table 7 of published application US2006/0140934 (U.S. Ser. No. 11/234,731, filed 2005, 9/23, entitled Modified Fc Molecules, the entirety of which is incorporated herein by reference). One of ordinary skill in the art recognizes that each of these references enables the use of different peptide libraries to select different peptides than are actually disclosed therein, following the disclosed procedures.

The term "myostatin binding peptide" includes peptides that can be identified or obtained as described in U.S. serial No. 10/742,379 (filed 12/19/2003, which is incorporated herein by reference in its entirety) and the peptides presented in table 8 of published application US2006/0140934 (U.S. serial No. 11/234,731, filed 2005 9/23/2005, entitled Modified Fc Molecules, which is incorporated herein by reference in its entirety). One of ordinary skill in the art recognizes that each of these references enables the use of different peptide libraries to select different peptides than are actually disclosed therein, following the disclosed procedures.

The terms "BAFF-antagonist peptide" and "BAFF binding peptide" include peptides that can be identified or obtained as described in U.S. patent application No. 2003/0195156a1 (incorporated herein by reference in its entirety) as well as those peptides that appear in table 9 of published application US2006/0140934 (U.S. serial No. 11/234,731, filed 2005, 9/23, entitled Modified Fc Molecules, which is incorporated herein by reference in its entirety). One of ordinary skill in the art recognizes that the foregoing references enable the selection of different peptides from those actually disclosed therein using different peptide libraries in accordance with the disclosed procedures.

The foregoing are intended as non-limiting examples only of pharmacologically active polypeptides that may be usefully conjugated or fused to the immunoglobulins of the present invention, including antibodies and antibody fragments. Any comprising a pharmacologically active polypeptide moiety may be used in the scope of the present invention, including polypeptides having the so-called avimer structure (see, e.g., Kolkman et al, Novel Proteins with Targeted Binding, US 2005/0089932; Baker et al, IL-6Binding Proteins, US 2008/0281076; Stemmer et al, Protein scans and Uses Thereof, US2006/0223114 and US 2006/0234299).

It is known in the art to use useful preclinical animal models for validating a drug for a therapeutic indication of interest (e.g., "an adaptive-transfer model of a peroxidic disease", J. bone Mineral Res.19: 155 (2004); Gruner et al "an ultrasonic pulmonary flow meter-based animal model of an artificial disease", Blood 105: 1492-99 (2005); Braun et al "a pulmonary amyloid model, an ortho-clinical model, and Murin" for Valverde et al)e strokeemodel ", WO2009/115609a 1). For example, the adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis has been described for investigative studies on immune diseases such as multiple sclerosis (Beeton et al, J.Immunol.166: 936 (2001); Beeton et al, PNAS 98: 13942 (2001); Sullivan et al, example 45 of WO2008/088422A2, incorporated herein by reference in its entirety). In the AT-EAE model, animals treated with an effective amount of the pharmaceutical composition of the invention are expected to have significantly reduced disease severity and increased survival, while untreated animals are expected to develop severe disease and/or die. For the running AT-EAE model, the cerebrospinal CD4+ rat T cell line, PAS, was specific for Myelin Basic Protein (MBP) derived from dr. The maintenance of these cells in vitro and their use in AT-EAE models has been previously described [ Beeton et al, (2001) PNAS98, 13942 ]. PAST cells were maintained in vitro by alternating rounds of antigen stimulation or activation with MBP and irradiated thymocytes (2 days) and proliferation with T cell growth factor (5 days). Activated PAS T cells (3X 10)⁵/ml) involves mixing the cells with 10. mu.g/ml MBP and 15X 10⁶Irradiated (3500rad) thymocytes were incubated for 2 days per ml of syngeneic. On day 2 after in vitro activation, 10-15X 10 was injected by tail vein⁶Live PAS T cells were injected into female Lewis rats (Charles River Laboratories) 6-12 weeks old. Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS) or test pharmaceutical compositions were performed from day-1 to 3, where day-1 represents 1 day prior to injection of PAS T cells (day 0). In vehicle-treated rats, acute EAE is expected to develop 4 to 5 days after PAS T cell injection. Typically, serum was collected by tail vein bleeding on day 4 and cardiac puncture on day 8 (end of study) for analysis of inhibitor levels. Rats are typically weighed on days-1, 4, 6 and 8. Animals can be scored blindly from the day of cell transfer (day 0) to day 3 once a day and from day 4 to day 8 twice a day. Clinical signs were assessed as a total score of the degree of paresis in the extremities and tails. Clinical scoring: 0, 0.5, 1.0, 2.0, mild lower limb paralysis (digital tail), no physical signs, distal tip flaccidity (digital tail), and mild lower limb paralysis (digital tail) Or, ataxia, 3.0 ═ moderate paralysis of lower limbs, 3.5 ═ one paralysis of hind limbs, 4.0 ═ complete paralysis of hind limbs, 5.0 ═ complete paralysis and incontinence of hind limbs, 5.5 ═ paralysis of four limbs, 6.0 ═ dying state or death. Rats that achieved a score of 5.0 were typically euthanized.

Preparation of antibody embodiments of immunoglobulins

Polyclonal antibodiesPolyclonal antibodies are preferably generated in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Alternatively, the antigen may be injected directly into the lymph nodes of the animal (see Kilparick et al, Hybridoma, 16: 381-389, 1997). Improved antibody responses may be obtained by conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent such as maleimidobenzoyl sulfosuccinimide ester (conjugated through a cysteine residue), N-hydroxysuccinimide (conjugated through a lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art.

Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 μ g of protein or conjugate (for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals were boosted with an initial amount of 1/5 to 1/10 of peptide or conjugate in freund's complete adjuvant and by subcutaneous injection at multiple sites. At 7-14 days after the booster injection, the animals were bled and the serum was assayed for antibody titer. Animals were boosted until titers reached equilibrium. Preferably, the animal is boosted with the same antigen, but conjugated to a different protein and/or by a conjugate of a different cross-linking reagent. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum are useful for enhancing immune responses.

Monoclonal antibodiesThe immunoglobulin of the invention provided comprises a monoclonal antibody. Monoclonal antibodies can be prepared using any technique known in the art, for example, by immortalizing spleen cells harvested from transgenic animals after completion of an immunization program. Spleen cells can be immortalized using any technique known in the art, for example, by fusing them with myeloma cells to produce hybridomas. For example, monoclonal antibodies can be used as described by Kohler et al, Nature, 256: 495(1975), or may be prepared from predetermined sequences by recombinant DNA Methods as used herein (e.g., Cabilly et al, Methods of producing monoclonal antibodies, vectors and transformed host cells for use in heat, U.S. Pat. No. 6,331,415), including, for example, the "split DHFR" method, which optionally uses mammalian cell lines (e.g., CHO cells) of glycosylating antibodies to facilitate the general preparation of equimolar concentrations of light and heavy chains (see, for example, Page, Antibody production, EP0481790a2 and U.S. Pat. No. 5,545,403).

Generally, in a hybridoma method that is not used to prepare an immunoglobulin of the invention, but is used to prepare an antigen binding protein, a mouse or other suitable host mammal, such as a rat, hamster, or cynomolgus monkey, is immunized as described herein to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion reagent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)).

In some cases, hybridoma cell lines are generated by: immunizing a transgenic animal having a human immunoglobulin sequence with an immunogen; harvesting splenocytes from the immunized animal; fusing the harvested splenocytes with a myeloma cell line, thereby producing hybridoma cells; establishing a hybridoma cell line from the hybridoma cells and identifying the hybridoma cell line that produces an antibody that binds the antigen of interest. Such hybridoma cell lines and monoclonal antibodies produced by them are aspects of the invention.

The hybridoma cells, once prepared, are inoculated and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to culture medium. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human Monoclonal antibodies (Kozbor, J.Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal antibody production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987)). The myeloma cells used for hybridoma-producing fusion procedures are preferably enzyme-deficient in that they do not produce antibodies, have high fusion efficiency, and are not grown in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for mouse fusion include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7, and S194/5XXO Bul; examples of cell lines used for rat fusion include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B 210. Other cell lines used for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC 729-6.

The medium in which the hybridoma cells are grown is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can be, for example, byOr Scatchard analysis (Munson et al, anal. biochem., 107: 220 (1980); Fischer et al, A peptide-immunoglobulin-conjugate, WO2007/045463A1, example 10, which is incorporated herein by reference in its entirety).

After identification of the hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, the hybridoma cells can be cultured in an animal as a ascites tumor.

Hybridomas or mAbs can be further screened to identify mAbs having particular properties, e.g., inhibition of K ¹⁺Capacity to flow through kv1.x channels. Examples of such screening are provided in the examples below. Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as protein a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, or any other suitable purification technique known in the art.

Recombinant production of antibodiesThe invention provides isolated nucleic acids encoding any of the antibodies of the invention described herein (monoclonal and polyclonal), including antibody fragments, optionally operably linked to control sequences recognized by the host cell, vectors and host cells comprising the nucleic acids, as well as recombinant techniques for producing the antibodies, which can include culturing the host cell such that the nucleic acids are expressed, and optionally recovering the antibodies from the host cell culture or medium. Similar materials and methods are applicable to the preparation of polypeptide-based immunoglobulins.

The relevant amino acid sequence from the immunoglobulin or polypeptide of interest can be determined by direct protein sequencing, and appropriate coding nucleotide sequences can be designed according to the universal codon table. Alternatively, the genome or cDNA encoding a monoclonal antibody can be isolated from cells producing such antibody and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody).

Cloning of the DNA is performed using standard techniques (see, e.g., Sambrook et al (1989) Molecular Cloning: A Laboratory Guide, Vol.1-3, Cold Spring harborPress, which is incorporated herein by reference). For example, a cDNA library can be constructed by reverse transcribing polyA + mRNA, preferably membrane-associated mRNA, and screening the library using probes specific for human immunoglobulin polypeptide gene sequences. However, in one embodiment, Polymerase Chain Reaction (PCR) is used to amplify the cDNA (or portions of the full-length cDNA) encoding the immunoglobulin gene segment of interest (e.g., a light chain or heavy chain variable segment). The amplified sequences can be readily cloned into any suitable vector, for example, an expression vector, a minigene vector, or a phage display vector. It will be appreciated that the particular cloning method employed is not critical, so long as the sequence of a certain portion of the immunoglobulin polypeptide of interest can be determined.

One source of antibody nucleic acid is a hybridoma, which is produced by: b cells are obtained from animals immunized with the antigen of interest and fused to immortalized cells. Alternatively, the nucleic acid may be isolated from B cells (or whole spleen) of the immunized animal. Yet another source of antibody-encoding nucleic acids is a library of such nucleic acids, e.g., produced by phage display technology. Polynucleotides encoding peptides of interest (e.g., variable region peptides with desired binding properties) can be identified by standard techniques such as panning.

Although the sequence encoding the entire variable region of an immunoglobulin polypeptide can be determined; however, it is sometimes sufficient to sequence only a part of the variable region, for example, the CDR-encoding part. Sequencing was performed using standard techniques (see, e.g., Sambrook et al (1989) Molecular Cloning: Arabidopsis Guide, volumes 1-3, Cold Spring Harbor Press and Sanger, F. et al (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by reference). By comparing the sequence of the cloned nucleic acids to the sequences of published human immunoglobulin genes and cdnas, one will be able to readily determine from the sequenced regions (i) the germline segment usage of hybridoma immunoglobulin polypeptides (including the heavy chain isotype) and (ii) the sequences of the heavy and light chain variable regions, including sequences resulting from N region addition and somatic mutation processes. One source of immunoglobulin gene sequence Information is the National center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.

The isolated DNA may be operably linked to control sequences or placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin protein to direct the synthesis of monoclonal antibodies in the recombinant host cell. Recombinant production of antibodies is well known in the art.

Nucleic acids are operably linked when placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous and, in the case of secretory leader sequences, contiguous and in reading phase. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

Many vectors are known in the art. The carrier component may include one or more of the following: signal sequences (e.g., which may direct secretion of the antibody; e.g., ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGGCTGAGAGGTGCGCGCTGT// SEQ ID NO: 102, which encodes the VK-1 signal peptide sequence MDMRVPAQLLGLLLLWLRGARC// SEQ ID NO: 103), an origin of replication, one or more selectable marker genes (e.g., which may confer antibiotic or other drug resistance, complement auxotrophic deficiencies, or provide key nutrients not available in the culture medium), enhancer elements, promoters, and transcription termination sequences, all of which are well known in the art.

Cells, cell lines and cell cultures are often used interchangeably, and all of these designations herein include progeny. Transformants and transformed cells include the initial test cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that are screened for the same function or biological activity as the originally transformed cell are included.

Exemplary host cells include prokaryotic, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, e.g.gram-negative or gram-positive organisms, e.g.Enterobacteriaceae such asGenus Escherichia(Escherichia) (e.g. Escherichia)Escherichia coli(E.coli))、Enterobacter sp(Enterobacter)、Erwinia genus(Erwinia)、Klebsiella genus(Klebsiella)、Deformation of Bacillus genus(Proteus)、Salmonella(Salmonella) (for example,salmonella typhimurium(Salmonella typhimurium))、Serratia genus(Serratia) (e.g.,serratia marcescens(Serratia marcescans)) andshigella(Shigella), andbacillus genus(Bacillus) (for example)Bacillus subtilis(B. subtilis) andbacillus licheniformis(B.licheniformi))、Pseudomonas sp(Pseudomonas) andstreptomyces genus (Streptomyces). Eukaryotic microorganisms such as filamentous fungi or yeasts are suitable for cloning or expression of recombinant polypeptides or antibodiesReach the host.Saccharomyces cerevisiae(Saccharomyces cerevisiae) or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful herein, e.g.Pichia genus(Pichia) such asPichia pastoris(P.pastoris)、Schizosaccharomyces pombe(Schizosaccharomyces pombe); kluyveromyces (Kluyveromyces) genus,Arctomyces(Yarrowia)；Candida genus(Candida)；Trichoderma reesei(Trichoderma reesia)；Neurospora crassa(Neurospora crassa)；Prosperous and powerful Saccharomyces(Schwanniomyces), e.g.Schwann yeast West(Schwanniomyces occidentalis); and filamentous fungi, such as, for example,genus Neurospora(Neurospora)、Penicillium Belong to(Penicillium)、Genus campylobacter(Tolypocladium) andaspergillus fungi(Aspergillus) hosts, e.g.Aspergillus nidulans(A. nidulans) andaspergillus niger(A.niger)。

Host cells for expression of glycosylated immunoglobulins (including antibodies) may be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as: Spodoptera frugiperda(Spodoptera frugiperda) (carpenterworm)Aedes aegypti(Aedes aegypti) (mosquitoes), Aedes albopictus (mosquitoes),Fruit fly with black belly(D(osophila melanogaster) (fruit flies), andsilkworm (Bombyx mori)(Bombyx mori). A variety of viral strains for transfecting such cells are publicly available, e.g., Autographa californica: (A. ferox)Autographa californica) L-1 variants of NPV and Bombyx moriBombyx mori) Bm-5 strain of NPV.

Vertebrate host cells are also suitable hosts, and recombinant production of antigen binding proteins (including antibodies) from such cells has become a routine procedure. Examples of useful mammalian host cell lines are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, [ Graham et al, J.Gen Virol.36: 59(1977) ], baby hamster kidney cells (BHK, ATCC CCL10), mouse testicular support cells (TM4, Mather, biol. Reprod.23: 243-.

Host cells are transformed or transfected with the nucleic acids or vectors described above for the production of immunoglobulins and cultured in conventional nutrient media, suitably modified, for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Furthermore, the novel vectors and transfected cell lines with multiple copies of the transcription unit separated by a selectable marker are particularly useful for the expression of immunoglobulins.

The host cells used to produce the immunoglobulins of the present invention may be cultured in a variety of media. Commercially available media, such as Ham's F10(Sigma), minimal essential Medium (minimal essential Medium) ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma), are suitable for culturing host cells, furthermore, any of the media described in Ham et al, meth.Enz.58: 44(1979), Barnes et al, anal. biochem.102: 255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90103430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as the Medium for the host cells Protein or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin (Gentamycin)^TM) Drugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression, and will be apparent to one of ordinary skill in the art.

In culturing the host cell, the immunoglobulin may be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the immunoglobulin is produced intracellularly, as a first step, the particulate debris, host cells or lysed fragments are removed, for example by centrifugation or ultrafiltration.

The immunoglobulin (e.g., antibody or antibody fragment) can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or preferably affinity chromatography, and using the antigen or protein a or protein G of interest as an affinity ligand. Protein A can be used to purify proteins comprising polypeptides based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J.Immunol. meth.62: 1-13 (1983)). Protein G is recommended for all mouse isoforms as well as human gamma 3(Guss et al, EMBO J.5: 15671575 (1986)). The matrix to which the affinity ligand is attached is typically agarose, but other matrices are also available. Physically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. In the case where the protein contains C _HIn the case of 3 domains, Bakerbond ABX^TMResins (j.t.baker, phillips burg, n.j.) can be used for purification. Depending on the antibody to be recovered, other techniques for protein purification may also be used, such as alcohol precipitation, reverse phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

Chimerization, humanization, and Human engineering (Human Engineered) ^TM ) Monoclonal antibodies. Chimeric monoclonal antibodies in which the variable Ig domain of a rodent monoclonal Antibody is fused to a Human Constant Ig domain can be produced using standard procedures known in the art (see Morrison, S.L. et al (1984) Chimeric Human Antibody Molecules; use Antibody Binding domain with Human Constant Region Domains, Proc. Natl. Acad. Sci. USA81, 6841-. Various techniques have been described to humanize or modify antibody sequences to make them more human-like, for example by: (1) grafting of non-human Complementarity Determining Regions (CDRs) to human framework and constant regions (a process known in the art as humanization by "CDR grafting") or (2) grafting of the entire non-human variable domain, but either "masking" them with a human-like surface by replacing surface residues (a process known in the art as "veneering") or (3) modifying selected non-human amino acid residues to make them more human-like based on the possibility of each residue participating in antigen binding or antibody structure and its possibility for immunogenicity. See, e.g., Jones et al, Nature 321: 522525 (1986); morrison et al, proc.natl.acad.sci., u.s.a., 81: 68516855 (1984); morrison and Oi, adv.immunol., 44: 6592 (1988); verhoeyer et al, Science 239: 15341536 (1988); padlan, mol.immun.28: 489498 (1991); padlan, molecular. immunol.31 (3): 16917 (1994); and kertborough, c.a. et al, Protein eng.4 (7): 77383 (1991); co, M.S. et al (1994), J.Immunol.152, 2968-; studnicka et al, protein engineering 7: 805-814 (1994); each of these references is incorporated herein by reference in its entirety.

Various techniques have been described to humanize or modify antibody sequences to make them more human-like, for example by: (1) grafting of non-human Complementarity Determining Regions (CDRs) to human framework and constant regions (a process known in the art as humanization by "CDR grafting") or (2) grafting of the entire non-human variable domain, but "masking" them with a human-like surface by replacing surface residues (a process known in the art as "veneering") or (3) modifying selected non-human amino acid residues to make them more human-like based on the possibility of each residue to participate in antigen binding or antibody structure and its possibility for immunogenicity. See, e.g., Jones et al, Nature 321: 522525 (1986); morrison et al, proc.natl.acad.sci., u.s.a., 81: 68516855 (1984); morrison and Oi, adv.immunol., 44: 6592 (1988); verhoeyer et al, Science 239: 15341536 (1988); padlan, mol.immun.28: 489498 (1991); padlan, molecular. immunol.31 (3): 16917 (1994); and kertborough, c.a. et al, Protein eng.4 (7): 77383 (1991); co, M.S. et al (1994), J.Immunol.152, 2968-; studnicka et al, protein engineering 7: 805-814 (1994); each of these references is incorporated herein by reference in its entirety.

In one aspect of the invention, the light and heavy chain variable regions of the antibodies provided herein (see tables 2A-B) are grafted to the Framework Regions (FRs) from antibodies from the same or different phylogenetic species. To generate identical human FRs, FRs from several human heavy or light chain amino acid sequences can be aligned to identify identical amino acid sequences. In other embodiments, the FRs of a heavy or light chain disclosed herein are replaced with FRs from a different heavy or light chain. In one aspect, rare amino acids in the FR of the heavy and light chains of the antibody are not substituted, while other FR amino acids are substituted. A "rare amino acid" is a specific amino acid that is not normally present in an FR at the position where it is located. Alternatively, grafted variable regions from one heavy or light chain may be used with constant regions that differ from the constant regions of the particular heavy or light chain disclosed herein. In other embodiments, the grafted variable region is part of a single chain Fv antibody.

Antibodies can also be produced using transgenic animals that do not produce endogenous immunoglobulins and are engineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals with human Ig loci, wherein the animals do not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain loci. WO91/10741 also discloses a transgenic non-primate mammalian host capable of setting an immune response to an immunogen, wherein the antibody has primate constant and/or variable regions, and wherein the endogenous immunoglobulin coding locus is replaced or inactivated. WO96/30498 discloses the use of the Cre/Lox system to modify a mammalian immunoglobulin locus, for example to replace all or part of a constant or variable region to form a modified antibody molecule. WO94/02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. U.S. Pat. No. 5,939,598 discloses a method of making a transgenic mouse, wherein the mouse lacks an endogenous heavy chain and expresses an exogenous immunoglobulin locus comprising one or more heterologous constant regions.

Using the above transgenic animals, an immune response can be generated to the selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas secreting human-derived monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art and can be used to immunize transgenic mice as described, for example, in WO 96/33735. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effects of the corresponding protein. See also Jakobovits et al, proc.natl.acad.sci.usa, 90: 2551 (1993); jakobovits et al, Nature, 362: 255-258 (1993); bruggermann et al, Yeast in immunity, 7: 33 (1993); mendez, nat. genet.15: 146-156 (1997); and U.S. patent No. 5,591,669, U.S. patent No. 5,589,369, U.S. patent No. 5,545,807; and U.S. patent application No. 20020199213. U.S. patent application No. 20030092125 describes methods for biasing the immune response of an animal towards a desired epitope. Human antibodies can also be produced by in vitro activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).

Production of antibodies by phage display technology

The development of techniques for preparing libraries of recombinant human antibody genes and displaying the encoded antibody fragments on the surface of filamentous phage has provided another approach for producing human antibodies. Phage display is described, for example, in Dower et al, WO91/17271, McCafferty et al, WO92/01047 and Caton and Koprowski, proc.natl.acad.sci.usa, 87: 6450- > 6454(1990) (each of these references is incorporated herein by reference in its entirety). Antibodies prepared by phage technology are typically produced in bacteria as antigen binding fragments, such as Fv or Fab fragments, and therefore lack effector function. Effector functions can be introduced by one of two strategies: the fragments can be engineered to be full antibodies for expression in mammalian cells, or to be bispecific antibody fragments having a second binding site capable of triggering effector function.

Fd fragment of antibody (V)_H-C_H1) And light chain (V)_L-C_L) Individual clones are typically cloned by PCR and randomly recombined in a combinatorial phage display library, which can then be selected for binding to a particular antigen. The antibody fragment is expressed on the phage surface and selection of Fv or Fab by antigen binding (and thus phage comprises DNA encoding the antibody fragment) is accomplished by several rounds of antigen binding and reamplification (a procedure known as panning). Antibody fragments specific for the antigen are enriched and finally isolated.

Phage display Technology can also be used in a method for humanizing rodent monoclonal antibodies, referred to as "guided selection" (see Jespers, l.s. et al, Bio/Technology12, 899-903(1994)), so that the Fd fragment of a mouse monoclonal antibody can be displayed in combination with a library of human light chains, and the resulting hybrid Fab library can then be selected with antigen.

A variety of procedures have been described for obtaining human antibodies from phage display libraries (see, e.g., Hoogenboom et al, J.mol.biol., 227: 381 (1991); Marks et al, J.mol.biol, 222: 581-. In particular, in vitro selection and evolution of antibodies obtained from phage display libraries has become a powerful tool (see Burton, D.R. and Barbas III, C.F., adv. Immunol.57, 191-280 (1994); and Winter, G. et al, Annu. Rev. Immunol.12, 433-455 (1994); U.S. patent application No. 20020004215 and WO 92/01047; U.S. patent application No. 20030190317 and U.S. patent No. 6,054,287 published 2003, 9.a., U.S. patent No. 5,877,293).

Watkins, "Screening of phase-Expressed antibodies by means of Capture Lift," Methods in Molecular Biology, Antibody phase Display: methods and Protocols 178: 187-193 and U.S. patent application publication No. 20030044772, published 3/6/2003, describe methods for screening phage-expressed antibody libraries or other binding molecules by a capture ladder (capture lift), a method involving immobilization of candidate binding molecules to a solid support.

Other embodiments of the immunoglobulin: antibody fragments

As noted above, antibody fragments include portions of an intact full-length antibody, preferably the antigen-binding or variable regions of an intact antibody, and include linear antibodies and multispecific antibodies formed from antibody fragments. Non-limiting examples of antibody fragments include Fab, Fab ', F (ab') 2, Fv, Fd, domain antibodies (dAb), Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), single chain antibody fragments, maxibody (maxibody), diabodies, triabodies, tetrabodies, minibodies (minibodies), linear antibodies, chelating recombinant antibodies, triabodies (tribodies) or diabodies (bibodies), intrabodies, nanobodies, Small Modular Immunopharmaceuticals (SMIPs), antibody binding domain immunoglobulin fusion proteins, camelized antibodies (camelized antibodies), antibodies comprising VHH, or muteins or derivatives thereof, and polypeptides comprising at least a portion (e.g., a CDR sequence) of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, so long as the antibody retains the desired biological activity.

Other antibody fragments include those consisting of V_HDomain antibody (dAb) fragments of domains (Ward et al, Nature 341: 544-546, 1989).

A "linear antibody" comprises a pair of Fd segments (V) in tandem_H-C_H1-V_H-C_H1) Which form a pair of antigen binding regions. Linear antibodies may be bispecific or monospecific (Zapata et al Protein Eng.8: 1057-62 (1995)).

Olafsen et al, Protein Eng Des Sel.2004, 4 months; 17(4): 315-23, "minibodies" consisting of scFv fused to CH3 via a peptide linker (no hinge) or via an IgG hinge have been described.

The term "macroantibody" refers to a bivalent scFvs covalently attached to the Fc region of an immunoglobulin, see, e.g., Fredericks et al, Protein Engineering, Design & Selection, 17: 95-106(2004) and Powers et al, Journal of Immunological Methods, 251: 123-135(2001).

Functional heavy chain antibodies lacking a light chain are naturally present in certain animal species, such as nurse shark, bearshark and camelids, such as camel, dromedary, alpaca and llama. In these animals the antigen binding site is reduced to a single domain, VH_HA domain. These antibodies use only the heavy chain variable region to form the antigen binding region, i.e., these functional antibodies are homodimers of heavy chains having only the structure H2L2 (referred to as "heavy chain antibodies" or "hcabs"). It is reported that camelized V _HHAnd IgG2 and IgG3 constant regions comprising a hinge, CH2, and CH3 domains and lacking a CH1 domain. Typically only V_HAre difficult to produce in soluble form, but when the framework residues become more similar to VH_HImprovements in solubility and specific binding can be obtained (see, e.g., Reichman et al, J Immunol Methods1999, 231: 25-38.). Camelization of V has been found_HHThe domains bind with high affinityAntigens (Desmyter et al, J.biol.chem.276: 26285-90, 2001) and high stability in solution (Ewert et al, Biochemistry 41: 3628-36, 2002). Methods for producing antibodies with camelized heavy chains are described, for example, in U.S. patent publication nos. 2005/0136049 and 2005/0037421. Alternative scaffolds may be prepared from human variable domains that more closely match shark V-NAR scaffolds and may provide a framework for long penetrating loop structures.

Since the variable domain of the heavy chain antibody is the smallest fully functional antigen-binding fragment with a molecular weight of only 15kDa, this entity is called a nanobody (cortex-Retamozo et al, Cancer Research 64: 2853-57, 2004). Nanobody libraries can be generated from immunized monomodal camels as described in Conrath et al, (Antimicrob AgentsChemother 45: 2807-12, 2001).

Intrabodies are single chain antibodies that display intracellular expression and can manipulate intracellular protein function (Biocca et al, EMBO J.9: 101-108, 1990; Colby et al, Proc Natl Acadsi U S A.101: 17616-21, 2004). Intrabodies comprising cell signal sequences that retain the antibody construct in the intracellular region can be prepared as described in Mhashilkar et al (EMBO J14: 1542-51, 1995) and Wheeler et al (FASEB J.17: 1733-5.2003). Transmembrane antibodies are cell-penetrating antibodies in which a Protein Transduction Domain (PTD) is fused to a single chain variable fragment (scFv) antibody, Heng et al (Med Hypotheses.64: 1105-8, 2005).

The invention also encompasses antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for a target protein. These constructs are single chain polypeptides comprising an antigen binding domain fused to an immunoglobulin domain necessary to perform the effector function of an antibody. See, e.g., WO03/041600, U.S. patent publication 20030133939, and U.S. patent publication 20030118592.

Various techniques have been developed for the preparation of antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies, but can also be produced directly by recombinant host cells. See, e.g., Better et al, Science 240: 1041-1043 (1988); skerra et al Science 240: 1038-1041 (1988); carter et al, Bio/Technology 10: 163-167(1992).

Other embodiments of the immunoglobulin: multivalent antibodies

In some embodiments, it may be desirable to produce multivalent or even multispecific (e.g., bispecific, trispecific, etc.) monoclonal antibodies. Such an antibody may have binding specificity for at least two different epitopes of the target antigen, or it may bind to two different molecules, e.g., bind to the target antigen and bind to a cell surface protein or receptor. For example, a bispecific antibody may comprise a scaffold (arm) that binds to a target and another scaffold that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3), or an Fc receptor of IgG (fcyr), such as fcyri (CD64), fcyrii (CD32), and fcyriii (CDl6), thereby focusing cellular defense mechanisms to cells expressing the target. As another example, bispecific antibodies can be used to localize cytotoxic agents locally to cells expressing a target antigen. These antibodies have a target binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-60, vinca alkaloid, ricin a chain, methotrexate, or radioisotope hapten). Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

In addition, the immunoglobulins (e.g., antibodies and antibody fragments) and conjugates of the invention can also be constructed to fold into multivalent forms, which can improve half-life in blood. The multivalent forms can be prepared by techniques known in the art.

Bispecific or multispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heterologous conjugate may be coupled to avidin and the other to biotin. Heteroconjugate antibodies are also prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with various crosslinking techniques. Another method was designed to make tetramers by adding a streptavidin coding sequence at the C-terminus of scFv. Streptavidin is composed of four subunits, so when scFv-streptavidin is folded, the four subunits associate to form a tetramer (Kipriyanov et al, Hum antibodies hybrids 6 (3): 93-101(1995), the disclosure of which is incorporated by reference in its entirety throughout the day).

According to another method for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. One interface includes C of the antibody constant domain _H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A compensatory "cavity" of the same or similar size to the large side chain described above is created at the interface of the second antibody molecule by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. See WO96/27011 published on 9/6 1996.

Techniques for generating bispecific or multispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific or tripartite antibodies can be prepared using chemical ligation. Brennan et al, Science 229: 81(1985) describes a procedure in which intact antibodies are proteolytically cleaved to form F (ab')₂And (3) fragment. These fragments are reduced in the presence of the dimercaptoxide complexing agent sodium arsenite to stabilize the vicinal dimercaptotes and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab ' -TNB derivatives is then converted to the Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies prepared can be used as reagents for the selective immobilization of enzymes. Better et al, Science 240: 1041-. For example, Fab' -SH fragments can be recovered directly from E.coli and processed Chemical coupling to form bispecific antibodies (Carter et al, Bio/Technology 10: 163-.

Shalaby et al, j.exp.med.175: 217-225(1992) describes fully humanized bispecific antibodies F (ab')₂And (3) preparing molecules. Each Fab' fragment was secreted separately from e.coli and subjected to in vitro directed chemical coupling to form bispecific antibodies.

Various techniques have also been described for the preparation and isolation of bispecific or multispecific antibody fragments directly from recombinant cell cultures. For example, bispecific antibodies have been prepared using leucine zippers such as GCN4 (see generally Kostelny et al, J.Immunol.148 (5): 1547) -1553(1992), where leucine zipper peptides from Fos and Jun proteins are joined by gene fusion to Fab' portions of two different antibodies. The antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to prepare antibody homodimers.

The diabody described above is an example of a bispecific antibody. See, e.g., Hollinger et al, proc.natl.acad.sci.usa, 90: 6444-6448(1993). Bivalent diabodies can be stabilized by disulfide bonds.

Stable monospecific or bispecific Fv tetramers may also be prepared by (scFv)₂)₂Non-covalent association in the configuration or as a double-four chain antibody. Alternatively, two different scfvs may be linked one after the other to form a bis-scFv.

Another strategy for making bispecific antibody fragments by using single chain fv (sFv) dimers has also been reported. See Gruber et al, j.immunol.152: 5368(1994). One approach has been to link two scFv antibodies using a linker or disulfide bond (Malender and Voss, J.biol.chem.269: 199-2061994; WO94/13806 and U.S. Pat. No. 5,989,830, the disclosures of which are incorporated herein by reference in their entirety).

AlternativelyBispecific antibodies can be Protein Eng.8(10) such as Zapata: "Linear antibody" prepared as described in 1057-. Briefly, these antibodies comprise a pair of tandem Fd segments (V)_H-C_H1-V_H-C_H1) Which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two titers are also contemplated. For example, trispecific antibodies can be prepared. (Tutt et al, J.Immunol.147: 60 (1991)).

A "chelating recombinant antibody" is a bispecific antibody that recognizes adjacent and non-overlapping epitopes of a target antigen, and is flexible enough to bind both epitopes simultaneously (Neri et al, J Mol biol.246: 367-73, 1995).

The preparation of bispecific Fab-scFv ("diabodies") and trispecific Fab- (scFv) (2) ("triabodies") is described in Schoonjans et al (J Immunol.165: 7050-57, 2000) and Willems et al (J Chromatogr B Analyt Technol Biomed Life Sci.786: 161-76, 2003). For diabodies or triabodies, scFv molecules are conjugated to VL-CL (L) and VH-CH₁(Fd), e.g., to make a triabody in which two scFv are fused to the C-terminus of a Fab, and in a diabody in which one scFv is fused to the C-terminus of a Fab.

In yet another approach, dimers, trimers and tetramers are prepared after the introduction of free cysteine into the parent protein. Peptide-based crosslinkers with variable numbers (2 to 4) of maleimide groups are used to crosslink proteins of interest with free cysteines (Cochran et al, Immunity12 (3): 241-50(2000), the disclosure of which is incorporated herein in its entirety).

Other embodiments of immunoglobulins

The immunoglobulins of the present invention also include peptide antibodies. The term "peptide antibody" refers to a molecule comprising an antibody Fc domain attached to at least one peptide. The preparation of peptide antibodies is generally described in PCT publication WO00/24782, published 5/4/2000. Any of these peptides may be linked back and forth (i.e., sequentially) with or without a linker. The peptide containing the cysteinyl residue may be cross-linked to another Cys-containing peptide, either or both of which may be linked to a vehicle. Any peptide having more than one Cys residue may also form an intrapeptide disulfide bond. Any of these peptides can be derivatized, e.g., the carboxy terminus can be capped with an amino group, the cysteine can be capped, or the amino acid residue can be substituted with moieties other than amino acid residues (see, e.g., Bhatnagar et al, J.Med.chem.39: 3814-9(1996) and Cuthbertson et al, J.Med.chem.40: 2876-82(1997), both of which are incorporated herein by reference in their entirety). Similar to affinity maturation of antibodies, the peptide sequence can be optimized or altered by alanine scanning or random or directed mutagenesis and then screened to identify the best binders. Lowman, ann.rev.biophysis.biomol.struct.26: 401-24(1997). A variety of molecules can be inserted into the immunoglobulin structure, e.g., within the peptide portion itself or between the peptide and the vehicle portion of the immunoglobulin, while retaining the desired activity of the immunoglobulin. For example, molecules such as Fc domains or fragments thereof, polyethylene glycol or other related molecules, e.g., dextran, fatty acids, lipids, cholesterol groups, small molecule carbohydrates, peptides, detectable moieties as described herein (including fluorescent reagents, radioactive labels such as radioisotopes), oligosaccharides, oligonucleotides, polynucleotides, interfering (or other) RNAs, enzymes, hormones, and the like, can be readily inserted. Other molecules suitable for insertion in this manner will be understood by those skilled in the art and are encompassed within the scope of the present invention. This includes the insertion of, for example, a desired molecule between two consecutive amino acids, optionally connected by a suitable linker.

JointA "linker" or "linker moiety", as used interchangeably herein, refers to a biologically acceptable peptidyl or non-peptidyl organic moiety covalently bound to amino acid residues of a polypeptide chain (e.g., an immunoglobulin HC or an immunoglobulin LC or an immunoglobulin Fc domain) comprised in a composition of the inventionA group which covalently links or conjugates the polypeptide chain to another Peptide or polypeptide chain in a molecule, or to a Therapeutic moiety such as a biologically active small molecule or oligopeptide, or to a half-life extending moiety, see, for example, Sullivan et al, Toxin Peptide Therapeutic Agents, US 2007/0071764; sullivan et al, Toxin Peptide Therapeutic Agents, PCT/US2007/022831 (published as WO 2008/088422); and U.S. provisional application serial No. 61/210,594 (filed 3/20/2009), which are all incorporated herein by reference in their entirety.

The presence of any linker moiety in the immunoglobulins of the present invention is optional. When present, the chemical structure of the linker is not critical, as it primarily serves as a spacer for positioning, linking, ligating or optimizing the presentation or position of one or more functional moieties with respect to the immunoglobulin molecule of the invention. The presence of a linker moiety may be used to optimize the pharmacological activity of some embodiments of the immunoglobulins (including antibodies and antibody fragments) of the present invention. The linker is preferably composed of amino acids linked together by peptide bonds. The linker moiety, if present, may be independently the same or different from any other linker or linkers that may be present in the immunoglobulin of the invention.

As noted above, the linker moiety, if present (whether within the primary amino acid sequence of an immunoglobulin, or as a linker for attaching a therapeutic moiety or half-life extending moiety to an immunoglobulin of the invention), may be "peptidyl" in nature (i.e., composed of amino acids linked together by peptide bonds) and constitute preferably from 1 up to about 40 amino acid residues in length, more preferably from 1 up to about 20 amino acid residues, and most preferably from 1 to about 10 amino acid residues. Preferably, but not necessarily, the amino acid residues in the linker are from 20 canonical amino acids, more preferably from cysteine, glycine, alanine, proline, asparagine, glutamine and/or serine. Even more preferably, the peptidyl linker is composed mainly of sterically unhindered amino acids, for exampleSuch as glycine, serine and alanine, linked by peptide bonds. It is also desirable to select peptidyl linkers that avoid rapid proteolytic metabolic turnover in the in vivo circulation, if present. Some of these amino acids may be glycosylated, as is well understood by those skilled in the art. For example, a useful linker sequence constituting a sialylation site is X ₁X₂NX₄X₅G (SEQ ID NO: 148), wherein X₁、X₂、X₄And X₅Each independently any amino acid residue.

In other embodiments, the 1 to 40 amino acids of the peptidyl linker moiety are selected from the group consisting of glycine, alanine, proline, asparagine, glutamine and lysine. Preferably, the linker is composed mainly of sterically unhindered amino acids such as glycine and alanine. Thus, preferred linkers include polyglycine, polyserine and polyalanine, or a combination of any of these. Some exemplary peptidyl linkers are poly (Gly)_1-8In particular (Gly)₃、(Gly)₄(SEQ IDNO：149)、(Gly)₅(SEQ ID NO: 150) and (Gly)₇(SEQ ID NO: 151) and poly (Gly)₄Ser (SEQ ID NO: 152), poly (Gly-Ala)_2-4Hemo (Ala)_1-8. Other specific examples of peptide-based linkers include (Gly)₅Lys (SEQ ID NO: 154) and (Gly)₅LysArg (SEQ ID NO: 155). Other examples of useful peptidyl linkers are: other examples of useful peptidyl linkers are:

(Gly)₃Lys(Gly)₄(SEQ ID NO：159)；

(Gly)₃AsnGlySer(Gly)₂(SEQ ID NO：156)；

(Gly)₃Cys(Gly)₄(SEQ ID NO: 157); and

GlyProAsnGlyGly(SEQ ID NO：158)。

to explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄Meaning Gly-Gly-Gly-Lys-Gly-Gly-Gly (SEQ ID NO: 159). Other combinations of Gly and Ala are also useful.

Commonly used linkers include those that may be identified herein as "L5" (GGGGGGS; or "G ₄S "; SEQ ID NO: 152) "L10" (GGGGSGGGGS; SEQ ID NO: 153) "L25" (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 146) and any of the joints used in the working examples below.

In some embodiments of the compositions of the invention comprising a peptide linker moiety, an acidic residue, such as a glutamic acid or aspartic acid residue, is placed in the amino acid sequence of the linker moiety. Examples include the following peptide linker sequences:

GGEGGG(SEQ ID NO：160)；

GGEEEGGG(SEQ ID NO：161)；

GEEEG(SEQ ID NO：162)；

GEEE(SEQ ID NO：163)；

GGDGGG(SEQ ID NO：164)；

GGDDDGG(SEQ ID NO：165)；

GDDDG(SEQ ID NO：166)；

GDDD(SEQ ID NO：167)；

GGGGSDDSDEGSDGEDGGGGS(SEQ ID NO：168)；

WEWEW(SEQ ID NO：169)；

FEFEF(SEQ ID NO：170)；

EEEWWW(SEQ ID NO：171)；

EEEFFF(SEQ ID NO：172)；

WWEEEWW (SEQ ID NO: 173); or

FFEEEFF(SEQ ID NO：174)。

In other embodiments, the linker constitutes a phosphorylation site, e.g., X₁X₂YX₄X₅G (SEQ ID NO: 175), wherein X₁、X₂、X₄And X₅Each independently any amino acid residue; x₁X₂SX₄X₅G (SEQ ID NO: 176), wherein X₁、X₂、X₄And X₅Each independently any amino acid residue; or X₁X₂TX₄X₅G (SEQ ID NO: 177), wherein X₁、X₂、X₄And X₅Each independently any amino acid residue.

The joints shown herein are exemplary; peptidyl linkers within the scope of the invention may be much longer and may contain other residues. The peptidyl linker may comprise, for example, a cysteine, another thiol, or a nucleophile for conjugation to the half-life extending moiety. In another embodiment, the linker comprises a cysteine or homocysteine residue for conjugation to a maleimide, iodoacetamide or thioester, functionalized half-life extending moiety, or other 2-amino-ethanethiol or 3-amino-propanethiol moiety.

Another useful peptide-based linker is a larger flexible linker comprising random Gly/Ser/Thr sequences, such as: GSGSATGGSGSTASSGSGSATH (SEQ ID NO: 178) or HGSGSATGGSGSTASSGSGSAT (SEQ ID NO: 179), which is estimated to be about 1kDaPEG molecular size. In addition, useful peptidyl linkers may be comprised of amino acid sequences known in the art for forming rigid helical structures (e.g., rigid linker: -AEAAAKEAAAKEAAAKAGG-) (SEQ ID NO: 180). In addition, the peptidyl linker may also comprise a non-peptidyl segment, e.g., of the formula-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-a 6 carbon aliphatic molecule represented. Peptidyl linkers can be altered to form derivatives as described herein.

Optionally, a non-peptidyl linker moiety is also used to conjugate the half-life extending moiety to the peptide moiety of the toxin peptide analog conjugated to the half-life extending moiety. For example, an alkyl linker such as-NH- (CH) may be used₂)_s-c (o) -, wherein s is 2-20. These alkyl linkers may be further substituted with any non-hindered group, such as lower alkyl (e.g., C)₁-C₆) Lower acyl, halogen (e.g., Cl, Br), CN, NH₂Phenyl, and the like. Exemplary non-peptidyl linkers are polyethylene glycol (PEG) linkers (e.g., as shown below):

(I)

Wherein n is such that the linker has a molecular weight of from about 100 to about 5000 daltons (Da), preferably from about 100 to about 500 Da.

In one embodiment, the non-peptidyl linker is an aryl group. Can be as described in the art, e.g., Sullivan et al, Toxin Peptide Therapeutic Agents, US 2007/0071764; sullivan et al, Toxin Peptide Therapeutic Agents, PCT/US2007/022831 (published as WO 2008/088422); and the linker to form the derivative in the same manner as described in U.S. provisional application serial No. 61/210,594 (filed 3/20/2009), which are all incorporated herein by reference in their entirety.

In addition, PEG moieties can be attached to the N-terminal amine or selected side chain amines by reductive alkylation using PEG aldehydes or acylation using hydroxysuccinimide esters or carbonates of PEG, or by thiol conjugation.

"aryl" is phenyl or phenyl fused to a saturated, partially saturated or unsaturated 3-, 4-or 5-membered carbon bridge position, said phenyl or bridge being interrupted by 0, 1, 2 or 3 substituents selected from C_1-8Alkyl radical, C_1-4Haloalkyl or halogen.

"heteroaryl" is an unsaturated 5, 6 or 7 membered monocyclic or partially saturated or unsaturated 6-, 7-, 8-, 9-, 10-or 11 membered bicyclic ring wherein at least one ring is unsaturated, and said monocyclic and bicyclic rings contain 1, 2, 3 or 4 selected from Atoms from N, O and S, wherein the ring is substituted with 0, 1, 2 or 3 atoms selected from C_1-8Alkyl radical, C_1-4Haloalkyl and halogen.

The non-peptidyl moiety, e.g., the non-peptidyl linker or the non-peptidyl half-life extending moiety, of the composition of matter of the invention may be synthesized by conventional organic chemistry reactions.

The foregoing is merely exemplary and is not an exhaustive discussion of the types of linkers that may optionally be employed in accordance with the present invention.

Preparation of immunoglobulin variantsAs mentioned above, recombinant DNA-and/or RNA-mediated protein expression and protein engineering techniques, or any other method of producing peptides, are suitable for use in the preparation of the compositions of the invention. For example, the polypeptide may be produced in a transformed host cell. Briefly, a recombinant DNA molecule or construct encoding a peptide is prepared. Methods for preparing such DNA molecules are well known in the art. For example, sequences encoding peptides can be excised from DNA using suitable restriction enzymes. Any of a number of available and well known host cells can be used in the practice of the present invention. The choice of a particular host depends on a number of factors recognized in the art. Such factors include, for example, compatibility with the chosen expression vector, toxicity of the peptide encoded by the DNA molecule, conversion rate, ease of peptide recovery, expression characteristics, biosafety and cost. The balance of these factors must be achieved with the following recognition: not all hosts may be equally efficient for expression of a particular DNA sequence. Within these general guidelines, microbial host cells useful in the culture medium include bacteria (e.g., bacteria) Escherichia coli) Yeast (e.g. yeast)Saccharomyces) And other fungal cells, insect cells, plant cells, mammalian (including human) cells such as CHO cells and HEK-293 cells, as well as other host cells referred to herein or known in the art. Modification can also be performed at the DNA level. The DNA sequence encoding the peptide may be altered to codons more compatible with the host cell of choice. For theEscherichia coliOptimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent limitsRestriction sites which may facilitate DNA processing in the host cell of choice. The transformed host is then cultured and purified. The host cell may be cultured under conventional fermentation conditions such that the desired compound is expressed. These fermentation conditions are well known in the art. In addition, the DNA optionally also encodes a coding region 5' to the fusion protein, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed immunoglobulin. For further examples of suitable recombinant methods and exemplary DNA constructs for recombinant expression of the compositions of the invention by mammalian cells, see, e.g., Sullivan et al, Toxin Peptide Therapeutic Agents, US 2007/0071764; sullivan et al, Toxin Peptide Therapeutic Agents, PCT/US2007/022831 (published as WO 2008/088422); and U.S. provisional application serial No. 61/210,594 (filed 3/20/2009), the present compositions comprise a dimeric Fc fusion protein ("peptide antibody") or a chimeric immunoglobulin (light chain + heavy chain) -Fc heterotrimer ("half-antibody") conjugated to a specific binding agent of the present invention, which are all incorporated herein by reference in their entirety.

Amino acid sequence variants of the desired immunoglobulin can be prepared by introducing appropriate nucleotide changes into the encoding DNA or by peptide synthesis. Such variants include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of an immunoglobulin or antibody. Any combination of deletions, insertions, and substitutions are made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes can also alter post-translational processing of the immunoglobulin, for example, changing the number or position of glycosylation sites. In some cases, the preparation of immunoglobulin variants is intended to modify those amino acid residues directly involved in epitope binding. In other embodiments, for the purposes of the discussion herein, it may be desirable to modify residues that are not directly involved in epitope binding or residues that are not involved in epitope binding in any way. Mutagenesis within any of the CDR regions and/or framework regions is contemplated. One skilled in the art can design amino acid sequences of immunoglobulins (including antibodies or antibody fragments) using covariance analysis techniquesUseful modifications of (1). (e.g., Choulier et al, covariane Analysis of Protein Families: The Case soft Variable Domains of Antibodies, Proteins: Structure, Function, and genetics 41: 475-484 (2000); Demarest et al, Optimization of The Antibodies C _H3 Domain by resource Analysis of IgG Sequences, J.mol.biol.335: 41-48 (2004); hugo et al, VL position34is a key determination for the engineering of stable antibodies with fast authentication rates, protein engineering16 (5): 381-86 (2003); aurora et al, Sequence covarian networks, methods and uses therof, US2008/0318207a 1; glaser et al, Stabilized polypeptide compounds, US2009/0048122A 1; urech et al, Sequence based engineering and optimization of single chain antibodies, WO2008/110348A 1; borras et al, Methods of modifying antibodies, and, andemodified antibodies with improved functional properties, WO2009/000099A 2). Such modifications as determined by covariance analysis may improve the potency, pharmacokinetic, pharmacodynamic and/or manufacturability characteristics of the immunoglobulin.

Nucleic acid molecules encoding amino acid sequence variants of an immunoglobulin or antibody are prepared by a variety of methods known in the art. Such methods include oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant forms of immunoglobulins.

Substitution mutagenesis within any of the hypervariable or CDR regions or framework regions is contemplated. A useful method for identifying certain residues or regions of an immunoglobulin at preferred positions for mutagenesis is referred to as "alanine scanning mutagenesis", e.g., Cunningham and Wells Science, 244: 1081-1085 (1989). In this regard, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. Those amino acid positions exhibiting functional sensitivity to substitution are then refined by introducing additional or other variants at or for the substitution site. Thus, where the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region and screened for the desired activity of the expressed variant.

Some embodiments of the immunoglobulins of the present invention may also be prepared by synthetic methods. Solid phase synthesis is the preferred technique for preparing individual peptides because it is the most cost effective method for preparing small peptides. For example, well-known solid phase synthesis techniques include the use of protecting groups, linkers, and solid phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid phase peptide synthesis. Suitable techniques are well known in The art (e.g., Merrifield (1973), Chem. polypeptides, pp.335-61 (edited by Katsoyannis and Panayotis), Merrifield (1963), J.Am.Chem.Soc.85: 2149; Davis et al (1985), biochem. Intl.10: Stewart and Young (1969), Solid phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al (1976), The Proteins (3 rd edition) 2: 105- "253; and Erickson et al (1976), The Proteins (3 rd edition) 2: 257. 527; protective group Peptides Synthesis", 3 rd edition, T.W.and P.G.M.M.M.M.M.M.P.S.P.P.P.S.P.P.P.S.P.S.P.S.P.S.S.P.S.S.P.S.S.S.S.P.S.S.S.P.S.S.S.S.S.S.P.S.P.S.S.S.P.S.S.S.P.S.S.S.P.S.S.S.S.P.S.S.P.S.S.S.S.P.P.S.S.S.S.P.S.S.S.P.P.S.P.S.P.P.S.P.P.S.S.S.S.S.S.S.P.S.S.P.P.S.S.P.P.S.P.P.S.S.S.S.P.S.P.P.P.P.P.P.P.S.S.P.P.P.S.P.P.P.S.S.P.S.S.P.P.P.P.S.S.S.P.P.P.P.S.S.P.P.P.S.P.P.P.P.P.P.S.P.P.P.P.P.P.P., 2 nd edition ", edited by m.bodanszky and a.bodanszky, Springer-Verlag, 1994; "ProtectingGroups," p.j.kocienski editions, Georg Thieme Verlag, Stuttgart, Germany, 1994; "Fmoc Solid Phase Peptide Synthesis, A Practical Approach," edited in W.C. Chan and P.D. white, Oxford Press, 2000; fields et al, synthetic peptides: a User's Guide, 1990, 77-183). For additional examples of synthetic and purification methods known in the art that are suitable for use in preparing the subject compositions of the invention, see, e.g., Sullivan et al, Toxin Peptide Therapeutic Agents, US2007/0071764 and Sullivan et al, Toxin Peptide Therapeutic Agents, PCT/US2007/022831 (published as WO2008/088422A 2), both of which are incorporated herein by reference in their entirety.

In the further description of the immunoglobulins and any of the variants herein, the one letter abbreviation system is often used to indicate the class of 20 "canonical" amino acid residues that are typically incorporated into naturally occurring peptides and proteins (table 3). The meaning of such single letter abbreviations is fully interchangeable with three letter abbreviations or non-abbreviated amino acid names. In the one-letter abbreviation system used herein, upper case letters denote L-amino acids, and lower case letters denote D-amino acids. For example, the abbreviation "R" represents L-arginine and the abbreviation "R" represents D-arginine.

TABLE 3 one-letter abbreviations for the canonical amino acids.

The three letters are abbreviated in parentheses.

Amino acid substitutions in an amino acid sequence are generally indicated herein by the single letter abbreviation for the amino acid residue at a particular position followed by the numerical one-letter symbol for the amino acid position relative to the initial sequence of interest and then followed by the substituted amino acid residue. For example, "T30D" represents the substitution of an aspartic acid residue for a threonine residue at amino acid position 30 relative to the initial sequence of interest. As another example, "W101F" represents the substitution of a phenylalanine residue for a tryptophan residue at amino acid position 101 relative to the initial sequence of interest.

Within the scope of the present invention, non-canonical amino acid residues may be incorporated into polypeptides by employing known protein engineering techniques using recombinant expression cells. (see, e.g., Link et al, Non-symmetrical amino acids in protein engineering, Current Opinion in Biotechnology, 14 (6): 603-609 (2003)). The term "non-canonical amino acid residue" refers to a D-or L-shaped amino acid residue that is not among the 20 canonical amino acids typically incorporated into naturally occurring proteins, e.g., beta-amino acids, high amino acids, cyclic amino acids, and amino acids with derivatized side chains. Examples include (L-form or D-form) beta-alanine, beta-aminopropionic acid, pipecolic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid; n α -ethylglycine, N α -ethylasparagine, hydroxylysine, allohydroxylysine, isodesmosine, alloisoleucine, ω -methylarginine, N α -methylglycine, N α -methylisoleucine, N α -methylvaline, γ -carboxyglutamine, epsilon-N, N-trimethyllysine, epsilon-N-acetyl lysine, O-phosphoserine, N α -acetyl serine, N α -formylmethionine, 3-methylhistidine, 5-hydroxylysine and other similar amino acids, and those listed in table 4 below, as well as derivatized forms of any of these as described herein. Table 4 contains some exemplary non-canonical amino acid residues used according to the invention and the associated abbreviations commonly used herein, but practitioners of the art will understand that different abbreviations and nomenclature may apply to the same substance and appear interchangeably herein.

Table 4 non-canonical amino acids that can be used according to the invention for amino acid addition, insertion or substitution into the peptide sequence. Both abbreviations are understood to be applicable if the abbreviations listed in table 4 differ from another abbreviation disclosed elsewhere herein for the same substance. The amino acids listed in Table 4 may be in the L-form or D-form.

The nomenclature and symbolization of amino acids and peptides by the UPAC-IUB Joint Biochemical nomenclature Commission (JCBN) has been published in the following documents: biochem.j., 1984, 219, 345-373; eur.j.biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; lnat.j.pept.prot.res., 1984, 24, after page 84; biol. chem., 1985, 260, 14-42; pure apple chem., 1984, 56, 595-624; amino Acids and Peptides, 1985, 16, 387- > 410; biochemical Nomenclature and Related Documents, 2 nd edition, Portland Press, 1992, pages 39-69.

One or more available modifications to the peptide domain of the immunoglobulin of the invention may include amino acid additions or insertions, amino acid deletions, peptide truncations, amino acid substitutions, and/or chemical derivatization of amino acid residues by known chemical techniques. For example, the amino acid sequence so modified includes at least one amino acid residue inserted or substituted therein relative to the amino acid sequence of the native sequence of interest, wherein the inserted or substituted amino acid residue has a side chain comprising a nucleophilic or electrophilic reactive functional group through which the peptide is conjugated to the linker and/or half-life extending moiety. Useful examples of such nucleophilic or electrophilic reactive functional groups according to the present invention include, but are not limited to, thiol, primary amine, seleno, hydrazide, aldehyde, carboxylic acid, ketone, aminooxy, masked (protected) aldehyde or masked (protected) keto functional groups. Examples of amino acid residues having a side chain comprising an electrophilic reactive functional group include, but are not limited to, a lysine residue, a homolysine, an α, β -diaminopropionic acid residue, an α, γ -diaminobutyric acid residue, an ornithine residue, cysteine, homocysteine, a glutamic acid residue, an aspartic acid residue, or a selenocysteine residue.

Amino acid residues are generally classified according to different chemical and/or physical properties. The term "acidic amino acid residue" refers to a D-or L-shaped amino acid residue having a side chain comprising an acidic group. Exemplary acidic residues include aspartic acid and glutamic acid residues. The term "alkyl amino acid residue" refers to a residue having a C which may be straight, branched or cyclic_1-6D-or L-form amino acid residues of alkyl side chains, including the amino acid amine of proline, wherein C_1-6Alkyl is substituted by 0, 1, 2 or 3 groups selected from C_1-4Haloalkyl, halo, cyano, nitro, -C (═ O) R^b、-C(＝O)OR^a、-C(＝O)NR^aR^a、-C(＝NR^a)NR^aR^a、-NR^aC(＝NR^a)NR^aR^a、-OR^a、-OC(＝O)R^b、-OC(＝O)NR^aR^a、-OC_2-6Alkyl radical NR^aR^a、-OC_2-6Alkyl OR^a、-SR^a、-S(＝O)R^b、-S(＝O)₂R^b、-S(＝O)₂NR^aR^a、-NR^aR^a、-N(R^a)C(＝O)R^b、-N(R^a)C(＝O)OR^b、-N(R^a)C(＝O)NR^aR^a、-N(R^a)C(＝NR^a)NR^aR^a、-N(R^a)S(＝O)₂R^b、-N(R^a)S(＝O)₂NR^aR^a、-NR^aC_2-6Alkyl radical NR^aR^aand-NR^aC_2-6Alkyl OR^aSubstituted with the substituent(s); wherein R is^aEach occurrence independently is H or R^b(ii) a And R is^bIndependently in each case 0, 1, 2 or 3 substituents selected from halo, C_1-4Alkane, C_1-3Alkyl halides, -OC_1-4Alkane, -NH₂、-NHC_1-4Alkane, and-N (C)_1-4Alkyl) C_1-4Alkyl substituted C_1-6An alkyl group; or any protonated form thereof, including alanine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamate, cysteine, methionine, hydroxyproline, but these residues do not contain an aryl or aromatic group. The term "aromatic amino acid residue" refers to a D-or L-shaped amino acid residue having a side chain comprising an aromatic group. Exemplary aromatic residues include tryptophan, tyrosine, 3- (1-naphthyl) alanine, or phenylalanine residues. The term "basic amino acid residue" refers to a D-or L-shaped amino acid residue having a side chain comprising a basic group. Exemplary basic amino acid residues include histidine, lysine, homolysine, ornithine, arginine, N-methyl-arginine, ω -aminoarginine, ω -methyl-arginine, 1-methyl-histidine, 3-methyl-histidine and homoarginine (hR) residues. The term "hydrophilic amino acid residue" refers to a D-or L-shaped amino acid residue having a side chain comprising a polar group. Exemplary hydrophilic residues include cysteine, serine, threonine, histidine, lysine, asparagine, aspartic acid, glutamic acid, glutamine and citrulline (Cit) residues. The term "lipophilic amino acid residue" refers to a D-or L-shaped amino acid residue having a side chain comprising an uncharged aliphatic or aromatic group. Exemplary lipophilic side chains include phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine. Alanine (a) is amphiphilic-it can act as a hydrophilic or lipophilic residue. Thus, alanine is included within the definition of both "lipophilic residues" and "hydrophilic residues". The term "non-functionalized amino acid residue" refers to a residue having an acid-free sequence D-or L-form amino acid residues of side chains of a basic or aromatic group. Exemplary neutral amino acid residues include methionine, glycine, alanine, valine, isoleucine, leucine, and norleucine (Nle) residues.

Other useful embodiments may result from conservative modifications of the amino acid sequence of the polypeptides disclosed herein. Conservative modifications will result in peptides conjugated with half-life extending moieties having similar functionalized, physical and chemical properties to those of the conjugated (e.g., PEG conjugated) peptide from which these modifications were made. Such conservatively modified forms of the conjugated polypeptides disclosed herein are also contemplated embodiments of the invention.

In contrast, substantial modification of the functionalization and/or chemical properties of a peptide can be achieved by selecting amino acid sequence substitutions that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the region of the substitution, e.g., the a-helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.

For example, a "conservative amino acid substitution" may involve the substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any native residues in the polypeptide may also be substituted with alanine as has been described previously for "alanine scanning mutagenesis" (see, e.g., MacLennan et al, Acta Physiol. Scan. Suppl., 643: 55-67 (1998); Sasaki et al, 1998, adv. Biophys.35: 1-24(1998), which discusses alanine scanning mutagenesis).

Such substitutions (whether conservative or non-conservative) may be determined by one of skill in the art when the desired amino acid substitution is desired. For example, amino acid substitutions can be used to identify important residues of a peptide sequence, or to increase or decrease the affinity of a peptide or vehicle-conjugated peptide molecule described herein.

Naturally occurring residues can be divided into several classes based on general side chain properties:

1) hydrophobicity: norleucine (Nor or Nle), Met, Ala, Val, Leu, Ile;

2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

3) acidity: asp and Glu;

4) alkalinity: his, Lys, Arg;

5) residues that influence chain orientation: gly, Pro; and

6) aromatic: trp, Tyr, Phe.

Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other amino acid moieties in inverted or inverted form.

Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the toxin peptide analogs.

In making these changes, according to certain embodiments, the hydropathic index of the amino acid may be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. Their hydropathic and hydrophobic indices are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of amino acid hydropathic indices in conferring interactive biological function on proteins is understood in the art (see, e.g., Kvte et al, 1982, j.157：105-131). It is known that certain amino acids can be used to replace other amino acids having similar hydropathic indices or scores and still retain similar biological activity. In certain embodiments, when the alteration is made based on the hydropathic index, it comprises substituting amino acids having a hydropathic index within ± 2. In certain embodiments, those within ± 1 are included, and in certain embodiments, those within ± 0.5 are included.

It is also understood in the art that substitution of like amino acids can be made effectively based on hydrophilicity, particularly where the biofunctional protein or peptide formed thereby as disclosed herein is intended for use in immunological embodiments. In certain embodiments, the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In certain embodiments, changes based on similar hydrophilicity values include substitutions of amino acids having hydrophilicity values within ± 2, in certain embodiments within ± 1, and in certain embodiments within ± 0.5. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions".

Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, norleucine, alanine or methionine for another; substitution of one polar (hydrophilic) amino acid residue for another, such as substitution between arginine and lysine, substitution between glutamine and asparagine, substitution between glycine and serine; substitution of one basic amino acid residue, such as lysine, arginine or histidine, for another; or substitution of one acidic residue, such as aspartic acid or glutamic acid, for the other. The phrase "conservative amino acid substitution" also includes the replacement of a non-derivatized residue with a chemically derivatized residue, provided that the polypeptide exhibits the desired biological activity. Other exemplary amino acid substitutions that can be used in accordance with the present invention are shown in table 5 below.

TABLE 5 some useful amino acid substitutions.

Generally, an amino acid sequence variant of an immunoglobulin will have an amino acid sequence that is at least 60% amino acid sequence identity, or at least 65%, or at least 70%, or at least 75% or at least 80% identical, more preferably at least 85% identical, even more preferably at least 90% identical, and most preferably at least 95% identical (including, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) to the original immunoglobulin or antibody amino acid sequence of the heavy or light chain variable region. Herein, identity or homology with respect to the sequence is defined as: the percentage of amino acid residues that a candidate sequence is identical to the initial sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the immunoglobulin or antibody sequence will not be understood to affect sequence identity or homology.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include an immunoglobulin with an N-terminal methionyl residue or an immunoglobulin (including an antibody or antibody fragment) fused to an epitope tag or salvage receptor binding epitope. Other insertional variants of immunoglobulin or antibody molecules include, for example, fusion at the N-or C-terminus with a polypeptide that increases the serum half-life of the immunoglobulin.

Examples of epitope tags include flu HA-tag polypeptide and its antibody 12CA5[ Field et al, mol.cell.biol.8: 2159-2165(1988)](ii) a The C-myc tag and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [ Evan et al, mol. 3610-3616(1985)](ii) a And the herpes simplex virus glycoprotein d (gd) tag and antibodies thereto [ Paborsky et al, Protein Engineering3 (6): 547-553(1990)]. Other exemplary tags are polyhistidine sequences, typically about 6 histidine residues, which allow the isolation of such labeled compounds using nickel chelation. Other labels and tags, e.g.Labels (Eastman Kodak, Rochester, NY) are well known and commonly used in the art.

The following illustrates some specific, non-limiting embodiments of amino acid substitution variants of the immunoglobulins of the present invention (including antibodies and antibody fragments).

Any cysteine residues not involved in maintaining the correct conformation of the immunoglobulin may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the immunoglobulin to improve its stability (particularly where the immunoglobulin is an antibody fragment such as an Fv fragment).

In some cases, immunoglobulin variants are prepared with the aim of modifying those amino acid residues in the starting sequence which are directly involved in epitope binding. In other embodiments, for the purposes discussed herein, it may be desirable to modify residues that are not directly involved in epitope binding or residues that are not involved in epitope binding in any way. Mutagenesis within any of the CDR regions and/or framework regions is contemplated.

To determine the antigen binding protein amino acid residues important for epitope recognition and binding, alanine scanning mutagenesis can be performed to generate substitution variants. See, e.g., Cunningham et al, Science, 244: 1081-1085(1989), the disclosure of which is incorporated herein by reference in its entirety. In this method, individual amino acid residues are replaced one at a time with alanine residues and the resulting antibodies are screened for the ability to bind to their specific epitope relative to the unmodified polypeptide. Modified antigen binding proteins with reduced binding capacity are sequenced to determine altered residues, indicating their importance in binding or biological properties.

Substitutional variants of antigen binding proteins can be prepared by affinity maturation, in which random amino acid changes are introduced into the parent polypeptide sequence. See, e.g., Ouwehand et al, VoxSang74 (suppl 2): 223- > 232, 1998; rader et al, Proc.Natl.Acad.Sci.USA95: 8910-8915, 1998; dall' Acqua et al, curr, opin, struct, biol.8: 443-450, 1998, the disclosures of which are incorporated herein by reference in their entirety. Affinity maturation involves the preparation and screening of antigen binding proteins or variants thereof and selecting from the resulting variants those with modified biological properties such as increased binding affinity relative to the parent antigen binding protein. One convenient way to generate substitution variants is to use affinity maturation of phage display. Briefly, multiple hypervariable region sites were mutated to generate all possible amino substitutions at each site. The variants so produced are expressed on the surface of the particle in a monovalent manner as fusions to the gene III product of M13 packaged within each filamentous phage particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity). See, e.g., WO92/01047, WO93/112366, WO95/15388 and WO 93/19172.

Current methods of antibody affinity maturation belong to two mutagenesis categories: random and non-random. Error-prone PCR, mutagenic bacterial strains (Low et al, J.mol.Biol.260, 359-68, 1996) and saturation mutagenesis (Nishimiya et al, J.biol.Chem.275: 12813-20, 2000; Chowdhury, P.S.methods mol.biol.178, 269-85, 2002) are typical examples of random mutagenesis methods (Rajpal et al, Proc Natl Acad Sci U S.A.102: 8466-71, 2005). Non-random techniques typically use alanine scanning or site-directed mutagenesis to generate a limited set of specific muteins. Some of the methods are described in further detail below.

Affinity maturation by panning procedure-affinity maturation of recombinant antibodies is usually performed by several rounds of candidate antibody panning in the presence of progressively lower amounts of antigen. Each round of reduced amount of antigen selects the antibody with the highest affinity for the antigen, thereby obtaining high affinity antibodies from a large amount of starting material. Affinity maturation by panning is well known in the art and is described, for example, in Huls et al (Cancer Immunol Immunother.50: 163-71, 2001). Affinity maturation methods using phage display technology are described elsewhere herein and are known in the art (see, e.g., Daugherty et al, Proc Natl Acad Sci U S A.97: 2029-34, 2000).

Brooks-through mutagenesis-Brooks mutagenesis (LTM) (Rajpal et al, Proc Natl Acad Sci U S A.102: 8466-71, 2005) provides a method for rapid determination of antibody binding sites. For LTM, 9 amino acids representing the major side chain chemistries provided by the 20 natural amino acids were selected to carefully analyze the functional side chain contributions to binding at each position in all 6 CDRs of the antibody. LTM generates a single mutated position sequence within the CDR in which each "wild-type" residue is systematically substituted with one of the 9 selected amino acids. The mutated CDRs are combined to generate combinatorial single chain variable fragment (scFv) libraries that increase complexity and scale, but do not inhibit quantitative display of all mutated proteins. After forward selection, clones with improved binding were sequenced and the location of the beneficial mutation was determined.

Error-prone PCR-error-prone PCR involves randomization of nucleic acids between different selection rounds. Randomization occurs at a low rate due to the inherent error rate of the polymerase used but can be augmented by error-prone PCR (Zaccolo et al, J.mol.biol.285: 775-783, 1999) which uses a polymerase with a high inherent error rate during transcription (Hawkins et al, J.mol biol.226: 889-96, 1992). After the mutation cycle, clones with improved antigen affinity are selected using routine methods in the art.

Techniques utilizing gene shuffling and directed evolution can also be used to prepare and screen antigen binding proteins or variants thereof for a desired activity. For example, Jermutus et al, Proc Natl Acad Sci U sa, 98 (1): 75-80(2001) shows that combining ribosome display based customized in vitro selection strategies with in vitro differentiation by DNA shuffling allows evolution of the scFv dissociation rate or thermodynamic stability; fermer et al, Tumour biol.2004, 1-4 months; 25(1-2): 7-13 reports the use of phage display binding to DNA shuffling to improve affinity by nearly 3 orders of magnitude. Dougherty et al, Proc Natl Acad Sci U S A.2000, 29/2; 97(5): 2029-2034 reports that (i) functional clones appear at an unexpectedly high frequency in hypermutation libraries, (ii) gain-of-function mutants are well represented in such libraries, and (iii) most scFv mutations result in higher affinities corresponding to residues far from the binding site.

Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen, or to simulate these contact points using computer software. These contact residues and adjacent residues are candidate residues for substitution according to the techniques set forth herein. Once such variants are produced, they are subjected to screening as described herein, and antibodies having superior properties in one or more relevant assays may be selected for further development.

Immunoglobulins with modified carbohydrates

Immunoglobulin variants may also be prepared that have a modified glycosylation pattern relative to the parent polypeptide, such as the addition or deletion of one or more carbohydrate moieties that bind to the immunoglobulin, and/or the addition or deletion of one or more glycosylation sites in the immunoglobulin.

Glycosylation of polypeptides (including antibodies) is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. The presence of any of these tripeptide sequences in a polypeptide forms a potential glycosylation site. Thus, N-linked glycosylation sites can be added to an immunoglobulin in such a way that: the amino acid sequence is altered such that it comprises one or more of these tripeptide sequences. O-linked glycosylation refers to the attachment of one of the saccharides N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. O-linked glycosylation sites can be added to an immunoglobulin by inserting or substituting one or more serine or threonine residues into the original immunoglobulin or antibody sequence.

Altered effector function

Cysteine residues may be removed from or introduced into the Fc region of an antibody or Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in this region. The homodimeric immunoglobulin so formed may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp med.176: 1191-1195(1992) and shop, B.J.Immunol.148: 2918-2922(1992). Homodimeric immunoglobulins or antibodies may also be used as described by Wolff et al, Cancer Research 53: 2560-2565 (1993). Alternatively, immunoglobulins can be engineered that have dual Fc regions and may therefore have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-cancer robust design 3: 219-230(1989).

It is also contemplated that one or more of the N-terminal 20 amino acid residues (e.g., signal sequence) of the heavy or light chain are removed.

It may also be desirable to modify to increase serum half-life, for example, by: incorporation or addition of a salvage receptor binding epitope (e.g., by mutating the appropriate region, or by incorporating the epitope into a peptide tag that is then fused to either end or middle of the immunoglobulin, e.g., by DNA or peptide synthesis) (see, e.g., WO96/32478) or the addition of molecules such as PEG or other water-soluble polymers, including polysaccharide polymers.

The salvage receptor binding epitope preferably constitutes a region in which any one or more amino acid residues from one or both loops of the Fc domain are transferred to a similar position in the immunoglobulin or fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferably, the epitope is removed from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or VH region of an immunoglobulin or antibody, or more than one such region. Alternatively, the epitope is removed from the CH2 domain of the Fc region and transferred to the C of an immunoglobulin fragment_LRegion or V_LZone or both zones. See also international applications WO97/34631 and WO96/32478, which describe Fc variants and their interaction with salvage receptors.

Other sites and amino acid residues in the constant region responsible for Complement Dependent Cytotoxicity (CDC) (e.g., the C1q binding site) and/or antibody dependent cytotoxicity (ADCC) have been identified [ see, e.g., molecular. immunol.29 (5): 633-9 (1992); shields et al, j.biol.chem., 276 (9): 6591 and 6604 (2001); lazar et al, proc.nat' 1.acad.sci.103 (11): 4005(2006), which describe the effect of a mutation at a particular location, each of which is incorporated herein by reference in its entirety. Mutation of residues within the Fc receptor binding site may result in altered (i.e., enhanced or diminished) effector function, such as altered Fc receptor affinity, altered ADCC or CDC activity, or altered half-life. As described above, potential mutations include insertions, deletions, or substitutions of one or more residues, including substitutions with alanine, conservative substitutions, non-conservative substitutions, or substitutions with a corresponding amino acid residue from the same position in a different subclass (e.g., the replacement of an IgG1 residue at the same position with a corresponding IgG2 residue).

The invention also encompasses the preparation of immunoglobulin molecules (including antibodies and antibody fragments) having altered carbohydrate structures resulting in altered effector activity, including antibody molecules with absent or reduced fucosylation that exhibit improved ADCC activity. Various ways of achieving this are known in the art. ADCC effector activity is mediated, for example, by binding an antibody molecule to the Fc γ RIII receptor, which binding has been shown to rely on an N-linked glycosylated carbohydrate structure at Asn-297 of the CH2 domain. The non-fucosylated antibodies bind the receptor with increased affinity and trigger Fc γ RIII-mediated effector functions more efficiently than native fucosylated antibodies. For example, recombinant production of non-fucosylated antibodies in CHO cells that have been knocked out for alpha-1, 6-fucosyltransferase yields antibodies with 100-fold enhanced ADCC activity (Yamane-Ohnuki et al, Biotechnol Bioeng.2004, 9/5; 87 (5): 614-22). A similar effect can be achieved by: for example, by siRNA or antisense RNA treatment to reduce the activity of this or other enzymes in the fucosylation pathway, engineering cell lines to knock out the enzyme or enzymes, or culturing with selective glycosylation inhibitors (Rothman et al, Mol Immunol.1989, 12 months; 26 (12): 1113-23). Some host cell lines, such as Lec13 or the rat hybridoma YB2/0 cell line, naturally produce antibodies with lower levels of fucosylation. Shields et al, J Biol chem.2002, 26 months 7; 277(30): 26733-40; shinkawa et al, J Biol chem.2003, 31.1 month; 278(5): 3466-73. Increasing the level of bisecting carbohydrate, for example by recombinant production of antibodies in cells overexpressing GnTIII enzymes, has also been determined to increase ADCC activity. Umana et al, Nat biotechnol.1999 month 2; 17(2): 176-80. It has been predicted that the absence of only one of the two fucose residues may be sufficient to increase ADCC activity. (Ferrara et al, J Biol chem.2005, 12.5).

Other covalent modifications of immunoglobulins

Other specific covalent modifications of immunoglobulins are also encompassed within the scope of the present invention. Covalent modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of immunoglobulins or antibodies, as appropriate. Other types of covalent modifications can be introduced by reacting targeted amino acid residues with organic derivatizing agents that are capable of reacting with selected side chains or N-terminal or C-terminal residues.

Most commonly, the cysteinyl residue is reacted with an α -haloacetate (and corresponding amines) such as chloroacetic acid or chloroacetamide to produce a carboxymethyl or carboxyamidomethyl derivative. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, α -bromo- β (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuriyl-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1, 3-diazole.

Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH5.5-7.0 because this agent is relatively specific for histidyl side chains. Para-bromophenacyl bromide may also be used; the reaction is preferably carried out at pH6.0 in 0.1M sodium arsenate.

Lysyl and amino terminal residues are reacted with succinic acid or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residue. Other suitable reagents for derivatizing alpha amino acid-containing residues include imidates such as methyl picoliniminate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, orthomethylisourea, 2, 4-acetylacetone, and transaminases catalyzed reactions with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents of phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione and ninhydrin. High pK due to guanidine functionality_aDerivatization of arginine residues requires that the reaction be carried out under alkaline conditions. In addition, these reagents can react with lysine groups as well as arginine epsilon amino groups.

With the specific interest in introducing spectroscopic tags into tyrosyl residues, specific modifications of tyrosyl residues can be carried out by reacting them with aromatic diazo compounds or tetranitromethane. More commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Use of ¹²⁵I or¹³¹I iodination of tyrosyl residues to prepare labeled proteins for radioimmunoassay.

Pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R-n.dbd.c.dbd.n-R ', where R and R' are different alkyl groups) such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azocation-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl or glutamyl is converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are often deamidated to form the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. Deamidated forms of these residues are within the scope of the invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha amino groups of lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86 (1983)), acetylation of the amino terminus of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification involves chemical or enzymatic coupling of glycosides to immunoglobulins (e.g., antibodies or antibody fragments). The advantage of these procedures is that they do not require the production of immunoglobulins in host cells that have the glycosylation capability to carry out N-linked or O-linked glycosylation. Depending on the coupling mode used, one or more sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO87/05330 published on 11.9.1987 and Aplin and Wriston, CRC Crit. Rev. biochem., pp.259-306 (1981).

Removal of any carbohydrate moieties present on the immunoglobulin may be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the immunoglobulin to the compound trifluoromethanesulfonic acid or equivalent compound. This treatment results in the cleavage of most or all of the saccharides except the linked saccharide (N-acetylglucosamine or N-acetylgalactosamine) leaving the immunoglobulin intact. Hakimuddin et al, arch, biochem, biophysis, 259: 52(1987) and Edge et al, anal. biochem., 118: 131(1981) describe chemical deglycosylation. Enzymatic cleavage of carbohydrate moieties on immunoglobulins can be achieved by using a variety of endo-and exoglycosidases, such as Thotakura et al meth.enzymol.138: 350 (1987).

Another type of covalent modification of the immunoglobulins of the present invention (including antibodies and antibody fragments) involves linking the immunoglobulin to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyalkylene oxide, or polysaccharide polymers such as dextran. Such methods are known in the art, see, e.g., U.S. Pat. nos. 4,640,835; 4,496,689, respectively; 4,301,144, respectively; 4,670,417, respectively; 4,791,192, respectively; 4,179,337; 4,766,106; 4,179,337; 4,495,285, respectively; 4,609,546 or EP 315456.

Isolated nucleic acids

Another aspect of the invention is an isolated nucleic acid encoding an immunoglobulin of the invention, such as but not limited to an isolated nucleic acid encoding an antibody or antibody fragment of the invention. Such nucleic acids are prepared by recombinant techniques known in the art and/or disclosed herein.

In other embodiments, the isolated nucleic acid encodes an immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

(a) the heavy chain variable region comprises SEQ ID NO: 323 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188 or SEQ ID NO: 190; or

(b) The heavy chain variable region comprises SEQ ID NO: 321 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188 or SEQ ID NO: 190; or

(c) The heavy chain variable region comprises SEQ ID NO: 325 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 182. SEQ ID NO: 188 or SEQ ID NO: 190.

And in some embodiments, the isolated nucleic acid encodes an immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

(a) the light chain variable region comprises SEQ ID NO: 196 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 335. SEQ ID NO: 349. SEQ ID NO: 351. SEQ ID NO: 353. SEQ ID NO: 355 or SEQ ID NO: 359; or

(b) The light chain variable region comprises SEQ ID NO: 204 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349 or SEQ ID NO: 355 an amino acid sequence; or

(c) The light chain variable region comprises SEQ ID NO: 202 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349 amino acid sequence; or

(d) The light chain variable region comprises SEQ ID NO: 192 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 357. SEQ ID NO: 359 or SEQ ID NO: 369 amino acid sequence; or

(e) The light chain variable region comprises SEQ ID NO: 194 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 335. SEQ ID NO: 349 or SEQ ID NO: 351.

In other embodiments, the isolated nucleic acid encodes an immunoglobulin comprising an immunoglobulin heavy chain and an immunoglobulin light chain, wherein:

(a) the heavy chain variable region comprises SEQ ID NO: 323; and the light chain variable region comprises SEQ ID NO: 188; or

(b) The light chain variable region comprises SEQ ID NO: 196; and the heavy chain variable region comprises SEQ ID NO: 353; or

(c) The light chain variable region comprises SEQ ID NO: 202; and the heavy chain variable region comprises SEQ ID NO: 349 amino acid sequence; or

(d) The heavy chain variable region comprises SEQ ID NO: 325; and the light chain variable region comprises SEQ ID NO: 190.

Or in some embodiments, the isolated nucleic acid encodes an immunoglobulin comprising:

Comprises the amino acid sequence of SEQ ID NO: 113, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; and a polypeptide comprising SEQ ID NO: 110, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; or

Comprises the amino acid sequence of SEQ ID NO: 125, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 122, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; or

Comprises the amino acid sequence of SEQ ID NO: 101, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 98, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; or

Comprises the amino acid sequence of SEQ ID NO: 119, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 116, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both.

The invention also relates to vectors, including expression vectors, comprising any of the isolated nucleic acids of the invention. Also encompassed within the invention are isolated host cells comprising the expression vectors, which host cells are prepared by molecular biology techniques known in the art and/or disclosed herein.

The invention also relates to a method comprising the following steps:

culturing the host cell in a culture medium under conditions that allow expression of the immunoglobulin encoded by the expression vector; and

recovering the immunoglobulin from the culture medium. Recovery of immunoglobulins is carried out by known antibody purification methods such as, but not limited to, the antibody purification techniques disclosed in example 1 herein and elsewhere.

Gene therapy

Delivery of therapeutic immunoglobulins to appropriate cells can be carried out by ex vivo, in situ or in vivo gene therapy using any suitable method known in the art. For example, for in vivo therapy, nucleic acids encoding the desired immunoglobulin or antibody may be injected directly into the subject, alone or with a carrier, liposome, or pellet, and in some embodiments, at the site where expression of the immunoglobulin compound is desired. For ex vivo treatment, cells of the subject are removed, nucleic acids are introduced into these cells, and the altered cells are infused directly back into the subject or, for example, encapsulated within a porous membrane implanted into the patient. See, for example, U.S. Pat. Nos. 4,892,538 and 5,283,187.

There are a variety of techniques that can be used to introduce nucleic acids into living cells. These techniques vary depending on whether the nucleic acid is transferred to cells cultured in vitro or in vivo in the intended host. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, chemical treatment, DEAE-dextran, and calcium phosphate precipitation. Other in vivo nucleic acid transfer techniques include transfection with viral vectors (e.g., adenovirus, herpes simplex I virus, adeno-associated virus, or retrovirus) and lipid-based systems. Nucleic acids and transfection reagents are optionally associated with the microparticles. Exemplary transfection reagents include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, quaternary amine amphiphilic DOTMA ((dioleoyloxypropyl) trimethylammonium bromide, commercialized as Lipofectin by GIBCO-BRL) (Felgner et al, (1987) Proc. Natl. Acad. Sci. USA84, 7413-; lipophilic glutamic acid diesters with pendant trimethylammonium heads (Ito et al (1990) biochem. biophysis. acta1023, 124-132); metabolizable parent lipids such as the cationic lipids dioctadecylamidoglycyl spermine (dioctyl amidoglycyl spermine) (DOGS, Transfectam, Promega) and dipalmitoylphosphatidyl ethanolpentyl spermine (DPPES) (J.P.Behr (1986) Tetrahedron Lett.27, 5861-42; J.P.Behr et al (1989) Proc.Natl.Acad.Sci.USA86, 6982-698); metabolizable quaternary ammonium salts (DOTB, N- (1- [2, 3-dioleoyloxy ] propyl) -N, N, N-trimethylammoniummethane (DOTAP) (Boehringer Mannheim), Polyethyleneimine (PEI), dioleate, ChoTB, ChoSC) (Leventis et al (1990) Biochim. Inter.22, 235-charge 241); 3 β [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (DC-Chol), Dioleoylphosphatidylethanolamine (DOPE)/3 β [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol DC-Chol (Gao et al, (1991) Biochim.Biophys.Acta1065, 8-14), spermine, spermidine, lipopolyamine (Behr et al, Bioconjugate Chem, 1994, 5: 382. cndot. 389), Lipophilic Polylysine (LPLL) (Zhou et al, (1991) Biochim.Biophys.Acta939, 8-18), [ [ (1, 1, 3, 3-tetramethylbutyl) tolyloxy ] ethoxy ] ethyl ] dimethylbenzylammonium hydroxide (DEBDA hydroxide) and excess phosphatidylcholine/cholesterol (Ballas et al, (1988) Biophys.Acta939, 8-18) mixed in a1, Cetyl trimethylammonium bromide (CTAB)/DOPE mixtures (Pinnadiwage et al, (1989) Biochim. Biophys. acta985, 33-37), lipophilic diesters of glutamic acid (TMAG) with DOPE, CTAB, DEBDA, didodecylammonium bromide (DDAB), and mixtures of stearic acid amines with phosphatidylethanolamine (Rose et al, (1991) Biotechnique10, 520-525), DDAB/DOPE (TransfectACE, GIBCO BRL), and lipids with galactooligosaccharides. Exemplary transfection-enhancing reagents that increase transfer efficiency include, for example, DEAE-dextran, polybrene, lysosomal dividing peptides (Ohmori N I et al, Biochem Biophys Res Commun Jun.27, 1997; 235 (3): 726-9), chondroitin-based proteoglycans, sulfated proteoglycans, polyethylenimine, polylysine (Pollard H et al J Biol Chem, 1998273 (13): 7507-11), integrin binding peptide CYGGRGDTP (SEQ ID NO: 235), linear dextran nonasaccharides, glycerol, cholesteryl groups constrained at the 3' -terminal internucleoside junction of the oligonucleotide (Letser, R.L.1989Proc Natl Acad Sci USA 86: (17): 6553-6), lysophospholipids, lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyl lysophosphatidylcholine.

In some cases, it may be desirable to deliver the nucleic acid with an agent that directs a vector comprising the nucleic acid to the target cell. These "targeting" molecules include antigen binding proteins specific for cell surface membrane proteins on the target cell or ligands for receptors on the target cell. Where liposomes are employed, proteins that bind cell surface membrane proteins associated with endocytosis can be used to target and/or aid in uptake. Examples of such proteins include capsid proteins and fragments thereof that tend to specific cell types, antigen binding proteins for proteins that undergo internalization in circulation, and proteins that target intracellular localization and increase intracellular half-life. In other embodiments, receptor-mediated endocytosis may be used. These methods are described, for example, in Wu et al, 1987 or Wagner et al, 1990. For a review of the currently known gene markers and gene therapy protocols, see Anderson 1992. See also WO93/25673 and the references cited therein. For additional reviews on gene therapy techniques, see Friedmann, Science, 244: 1275-1281 (1989); anderson, Nature, supplementary to volume 392, 6679, pages 25-30 (1998); verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455460(1992).

Administration and preparation of pharmaceutical formulations

The immunoglobulins or antibodies used in practicing the methods of the invention can be formulated into pharmaceutical compositions and medicaments comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that does not react with the immune system of the subject when combined with an immunoglobulin or antibody. Examples include, but are not limited to, any of a variety of standard pharmaceutical carriers such as sterile phosphate buffered saline solution, bacteriostatic water, and the like. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, and can include other proteins for enhanced stability, e.g., albumin, lipoproteins, globulins, and the like, subject to mild chemical modification, and the like.

Exemplary immunoglobulin concentrations in the formulations can range from about 0.1mg/mL to about 180mg/mL or from about 0.1mg/mL to about 50mg/mL, or from about 0.5mg/mL to about 25mg/mL, or from about 2mg/mL to about 10 mg/mL. Aqueous formulations of immunoglobulins may be prepared using pH buffered solutions, which may have a pH in the range of, for example, about 4.5 to about 6.5, or about 4.8 to about 5.5, or about 5.0. Examples of buffers suitable for use at a pH within this range include acetates (such as sodium acetate), succinates (such as sodium succinate), gluconates, histidines, citrates and other organic acid buffers. The buffer concentration may be from about 1mM to about 200mM, or from about 10mM to about 60mM, depending on, for example, the desired isotonicity of the buffer and formulation.

Tonicity agents which also stabilize the immunoglobulin may be included in the formulation. Exemplary tonicity agents include polyols such as mannitol, sucrose or trehalose. Preferably the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may be suitable. Exemplary concentrations of the polyol in the formulation may range from about 1% to about 15% w/v.

Surfactants may also be added to the immunoglobulin preparation to reduce aggregation of the formulated immunoglobulin and/or minimize particle formation and/or reduce adsorption in the preparation. Exemplary surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbate 20 or polysorbate 80) or poloxamers (e.g., poloxamer 188). Exemplary concentrations of the surfactant can range from about 0.001% to about 0.5%, or from about 0.005% to about 0.2%, or from about 0.004% to about 0.01% w/v.

In one embodiment, the formulation comprises the above-identified agents (i.e., immunoglobulin, buffer, polyol, and surfactant) and is substantially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium chloride. In another embodiment, preservatives may be included in the formulation at a concentration ranging, for example, from about 0.1% to about 2%, or from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16 th edition, Osol, a. editor (1980) may be included in the formulation, provided that they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include additional buffering agents; a cosolvent; antioxidants (including ascorbic acid and methionine); chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium.

Therapeutic formulations of immunoglobulins are prepared as lyophilized formulations or as aqueous solutions for storage by mixing the immunoglobulins with the desired degree of purity and optionally a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. editor (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, maltose, or dextrins); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^TM、PLURONICS^TMOr polyethylene glycol (PEG).

In one embodiment, a suitable formulation of the claimed invention comprises an isotonic buffer, such as a phosphate, acetate or Tris buffer, in combination with a tonicity agent such as a polyol, sorbitol, sucrose or sodium chloride which regulates the osmotic pressure and remains stable. An example of such a tonicity agent is 5% sorbitol or sucrose. In addition, the formulation may optionally contain 0.01% to 0.02% weight/volume of a surfactant, for example to prevent aggregation and to stabilize. The pH of the formulation may be in the range of 4.5 to 6.5 or 4.5 to 5.5. Other exemplary descriptions of pharmaceutical formulations for antibodies can be found in US2003/0113316 and U.S. patent No. 6,171,586, each of which is incorporated herein by reference in its entirety.

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be advantageous to also provide an immunosuppressant. Such molecules are present in suitable combinations in amounts effective for the intended purpose.

The active ingredients can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. edition (1980).

Suspension and crystal forms of the immunoglobulin are also contemplated. Methods for preparing suspensions and crystalline forms are known to those skilled in the art.

The formulation to be used for in vivo administration must be sterile. The compositions of the present invention may be sterilized by conventional, well known sterilization techniques. Sterilization is readily achieved, for example, by filtration through sterile filtration membranes. The resulting solution may be packaged for use or filtered under sterile conditions and lyophilized, the lyophilized preparation being mixed with the sterile solution prior to administration.

The polypeptides are generally stabilized for long-term storage using a freeze-drying process, particularly when the polypeptides are relatively unstable in liquid compositions. The lyophilization cycle generally consists of three steps: freezing, primary drying and secondary drying; williams and Polli, Journal of Parentereralscience and Technology, Vol.38, No. 2, pp.48-59 (1984). In the freezing step, the solution is cooled until it is sufficiently frozen. At this stage, bulk water in solution forms ice. During the primary drying stage, the ice sublimes, which is done by using a vacuum to reduce the chamber pressure below the vapor pressure of the ice. Finally, the adsorbed or bound water is removed at a reduced chamber pressure and elevated resting temperature in a secondary drying stage. This process produces a material known as a lyophilized cake. The lyophilized cake can then be reconstituted prior to use.

Standard reconstitution procedures for lyophilized materials are to add back a volume of pure water (usually equal to the volume removed during lyophilization), but sometimes dilute solutions of the antibacterial agent are used to prepare the drug for parenteral administration; chen, Drug Development and Industrial Pharmacy, Vol.18, pp.11 and 12, 1311-1354 (1992).

Excipients have been indicated to act in some cases as stabilizers for freeze-dried products; carpenter et al, Developments in Biological Standardization, Vol.74, pp.225-. For example, known excipients include polyols (including mannitol, sorbitol, and glycerol); sugars (including glucose and sucrose); and amino acids (including alanine, glycine, and glutamic acid).

In addition, polyols and sugars are also often used to protect polypeptides from damage caused by freezing and drying and to enhance stability during storage in the dry state. Generally, sugars, in particular disaccharides, are effective both during the freeze-drying process and during storage. Other classes of molecules, including mono-and disaccharides and polymers such as PVP, have also been reported as stabilizers for lyophilized products.

For injection, the pharmaceutical formulation and/or medicament may be a powder suitable for reconstitution with a suitable solution as described above. Examples of these include, but are not limited to, freeze-dried, spin-dried or spray-dried powders, amorphous powders, granules, precipitants or granules. For injection, the formulation may optionally comprise stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations of these.

Can be used for preparing sustained release preparations. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated polypeptides are held in the body for a longer period of time, they may denature or aggregate due to exposure to moisture at 37 ℃, resulting in a loss of biological activity and possibly a change in immunogenicity. Reasonable strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

The formulations of the present invention may be designed to be short acting, fast releasing, long acting or sustained releasing as described herein. Thus, the pharmaceutical formulations may also be formulated for controlled release or for slow release.

The specific dose can be adjusted according to the disease condition, age, body weight, general health condition, sex and diet of the subject, administration interval, administration route, excretion rate and combination of the drugs. The inclusion of an effective amount of any of the above dosage forms is well within the bounds of routine experimentation and thus is well within the scope of the present invention.

The immunoglobulin is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and if local treatment is desired, intralesional administration. Parenteral infusion includes intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or subcutaneous administration. Furthermore, the immunoglobulin is suitably administered by pulse infusion, particularly with a gradually decreasing dose of the immunoglobulin or antibody. Administration is preferably by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. Other methods of administration are contemplated, including topical administration, particularly transdermal, transmucosal, rectal, buccal or topical administration, for example, through a catheter placed near the desired site. Most preferably, the immunoglobulin of the invention is administered intravenously in a physiological solution in a dose range between 0.01mg/kg and 100mg/kg, at a frequency ranging from once a day to once a week to once a month (e.g., once a day, once every other day, once every two days, or 2, 3, 4, 5, or 6 times a week), preferably in a dose range of 0.1 to 45mg/kg, 0.1 to 15mg/kg, or 0.1 to 10mg/kg, at a frequency of 2 or 3 times a week, or at most 45mg/kg and once a month.

Embodiments or aspects of the invention may include, but are not limited to, the following:

1. an isolated immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

(a) the heavy chain variable region comprises SEQ ID NO: 323 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188 or SEQ ID NO: 190; or

(b) The heavy chain variable region comprises SEQ ID NO: 321 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188 or SEQ ID NO: 190; or

(c) The heavy chain variable region comprises SEQ ID NO: 325 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 182. SEQ ID NO: 188 or SEQ ID NO: 190.

2. An isolated immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

(a) the light chain variable region comprises SEQ ID NO: 196 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 335. SEQ ID NO: 349. SEQ ID NO: 351. SEQ ID NO: 353. SEQ ID NO: 355 or SEQ ID NO: 359; or

(b) The light chain variable region comprises SEQ ID NO: 204 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349 or SEQ ID NO: 355 an amino acid sequence; or

(c) The light chain variable region comprises SEQ ID NO: 202 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349 amino acid sequence; or

(d) The light chain variable region comprises SEQ ID NO: 192 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 357. SEQ ID NO: 359 or SEQ ID NO: 369 amino acid sequence; or

(e) The light chain variable region comprises SEQ ID NO: 194 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 335. SEQ ID NO: 349 or SEQ ID NO: 351.

3. The isolated immunoglobulin of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 323; and the light chain variable region comprises seq id NO: 188.

4. The isolated immunoglobulin of claim 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 196; and the heavy chain variable region comprises seq id NO: 353.

5. The isolated immunoglobulin of claim 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 202; and the heavy chain variable region comprises seq id NO: 349 amino acid sequence.

6. The isolated immunoglobulin of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 325; and the light chain variable region comprises seq id NO: 190.

7. The isolated immunoglobulin of claim 1 or claim 2, wherein the isolated immunoglobulin comprises an antibody or antibody fragment.

8. The isolated immunoglobulin of any one of claims 1-7, comprising IgG1, IgG2, IgG3, or IgG 4.

9. The isolated immunoglobulin of any of claims 1-8, comprising a monoclonal antibody.

10. The isolated immunoglobulin of any one of claims 1-9, comprising a human antibody.

11. The isolated immunoglobulin of claim 10, comprising:

(a) comprises the amino acid sequence of SEQ ID NO: 113, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; and a polypeptide comprising SEQ ID NO: 110, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; or

(b) Comprises the amino acid sequence of SEQ ID NO: 125, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 122, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; or

(c) Comprises the amino acid sequence of SEQ ID NO: 101, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 98, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; or

(d) Comprises the amino acid sequence of SEQ ID NO: 119, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus or both; and a polypeptide comprising SEQ ID NO: 116, or an immunoglobulin light chain comprising the aforementioned sequence lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both.

12. The isolated immunoglobulin of any of claims 1-11, further comprising 1-24 pharmacologically active chemical moieties conjugated thereto.

13. The isolated immunoglobulin of any of claims 1-12, wherein the pharmacologically active chemical moiety is a pharmacologically active polypeptide.

14. The isolated immunoglobulin of any one of claims 1-13, wherein the immunoglobulin is recombinantly produced.

15. The isolated immunoglobulin of claim 14, wherein the immunoglobulin comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is inserted into the primary amino acid sequence of the immunoglobulin heavy chain within an internal loop of the immunoglobulin heavy chain Fc domain.

16. The isolated immunoglobulin of claim 13 or 14, wherein the immunoglobulin comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is conjugated at the N-terminus or C-terminus of the immunoglobulin heavy chain.

17. The isolated immunoglobulin of claim 13 or 14, wherein the immunoglobulin comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is conjugated at the N-terminus or C-terminus of the immunoglobulin light chain.

18. The isolated immunoglobulin of claim 13 or 14, wherein the pharmacologically active polypeptide is a toxin peptide, an IL-6 binding peptide, a CGRP peptide antagonist, a bradykinin B1 receptor peptide antagonist, a PTH agonist peptide, a PTH antagonist peptide, an ang-1 binding peptide, an ang-2 binding peptide, a myostatin binding peptide, an EPO-mimetic peptide, an FGF21 peptide, a TPO-mimetic peptide, an NGF binding peptide, a BAFF antagonist peptide, GLP-1 or a peptidomimetic thereof, or GLP-2 or a peptidomimetic thereof.

19. The isolated immunoglobulin of claim 18, wherein the toxin peptide is ShK or an ShK peptide analog.

20. A pharmaceutical composition comprising an immunoglobulin according to any one of claims 1 to 19; and a pharmaceutically acceptable diluent, excipient or carrier.

21. An isolated nucleic acid encoding the immunoglobulin of any one of claims 1-11.

22. An isolated nucleic acid encoding the immunoglobulin of claim 3.

23. An isolated nucleic acid encoding the immunoglobulin of claim 4.

24. An isolated nucleic acid encoding the immunoglobulin of claim 5.

25. An isolated nucleic acid encoding the immunoglobulin of claim 6.

26. An isolated nucleic acid encoding the immunoglobulin of claim 11.

27. An isolated nucleic acid encoding the immunoglobulin of any one of claims 13-19.

28. A vector comprising the isolated nucleic acid of claim 21.

29. A vector comprising the isolated nucleic acid of any one of claims 22 to 26.

30. A vector comprising the isolated nucleic acid of claim 27.

31. The vector of claim 28, comprising an expression vector.

32. The vector of claim 29, comprising an expression vector.

33. The vector of claim 30, comprising an expression vector.

34. An isolated host cell comprising the expression vector of any one of claims 31-33.

35. A method, comprising:

(a) culturing the host cell of claim 34 in a culture medium under conditions that allow expression of the immunoglobulin encoded by the expression vector; and

(b) recovering the immunoglobulin from the culture medium.

36. The immunoglobulin of claim 1, wherein 30 micromolar of the immunoglobulin does not significantly bind 30 nanomolar of soluble human IL-17R (SEQ ID NO: 89) in an aqueous solution incubated under physiological conditions as determined by a surface plasmon resonance binding assay.

37. The immunoglobulin of claim 2, wherein 10 micromolar of the immunoglobulin does not significantly bind 10 nanomolar of soluble human TR2(SEQ ID NO: 82) in an aqueous solution incubated under physiological conditions as determined by a surface plasmon resonance binding assay.

The invention is illustrated by the following further examples, which are not intended to be limiting in any way.

Examples

Example 1

Production and screening of antibodies to human IL-17R

Cloning and engineeringAntibody 16429DNA sequences encoding the immunoglobulin heavy chain (comprising VH1) and light chain (comprising VL1) subunits of anti-huIL-17R antibodies were obtained from tokker et al (WO2008/054603a2) and cloned using standard recombinant techniques. To eliminate the binding capacity of these antibodies, Polymerase Chain Reaction (PCR) amplification was used to generate a series of site-directed mutagenic clones. The amino acids to be altered are selected based on position in the Complementarity Determining Regions (CDRs), changes from germline sequences, estimated solvent exposure, and aromatic and charge properties. The initial set of mutants were germline and alanine scanning mutants. Subsequently, the mutations were pooled and in some cases alanine scanning mutants were mutated to introduce a negative charge by replacing alanine with glutamic acid or to introduce a positive charge by replacing alanine with arginine.

A representative example of a PCR site-directed mutagenesis procedure is the introduction of alanine in place of the tryptophan of CDR3 of the light chain of anti-IL 17.

PCR amplification was performed as a 3-step process, with 5 'and 3' PCR for introduction of mutations and final overlap PCR for ligation of both ends of the mutated anti-IL 17R light chain. 5 ' PCR used forward primer 5'-AAG CTC GAG GTC GAC TAG ACC ACC ATG GAAGCC CCA GCG CAG-3' (SEQ ID NO: 31) and reverse primer 5'-GAA AGTGAG CGG AGC GTT ATC ATA CTG CTG ACA-3' (SEQ ID NO: 32). 3 ' PCR used forward primer 5'-TGT CAG CAG TAT GAT AAC GCT CCG CTCACT TTC-3' (SEQ ID NO: 33) and reverse primer 5'-AAC CGT TTAAAC GCGGCC GCT CAA CAC TCT CCC CTG TTG AA-3' (SEQ ID NO: 34). Overlapping PCR used forward primer 5'-AAG CTC GAG GTC GAC TAG ACC ACCATG GAA GCC CCA GCG CAG-3' (SEQ ID NO: 31) and reverse primer 5'-AAC CGT TTA AAC GCG GCC GCT CAA CAC TCT CCC CTGTTG AA-3' (SEQ ID NO: 34).

PCR was performed using Phusion HF DNA polymerase (Finnzyme). PCR reaction cycles for 5 'and 3' PCR consisted of denaturation of anti-IL-17R light chain DNA at 94 ℃ for 20 seconds, followed by 3 amplification cycles plus an additional 27 cycles, each amplification cycle consisting of 94 ℃ for 20 seconds; 30 seconds at 55 ℃; and 72 ℃ for 30 seconds, and 27 additional cycles each consisting of 94 ℃ for 20 seconds; 30 seconds at 60 ℃; and 30 seconds at 72 ℃. The reaction was then incubated at 72 ℃ for 7 minutes after the last PCR cycle to ensure complete extension. The PCR reaction cycle of overlapping PCRs consisted of denaturing the 5 'and 3' PCR DNA at 94 ℃ for 20 seconds, followed by 3 amplification cycles plus an additional 27 cycles, each amplification cycle consisting of 94 ℃ for 20 seconds; 60 seconds at 55 ℃; and 72 ℃ for 40 seconds, and 27 additional cycles each consisting of 94 ℃ for 20 seconds; 30 seconds at 60 ℃; and 72 ℃ for 40 seconds. The reaction was then incubated at 72 ℃ for 7 minutes after the last PCR cycle to ensure complete extension. The overlapping PCR products were cloned into the pTT5 expression vector (NRCC) and their sequences determined using an ABI DNA sequencing instrument (PerkinElmer). Further details on the development of constructs are found in example 5 and example 6 herein. Table 6 (below) contains details regarding the primers and templates used to clone the constituent subunits of various embodiments of the immunoglobulins and conjugates of the present invention according to the same PCR cycle conditions described in this paragraph.

Transient expression for production of recombinant monoclonal antibodies

Transient transfection was performed in HEK293-6E cells as follows. From the National Research Council (Mon)treal, Canada) obtained a human embryonic kidney 293 cell line (293-6E cells) stably expressing Epstein Barr virus nuclear antigen-1. Cells were maintained as serum-free suspension cultures using F17 medium (Invitrogen, Carlsbad, CA) supplemented with 6mM L-glutamine (Invitrogen, Carlsbad, CA), 1.1% F-68Pluronic (Invitrogen, Carlsbad, CA) and 250 μ g/ul Geneticin (Geneticin) (Invitrogen, Carlsbad, CA). Suspension cell cultures were maintained in Erlenmeyer flask cultures. In moist 5% CO₂The flask was shaken under an atmosphere at 37 ℃ and 65 rpm. Stock solutions (1mg/ml) of 25-kDa linear PEI (Polysciences, Warrington, Pa.) were prepared with water, acidified to pH2.0 with HCl until dissolved, then neutralized with NaOH, sterilized by filtration (0.2 μm), aliquoted, and stored at-20 ℃ until needed. Tryptone N1 was obtained from OrganoTechni s.a. (tekni science, QC, Canada). Stock solutions (20%, w/v) were prepared in Freestyle medium (Invitrogem, Carlsbad, CA), filter sterilized through a 0.2 μm filter, and stored at 4 ℃ until use. Transfection is usually performed on the 1L scale. Cells (293-6E) were cultured to a viable cell density of 1.1X10 ⁶Individual cells/ml, then transfection complexes were prepared at 1/10th volume of the final culture volume. For the 1-L transfection culture, transfection complexes were prepared in 100ml F17 basal medium, and 500. mu.g of plasmid DNA (heavy and light chain DNA, 1: 1 ratio) was first diluted with 100ml F17 medium. After 5 minutes incubation at room temperature, 1.5ml of PEI solution was added. The complex was gently vortexed and then incubated at room temperature for 15 minutes. Cells were transfected by adding the transfection complex mixture to cells in shake flask culture. 24 hours after transfection, tryptone N1 was added to the transfected culture to a final concentration of 0.5%, and the transfected culture was incubated in a humidified 5% CO₂The atmosphere was kept on a shaker at 37 ℃ and 65rpm for an additional 5 days, after which the culture was harvested. Conditioned media was harvested by centrifugation at 4000rpm and then sterile filtered through a 0.2 μm filter (Corning Inc.).

Purification of antibodiesUsing recombinant protein A Sepharose (GE Healthcare), the conditions were adjusted at 7 ℃ and 5 ml/minThe culture medium was applied directly to the column to purify the transiently expressed antibody. The column was then washed with 10 column volumes of Dulbecco's PBS containing no divalent cations and then eluted with 100mM acetic acid (pH 3.5). Eluted antibody was collected based on the chromatogram and pH was adjusted to 5.0 using 2M Tris base. The pooled antibodies were then filtered through a 0.8/0.22 μm syringe filter and then dialyzed against 10mM acetic acid, 9% sucrose (pH 5.0). The buffer exchanged antibody was then concentrated using a Vivaspin30kDa centrifugal concentrator (Sartorius) and the concentrated product was filtered through a 0.22 μm cellulose acetate filter.

Binding assaysThen select lead candidates based on lack of binding to the IL-17R extracellular domain as determined by BIAcore analysis. Antibody 16429 is a human antibody that specifically binds huIL-17R. A solution equilibrium binding assay was developed to evaluate the binding activity of a panel of antibodies to huIL-17R. Antibody 16429 was immobilized to BIACore, inc., Piscataway, NJ according to the manufacturer's instructions (BIACore, inc.)2000, the research level sensor chip CM5 surface. Briefly, carboxyl groups on the sensor chip surface were activated by injecting 60 μ L of a mixture containing 0.2M N-ethyl-N' - (dimethylaminopropyl) carbodiimide (EDC) and 0.05M N-hydroxysuccinimide (NHS). Antibody 16429 was diluted with 10mM sodium acetate (pH4.0) and injected onto the activated chip surface at 30. mu.L/min over 6 min. Excess reactive groups on the surface were deactivated by injection of 60 μ L of 1M ethanolamine. The final immobilization level was approximately 6600 Resonance Units (RU). As shown in FIG. 2A, IL-17R at 10nM was used in the absence of soluble antibody to establish a 100% binding signal of IL-17R to the immobilized 16429 antibody. To determine antibody binding in solution, antibody samples of 10nM, 100nM and 1000nM were incubated with 10nM IL-17R. Reduced IL-17R binding signal after antibody incubation indicates that the antibody is bound to Binding of IL-17R. Based on this assay, the 16435, 16438, 16439, 16440, 16441 and 16444 antibodies showed a significant reduction in binding capacity for IL-17R. As shown in FIG. 2B, the 30nM IL-17R and 30 μ M antibody samples were used to further show that the selected antibodies lost their IL-17R binding activity. Based on this assay, all 6 antibodies examined (16435, 16438, 16439, 16440, 16441 and 16444) showed no significant IL-17R binding activity up to 30 μ M antibody.

Cell-based activity assaysThe interaction of IL-17 with IL-17R on cells induces the production of a variety of factors by these cells, including growth-associated oncogene alpha (GRO-alpha). A cell-based characterization assay was developed to measure the released GRO-alpha using a sandwich ELISA. In this ELISA, GRO- α capture antibody is used to bind GRO- α and then biotinylated GRO- α detection antibody is used to detect the captured protein. Streptavidin conjugated to horseradish peroxidase (HR) was then added to detect the amount of bound biotinylated GRO- α detection antibody. The amount of bound HR was measured by absorbance evaluation at 450 nm. An increase in absorbance at 450nm indicates an increase in the amount of GRO- α produced. In this assay, Human Foreskin Fibroblasts (HFFs) were incubated with 5ng/ml IL-17 and 0.1. mu.M, 1. mu.M and 10. mu.M antibody samples. Conditioned cell culture media were then harvested and evaluated for GRO- α levels using a GRO- α sandwich ELISA. In this assay, all 6 experimental carrier antibodies (16435, 16438, 16439, 16440, 16441 and 16444) showed no significant blocking activity up to 10 μ M antibody condition (fig. 3).

Homogeneity analysisHomogeneity of the antibody produced by transient expression was analyzed using two size exclusion columns in series (TSK-GELG3000SWXL, 5mM size, 7.8x300mM, TosohBioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (ph6.8) flowing at 0.5 mL/min (fig. 4A-B). While all antibodies showed relatively low levels of high molecular weight species, 16439 and 16435 had the least and 16440 the most. Lead antibodies were further analyzed on 1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) using reducing (FIG. 6) and non-reducing loading buffer (FIG. 5)Product mass of the body. All candidates appeared very similar by both non-reducing and reducing SDS-PAGE; 16433, however, did show some additional high molecular mass material on reduced SDS-PAGE. The lead candidates were further selected based on SEC behavior, SDS-PAGE homogeneity, BIAcore binding analysis, cell-based assay results, and expression levels. Based on these criteria, 16435 and 16444 were selected for further evaluation.

Stable expression of antibodiesAntibody 16435 and 16444 expression libraries were established by transfecting CHO DHFR (-) host cells with the corresponding HC and LC expression plasmid sets using standard electroporation procedures. For each antibody molecule, 3-4 different transfections were performed to generate multiple pools. Following transfection, cells were cultured as a pool in serum-free, (-) GHT selective growth medium to allow selection and recovery of plasmid-containing cells. Cell banks cultured in (-) GHT selective medium were cultured until they reached > 85% viability. The selected cell pool was expanded with 150nm MTX. When the viability of the MTX expanded pools reached > 85% viability, the pools were screened to assess expression using a reduced 6 day batch preparative assay with enriched preparative media. The best pool was selected based on the 6 day assay titer and appropriate quality confirmation. Subsequently, an 11-day fed-batch method was used for scale-up preparation for antibody production, followed by harvesting and purification.

Titers were determined by HPLC assay (fig. 7A-B) and using Poros a column, 20 μm, 2.1x30mm (Applied Biosystems, part # 1-5024-12). Briefly, antibodies in conditioned media were filtered using Spin-X columns (Corning, part #8160) and then analyzed by HPLC, and a blank injection of 1XPBS (Invitrogen part # 14190-. In addition, conditioned media without antibody was injected prior to analysis to condition the column, and new columns were conditioned by injecting 100 μ g of control antibody in triplicate. After washing with PBS for 9 minutes at 0.6 ml/min, the antibodies were eluted with ImmunoPure IgG elution buffer (Pierce, part #21009) and observed for absorbance at 280 nm. Antibody titers were quantified according to a standard curve of control antibody concentration versus peak area. Control antibody stocks were prepared at a concentration of 4mg/ml, and then 5 standard antibody concentrations (0.1 μ g/μ l to 1.6 μ g/μ l) were prepared by diluting the antibody control stocks with a volume of PBS. By extending the standard curve, the lower limit of detection was 0.02. mu.g/. mu.l antibody, while the upper limit of quantification was 4. mu.g/. mu.l. The test antibody is assumed to have similar absorbance properties as the control; the titer can be adjusted, however, by multiplying the titer by the ratio of the extinction coefficient of the control antibody to the extinction coefficient of the test antibody. The titer assay results showed that after scaling up to the fed-batch method, the 16435 antibody showed slightly better expression than the 16444 carrier antibody.

Purification of stably expressed antibodiesStably expressed antibodies were purified by Mab Select Sure chromatography (GE life sciences) and using 8 column volumes of divalent cation free Dulbecco' sPBS as wash buffer and 100mM acetic acid (ph3.5) as elution buffer at 7 ℃. Elution peaks were collected based on the chromatogram and the pH was raised to about 5.0 using 2M Tris base. The eluted pool was then diluted with at least 3 volumes of water at 7 ℃, filtered through a 0.22- μ M cellulose acetate filter, loaded onto an SP-HP sepharose column (GE Life Sciences) and washed with 10 column volumes of S-Buffer a (S-Buffer a) (20mM acetic acid, ph5.0) and then eluted using a gradient of 20 column volumes to 50% S-Buffer B (S-Buffer B) (20mM acetic acid, 1M NaCl, ph 5.0). The collection was performed based on chromatogram and SDS-PAGE analysis, and then the material was concentrated about 6-fold using VivaFlow TFF cassette with 30kDa membrane and diafiltered with about 5 volumes of 10mM acetic acid, 9% sucrose (ph 5.0). The dialyzed material was then filtered through a 0.8/0.2- μm cellulose acetate filter and the concentration was determined by absorbance at 280 nm. Comparison of the ion exchange chromatograms of the 16435 and 16444 variants showed no significant differences (fig. 8A-B).

Analysis of stably expressed antibodiesAnalysis of variants using 1.0-mm Tris-glycine 4-20% SDS-page (novex) and reducing and non-reducing loading buffer also showed no significant differences between the variants (fig. 9A-B). However, two size exclusion columns in series (TSK-GEL G3000SWXL, 5mm size, 7.8X300mm, T)osoh bioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (ph6.8) flowing at 0.5 mL/min showed: 16444 has more high molecular weight species, while 16435 has a more prominent pre-peak (FIG. 10).

Antibodies were also analyzed for heat resistance by DSC using MicroCal VP-DSC, in which the sample was heated from 20 ℃ to 95 ℃ at a rate of 1 ℃ per minute. DSC directly measures the thermal changes that occur in biomolecules during controlled increases or decreases in temperature, thereby making it possible to study materials in their native state.

DSC measures the enthalpy of unfolding (Δ H) due to thermal denaturation. Biomolecules in solution are in equilibrium between their native (folded) conformation and their denatured (unfolded) state. The higher the thermal transition midpoint (Tm) at which 50% of the biomolecules are unfolded, the more stable the molecule. DSC was also used to determine the heat capacity for denaturation (Δ Cp) change (see fig. 11). The protein was incubated at 0.5mg/ml in 10mM sodium acetate, 9% sucrose (pH5.0) (FIG. 11). 16435 the antibody produces the most desirable melting profile with higher temperatures for the secondary transition.

Antibodies were analyzed by reduced and non-reduced CE-SDS (FIGS. 12A-D). All CE SDS experiments were performed using a Beckman PA800CE system (Fullerton, CA) equipped with UV diode detectors using 221nm and 220nm wavelengths. Separation analysis was performed using bare fused silica capillary 50 μm x 30.2cm. Buffer vial preparation and loading and capillary cassette mounting were performed as described in the Beckman Coulter manual for IgG purity/heterogeneity. The operating conditions for reduced and non-reduced CE-SDS were similar to those described in the Beckman Coulter manual for IgG purity/heterogeneity, with some modifications as outlined below. For non-reducing conditions, antibody samples (150. mu.g) were added to 20. mu.l of SDS reaction buffer and 5. mu.l of 70mM N-ethylmaleimide. Water was then added to give a final volume of 35. mu.l and to bring the protein concentration to 4.3 mg/ml. The SDS reaction buffer consisted of 4% SDS, 0.01M citrate phosphate buffer (Sigma), and 0.036M disodium phosphate. The prepared solution was vortexed thoroughly and heated at 45 ℃ for 5 minutes. Then will beThe prepared solution was mixed with another 115. mu.l of 4% SDS. After vortexing and centrifugation, the prepared solution was placed in a 200 μ L PCR vial and then loaded onto a PA800 instrument. The sample was injected at the anode with reverse polarity for 30 seconds using-10 kV and then the separation was performed at-15 kV with 20psi pressure across the capillary during 35 minutes of separation. For the reducing conditions, purification of H by addition ₂O antibody samples were diluted to 2.1mg/ml and 95. mu.l of antibody was added to 105. mu.L of SDS sample buffer (Beckman) containing 5.6% beta-mercaptoethanol. The prepared solution was then vortexed thoroughly and then heated at 70 ℃ for 10 minutes. After centrifugation, the supernatant was placed in a 200 μ Ι pcr vial and then loaded onto a PA800 instrument. The sample was injected at the anode with reverse polarity for 20 seconds using-5 kV and then the separation was performed at-15 kV with 20psi pressure across the capillary during 30 minutes of separation. Very similar plots were generated with both reduced and non-reduced CE-SDS, 16435 and 16444 antibodies (fig. 12A-D).

To measure the photosensitivity of the antibodies, they were incubated at room temperature under ambient laboratory fluorescent lighting or coated in aluminum foil for 3 days. The light exposed and dark control antibodies were then analyzed using two size exclusion columns in series (TSK-GEL G3000SWXL, 5mM size, 7.8X300mM, Tosohbioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (pH6.8) flowing at 0.5 mL/min. 16444 showed significantly stronger light sensitivity than 16435 based on SEC chromatography (FIG. 13). The antibodies were then analyzed by Hydrophobic Interaction Chromatography (HIC) using two Dionex ProPac HIC-10 columns in tandem, with mobile phase A being 1M ammonium sulfate, 20mM sodium acetate (pH5.0) and mobile phase B being 20mM sodium acetate, 5% acetonitrile (pH 5.0). The sample was eluted with a 0-100% linear gradient at 0.8 ml/min over 50 min while observing absorbance at 220 nm. 16435 had a narrower main peak based on HIC chromatography, indicating better product homogeneity (fig. 14). 16435 was selected as the major lead of this family of antibodies based on lower light sensitivity, better purification yield (1219mg/L vs 1008mg/L), better DSC profile, better SEC profile, and fewer mutations from the parent antibody.

TABLE 6 PCR primer sets and templates for cloning the indicated products.

Example 2

Production and screening of antibodies to human TRAIL R2

Cloning and engineeringAntibody 16449DNA sequences encoding the immunoglobulin heavy chain (comprising VH12) and light chain (comprising VL6) subunits of anti-huTR 2 antibody were obtained from Gliniak et al (U.S. patent No. 7,521,048) and cloned using standard recombinant techniques. To eliminate the binding capacity of these antibodies, Polymerase Chain Reaction (PCR) amplification was used to generate a series of site-directed mutagenic clones. The amino acids to be altered are selected based on position in the Complementarity Determining Regions (CDRs), changes from germline sequences, estimated solvent exposure, and aromatic and charge properties. The initial set of mutants were germline and alanine scanning mutants. Subsequently, the mutations were pooled and in some cases alanine scanning mutants were mutated to introduce a negative charge by replacing alanine with glutamic acid or to introduce a positive charge by replacing alanine with arginine. Further details on the development of constructs are found in example 5 and table 6 herein.

Transient expression for production of recombinant monoclonal antibodiesTransient transfection was performed in HEK293-6E cells as follows. Human embryonic kidney 293 cell lines (293-6E cells) stably expressing the nuclear antigen-1 of the Epstein Barr virus were obtained from the National research Committee (National research Council, Montreal, Canada). Supplemented with 6mM L-glutamine (Invitrogen, Carlsbad, Calif.), 1.1% F-68Pluronic (Invitrogen) Carlsbad, CA) and 250 μ g/ul geneticin (Invitrogen, Carlsbad, CA) were maintained in serum-free suspension culture in F17 medium (Invitrogen, Carlsbad, CA). Suspension cell cultures were maintained in Erlenmeyer flask cultures. In moist 5% CO₂The flask was shaken under an atmosphere at 37 ℃ and 65 rpm. Stock solutions (1mg/ml) of 25-kDa linear PEI (Polysciences, Warrington, Pa.) were prepared with water, acidified to pH2.0 with HCl until dissolved, then neutralized with NaOH, sterilized by filtration (0.2 μm), aliquoted, and stored at-20 ℃ until needed. Tryptone N1 was obtained from OrganoTechni s.a. (tekni science, QC, Canada). Stock solutions (20%, w/v) were prepared in Freestyle medium (Invitrogem, Carlsbad, CA), filter sterilized through a 0.2 μm filter, and stored at 4 ℃ until use. Transfection is usually performed on the 1L scale. Cells (293-6E) were cultured to a viable cell density of 1.1X10⁶Individual cells/ml, then transfection complexes were prepared at 1/10th volume of the final culture volume. For the 1-L transfection culture, transfection complexes were prepared in 100ml F17 basal medium, and 500. mu.g of plasmid DNA (heavy and light chain DNA, 1: 1 ratio) was first diluted with 100ml F17 medium. After 5 minutes incubation at room temperature, 1.5ml of PEI solution was added. The complex was gently vortexed and then incubated at room temperature for 15 minutes. Cells were transfected by adding the transfection complex mixture to cells in shake flask culture. 24 hours after transfection, tryptone N1 was added to the transfected culture to a final concentration of 0.5%, and the transfected culture was incubated in a humidified 5% CO ₂The atmosphere was kept on a shaker at 37 ℃ and 65rpm for an additional 5 days, after which the culture was harvested. Conditioned media was harvested by centrifugation at 4000rpm and then sterile filtered through a 0.2 μm filter (Corning Inc.).

Purification of antibodiesThe transiently expressed antibody was purified using recombinant protein a sepharose (GE Healthcare) and the conditioned media was loaded directly onto the column at 5 ml/min at 7 ℃. The column was then washed with 10 column volumes of Dulbecco's PBS containing no divalent cations and then eluted with 100mM acetic acid (pH 3.5). Based on chromatogramsThe eluted antibody was collected and the pH was adjusted to 5.0 using 2M tris base. The pooled antibodies were then filtered through a 0.8/0.22 μm syringe filter and then dialyzed against 10mM acetic acid, 9% sucrose (pH 5.0). The buffer exchanged antibody was then concentrated using a Vivaspin30kDa centrifugal concentrator (Sartorius) and the concentrated product was filtered through a 0.22 μm cellulose acetate filter.

BIAcore binding assayAntibody 16449 is a human antibody that specifically binds to Trail receptor 2(TR 2). A solution equilibrium binding assay was developed to assess the binding activity of a panel of antibodies to TR 2. Antibody 16449 was immobilized to CM5 sensor chip surface as described in example 1 above. The final immobilization level was approximately 8000 Resonance Units (RU). TR2(1nM) was used in the absence of antibody to establish a 100% TR2 binding signal without antibody binding in solution. To determine antibody binding in solution, serial dilutions of antibody samples in the range of 7pM to 10nM were incubated with 1nM TR 2. The reduced TR2 binding signal after antibody incubation indicates binding of the antibody to TR2 in solution. The results in fig. 15 indicate that all 3 new antibody constructs (16449, 1869, and 1870) retained TR2 binding activity similar to the original construct.

In other experiments, 10nM TR2 was incubated with 50nM and 1 μ M antibody samples in the assay as described above. 10nM TR2 was used to define the 100% binding signal. While several antibodies showed significant lack of binding at 50nM (16613, 1919, 1913, 1910, 1920 and 1922), none showed complete lack of binding at 1000nM (results are shown in figure 16). Additional point mutagenesis resulted in an antibody with lower affinity for TR2 (fig. 17). The two sites (heavy chain Y125 and light chain Y53) were shown to be sensitive to mutagenic abnormalities, particularly with charged substitutions at position Y125. The bialanine substitution resulted in variants with even further reduced binding affinity for TR2 (fig. 18). Combining alanine mutations with charged mutations in pairs, or a larger progression, resulted in several molecules that did not show significant binding to TR2 even at 10 μ M antibody (fig. 19). Based on these data, binding studies were further performed on 5 of the best variants (10186, 10184, 4341, 10183 and 4241) under 50 μ M antibody (fig. 20A-B). All variants except 10186 showed no significant binding to TR2 even at 50 μ M.

Cell-based activity assaysColo205 is a human colon cancer cell line sensitive to the presence of TRAIL. Binding of the positive control IgG1 anti-TR 2mAb molecule (antibody 16449) on the surface of Colo205 to TR2 resulted in apoptosis. A Colo 205-based cell assay was developed to verify the cell killing efficacy of the antibodies. The in vitro biological activity of antibody 16449 (anti-TRAIL-R2 antibody) was analyzed by its ability to induce apoptosis in the human ascites colorectal adenocarcinoma cell line Colo 205. According to the manufacturer's instructions, Apo-One is used ^TMHomogeneous caspase^TMThe-3/7 assay kit (Promega corporation, Madison, Wis.) detects caspase-3 activation, which is used as a positive marker for apoptosis. (see Niles et al, the apo-One^TM.Homogeneous Caspase^TM-3/7 assoy: a modulated "solution" for apoptosis detection, Cell Notes 2: 2-3(2001)). In this method, the luminescent caspase-3/7 reagent provides a sensitive and robust monitoring of anti-TRAIL-R2 induced caspase activation in Colo205 cells. The luminescence produced is proportional to the amount of caspase activity present. The luminescence of each sample was measured in a plate reader. The biological activity of the test sample is determined by comparing the test sample response to a reference standard response. To compare the samples to standard control antibodies, 200nM and 10. mu.M antibody samples were preincubated with 1 or 100. mu.g/ml protein G. This mixture was then added to the Colo205 culture. Figure 20C indicates that, unlike the control anti-TR 2mAb molecule, the antibody sample did not have the ability to kill cells even at very high concentrations (e.g., 30 μ g/mL of antibody).

Homogeneity analysisProduct quality of the lead antibody was analyzed on 1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) using reducing (FIG. 21B) and non-reducing loading buffer (FIG. 21A). All candidates appeared very similar by both non-reducing and reducing SDS-PAGE. Using 1 size exclusion column (Phenomenex SEC3000, 7.8X300mm) with The homogeneity of the antibodies was further analyzed with a mobile phase of 50mM sodium phosphate, 250mM NaCl (pH6.8) flowing at 1.0 mL/min (representative results are shown in FIG. 22). While all antibodies showed relatively low levels of high molecular weight species, 10185 and 10184 showed slightly more high molecular weight material. Lead candidates were selected based on SEC behavior, BIAcore binding analysis, cell-based assay results, estimated proteolysis susceptibility, and lower shift in calculated isoelectric point. Based on these criteria, 4241 and 4341 were selected for further evaluation.

Construct development for stable expressionStably expressed antibody libraries 4241 and 4341 were established by transfecting CHO DHFR (-) host cells with the corresponding HC and LC expression plasmid sets using standard electroporation procedures. For each antibody molecule, 3-4 different transfections were performed to generate multiple pools. After transfection, cells were cultured as a pool in serum-free (-) GHT selective growth medium to allow selection and recovery of plasmid-containing cells. Cell banks cultured in (-) GHT selective medium were cultured until they reached > 85% viability. The selected cell pool was amplified with 150nm Methotrexate (MTX). When the viability of the MTX expanded pools reached > 85% viability, the pools were screened to assess expression using a reduced 6 day batch preparative assay with enriched preparative media. The best pool was selected based on the 6 day assay titer and appropriate quality confirmation. Subsequently, an 11-day fed-batch method was used for scale-up preparation for antibody production, followed by harvesting and purification.

Titers were determined by HPLC using a Poros A column, 20 μm, 2.1X30mm (applied biosystems, part # 1-5024-12). Briefly, antibodies in conditioned media were filtered using Spin-X columns (Corning, part #8160) and then analyzed by HPLC, and a blank injection of 1X PBS (Invitrogen, part # 14190-. In addition, conditioned media without antibody was injected prior to analysis to condition the column, and new columns were conditioned by injecting 100 μ g of control antibody in triplicate. After washing with PBS for 9 minutes at 0.6 ml/min, the antibodies were eluted with ImmunoPure IgG elution buffer (Pierce, part #21009) and the absorbance at 280nm was measured.

Antibody titers were quantified according to a standard curve of control antibody concentration versus peak area. Control antibody stocks were prepared at a concentration of 4mg/ml, and 5 standard antibody concentrations (0.1 μ g/μ l to 1.6 μ g/μ l) were prepared by diluting the antibody control stock with a volume of PBS. By extending the standard curve, the lower limit of detection was 0.02. mu.g/. mu.l antibody, while the upper limit of quantification was 4. mu.g/. mu.l. The test antibody is assumed to have similar absorbance properties as the control; the titer can be adjusted, however, by multiplying the titer by the ratio of the extinction coefficient of the control antibody to the extinction coefficient of the test antibody. The titer assay results showed that 4241 antibody showed slightly better expression than 4341 antibody after scaling up to the fed-batch method (fig. 23A-B).

Purification of stably expressed antibodiesStably expressed antibodies were purified by Mab Select Sure chromatography (GE life sciences) and using 8 column volumes of divalent cation free Dulbecco' sPBS as wash buffer and 100mM acetic acid (ph3.5) as elution buffer at 7 ℃. Elution peaks were collected based on the chromatogram and the pH was raised to about 5.0 using 2M Tris base. The eluted pool was then diluted with at least 3 volumes of water at 7 ℃, filtered through a 0.22- μ M cellulose acetate filter, and then loaded onto an SP-HP sepharose column (GE Life Sciences) and washed with 10 column volumes of S-buffer a (20mM acetic acid, ph5.0), then eluted to 50% S-buffer B (20mM acetic acid, 1M NaCl, ph5.0) using a gradient of 20 column volumes. The collection was performed based on chromatogram and SDS-PAGE analysis, and then the material was concentrated about 6-fold using VivaFlow TFF cassette with 30kDa membrane and diafiltered with about 5 volumes of 10mM acetic acid, 9% sucrose (ph 5.0). The dialyzed material was then filtered through a 0.8/0.2- μm cellulose acetate filter and the concentration was determined by absorbance at 280 nm. The purified samples were analyzed using 1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) reducing loading buffer (FIGS. 24A-B). These data show that 4241 and 4341 antibodies have similar purification properties, with no steps resulting in unexpected sample loss.

Analysis of stably expressed antibodiesComparing the ion exchange chromatograms of the 4241 and 4341 variants (fig. 25) shows 4341 with a narrower main peak, indicating less heterogeneity than 4241. Analysis of the variants using 1.0-mm Tris-glycine 4-20% SDS-page (novex) and reducing and non-reducing loading buffer showed no significant differences between the variants (fig. 26A-B). Analysis using two size exclusion columns in series (TSK-GEL G3000SWXL, 5mM particle size, 7.8x300mM, TosohBioscience, 08541) with a mobile phase of 100mM sodium phosphate, 250mM NaCl (ph6.8) flowing at 0.5 mL/min also showed no significant difference between the 4241 and 4341 variants (fig. 27A-B). Antibody heat resistance was analyzed by DSC using MicroCal VP-DSC, where the sample was heated from 20 ℃ to 95 ℃ at a rate of 1 ℃ per minute. The protein was dissolved at a concentration of 0.5mg/ml in 10mM sodium acetate, 9% sucrose (pH5.0) (FIG. 28). 4241 antibody produced the most ideal melting profile, with a higher temperature for the secondary transition compared to antibody 4341.

4241 and 4341 antibodies were analyzed by reduced and non-reduced CE-SDS (FIGS. 29A-D). All CE SDS experiments were performed using a Beckman PA800CE system (Fullerton, CA) equipped with UV diode detectors using 221nm and 220nm wavelengths. Separation analysis was performed using bare fused silica capillary 50 μm × 30.2 em. Buffer vial preparation and loading and capillary cassette mounting were performed as described in the Beckman Coulter manual for IgG purity/heterogeneity. The operating conditions for reduced and non-reduced CE-SDS were similar to those described in the Beckman Coulter manual for IgG purity/heterogeneity, with some modifications as outlined below. For non-reducing conditions, antibody samples (150. mu.g) were added to 20. mu.l of SDS reaction buffer and 5. mu.l of 70mM N-ethylmaleimide. Water was then added to give a final volume of 35. mu.l and to bring the protein concentration to 4.3 mg/ml. The SDS reaction buffer consisted of 4% SDS, 0.01M citrate phosphate buffer (Sigma), and 0.036M disodium phosphate. The prepared solution was vortexed thoroughly and then heated at 45 ℃ for 5 minutes. The prepared solution was then mixed with another 115. mu.l of 4% SDS. After vortexing and centrifugation, the prepared solution was placed in 200. mu.l PC R vial, and then loaded onto the PA800 instrument. The sample was injected at the anode with reverse polarity for 30 seconds using-10 kV and then the separation was performed at-15 kV with 20psi pressure across the capillary during 35 minutes of separation. For the reducing conditions, purification of H by addition₂O antibody samples were diluted to 2.1mg/ml and 95. mu.l of antibody was added to 105. mu.l of SDS sample buffer (Beckman) containing 5.6% beta-mercaptoethanol. The prepared solution was then vortexed thoroughly and then heated at 70 ℃ for 10 minutes. After centrifugation, the supernatant was placed in a 200 μ l PCR vial and then loaded onto a PA800 instrument. The sample was injected at the anode with reverse polarity for 20 seconds using-5 kV and then the separation was performed at-15 kV with 20psi pressure across the capillary during 30 minutes of separation. The two antibodies showed no significant difference by CE-SDS analysis (fig. 29A-D).

Antibody homogeneity was also analyzed using high performance ion exchange chromatography (SP-5PW, 10 μ M particles, 7.5mM ID. times.7.5 cm, Tosohbioscience, 08541) using 20mM acetic acid (pH5.0) as buffer A and 20mM acetic acid, 1M NaCl (pH5.0) as buffer B, flowing at 1 mL/min with an 80 min linear gradient of 0-40% buffer B. In the high performance ion exchange chromatogram using this method, the purified 4241 or 4341 antibodies showed no significant difference (fig. 30). To measure the photosensitivity of the antibodies, they were incubated at room temperature under ambient laboratory fluorescent lighting or coated in aluminum foil for 3 days. The antibodies were then analyzed by hydrophobic interaction chromatography using two Dionex ProPac HIC-10 columns in tandem, with mobile phase A being 1M ammonium sulfate, 20mM sodium acetate (pH5.0) and mobile phase B being 20mM sodium acetate, 5% acetonitrile (pH 5.0). The sample was eluted with a 0-100% linear gradient at 0.8 ml/min over 50 min while observing absorbance at 220 nm. Based on HIC chromatograms in the presence and absence of light exposure, the two antibodies displayed no significant difference (fig. 31A-B). Based primarily on the more uniform ion exchange chromatographic peaks during purification, 4341 was selected as the primary lead for this family of antibodies.

Example 3

Human tissue cross-reactivity assessment

Preliminary non-GLP studies were conducted to determine the cross-reactivity of the antibodies of the invention with various Human tissues, following generally the guidelines set forth in the Manufacture and Testing of Monoclonal antibody products for Human Use (U.S. department of Health and Human services, Food and Drug administration, Center for biology evaluation and Research (1997)). If the antibody is intended for drug development, a more comprehensive test under GLP conditions is required. Tissue cross-reactivity of antibodies 16435 and 4341 was assessed using a frozen section of selected human tissue using the AlexaFluor 488-labeled form of the test article (Charles River Laboratories, preclinical services, Reno, NV). Normal Human Tissue from two unique individuals (unless otherwise indicated) was obtained from a special pathology Services Human Tissue Bank (special pathology Services Human Tissue Bank) collected by the National disease research exchange (NDRI, philiadelphia, PA), currine, Inc. Tissues tested included human cerebellum, lung, cerebral cortex, ovary (from mature female), eye, placenta, gastrointestinal tract (small intestine), skin (1 individual), heart, spleen, kidney (1 individual), thyroid, liver, testis. Fresh frozen human tissue sections and control bead blocks (human serum albumin [ HSA ] ]Beads) were cut on a cryostat and thawed and mounted onto capillary gap slides. Tissue and control bead slides were fixed in cold acetone for approximately 10 minutes at-10 ℃ to-25 ℃. The fixed slides were allowed to dry for at least 1 hour (to overnight). If stored frozen, the fixed slides are removed from the freezer the day before the experiment and allowed to thaw overnight before use. All following steps were performed at room temperature unless otherwise indicated. Slide glass and 1X Morphosave^TMIncubate for about 15 minutes to preserve tissue morphology, then wash 2 times in 1X Phosphate Buffered Saline (PBS) for about 5 minutes each. In order to block endogenous peroxidationSubstrate enzyme, slides were incubated in glucose oxidase solution for about 1 hour at about 37 ℃. The slides were then washed 2 times in 1X PBS for approximately 5 minutes each. Endogenous biotin was blocked by sequential incubations in avidin and biotin solutions (approximately 15 minutes each). After incubation in biotin, the tissue sections were blocked with blocking antibody solution for approximately 25 minutes. Alexa Fluor488-Ab16435 and Alexa Fluor488 anti-Ab 4341 were applied to the sections at optimal concentration (2.0. mu.g/mL) or 5 times the optimal concentration (10.0. mu.g/mL) for approximately 25 minutes. The slides were washed 3 times with wash buffer and then incubated with a secondary antibody (rabbit anti-Alexa Fluor488) for approximately 25 minutes. After incubation with the secondary antibody, the slides were washed 4 times with wash buffer, then incubated with a tertiary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG antibody) for approximately 25 minutes and binding was developed with Diaminobenzidine (DAB) chromogenic substrate. HSA beads were used as negative control. Tissues proved adequate for immunohistochemistry by staining with an antibody against CD31 (anti-CD 31), platelet endothelial cell adhesion molecule (PECAM-1). At concentrations of 2.0 or 10.0 μ g/mL, there was no specific staining of any human tissue examined for any of the antibodies tested.

Example 4

Antibody embodiments of the invention Pharmacokinetic (PK) studies in rats and cynomolgus monkeys

The pharmacokinetic profile of 16435, 16444, 4241 and 4341 carrier antibodies was determined in adult Sprague-Dawley rats (8-12 weeks old) by: subcutaneously injected at 5mg/kg and collected approximately 250. mu.L of blood from the tail vein to approximately 1008 hours at 0, 0.25, 1, 4, 24, 48, 72, 96, 168, 336, 504, 672, 840 and 1008 hours post-administrationIn a serum separation tube. After collection, each sample was maintained at room temperature and after 30-40 minutes of clotting time, the samples were centrifuged at 11,500rpm for about 10 minutes at 2-8 ℃ using a calibrated Eppendorf5417R centrifuge system (Brinkmann Instruments, inc., Westbury, NY).The collected sera were then transferred to pre-labeled (for each rat) cryo-storage tubes and stored at-60 ℃ to-80 ℃ for future analysis. To measure serum sample concentrations from PK study samples, the following methods were used: an 1/2 area black plate (Corning3694) was coated with 2. mu.g/ml of anti-hu Fc, antibody 1.35.1, in PBS and then incubated overnight at 4 ℃. The plates were then washed and washed with I-Block at 4 deg.C ^TM(Applied Biosystems) was blocked overnight. If samples need to be diluted, they are diluted with rat SD control serum. The standards and samples were then tested with I-Block^TM+ 5% BSA was diluted 1: 20 to 380. mu.l of dilution buffer. The plates were washed and 50- μ l samples of the pre-treated standards and samples were transferred to the antibody 1.35.1 coated plates and incubated at room temperature for 1.5 h. The plate was washed and 50. mu.l of 100ng/ml solution in I-Block was added^TMAnti-hu Fc antibody 21.1-HRP conjugate in + 5% BSA and incubated for 1.5 h. The plates were washed and then 50. mu.l of Pico substrate was added, immediately after which the plates were analysed photometrically. The pharmacokinetic profiles were not significantly different for any of the four antibodies (fig. 32), with AUC of antibodies 16435, 16444, 4241 and 4341_0-tSD is 18,492 + -2, 104 respectively; 21,021 + -2,832; 24,045 + -2, 480 and 24,513 + -972 μ g/h/mL.

In the case of cynomolgus monkey (cynomolgus monkey)Macaca fascicularis)) 16435 to assess in vivo parameters. Briefly, a single IV bolus dose of 16435(1mg/kg or 10mg/kg) was administered to mature male cynomolgus monkeys (n ═ 2 per group). Serum samples were taken at 0.25, 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 360, 408, 456, 504, 552, 600, 648 and 672 hours before dosing and at time points after antibody administration. The 16435 antibody level of the samples was determined by using an anti-IgG sandwich ELISA. Using non-compartmental methods (enterprise version 5.1.1, 2006,corona View, CA) analyzed time concentration data. The resulting pharmacokinetic profiles did not show any significant abnormalities (fig. 33).

Example 5

Cloning, purification and analysis of antibody 16435-ShK [1-35, Q16K ] fusion

Cloning and expressionComponents of a monovalent 16435-ShK fusion (antibody 3742) include:

(a)16435κLC(SEQ ID NO：109)；

(b)16435 IgG2 HC (SEQ ID NO: 112); and

(c)16435 IgG2-ShK[1-35，Q16K](SEQ ID NO：377)：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAQLYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//SEQ ID NO：377

the desired product antibody fusion (3742) is a complete antibody in which the ShK [1-35, Q16K ] peptide (SEQ ID NO: 76) is fused to the C-terminus of one heavy chain (see schematic in FIG. 34). Since two different heavy chains share a light chain, the ratio of heavy to light to heavy-ShK [1-35, Q16K ] is 1: 2: 1. The expected expression products are 16435IgG2, monovalent 16435IgG2-ShK [1-35, Q16K ] and divalent 16435IgG2-ShK [1-35, Q16K ]. Monovalent 16435IgG2-Shk fusion protein was isolated from the mixture using cation exchange chromatography, as described herein.

The construct pTT5-aKLH120.6-IgG2-HC-L10-ShK [1-35, Q16K ] encoding (SEQ ID NO: 389) was used as a template to generate an ShK [1-35, Q16K ] fragment, which was digested with StuI and NotI and purified using a PCR purification kit (Qiagen). Meanwhile, pDC324(SEQ ID NO: 111) was digested with StuI and NotI, treated with Calf Intestinal Phosphatase (CIP) and run on a 1% agarose gel. The larger fragment was excised and gel purified by Qiagen's gel purification kit. The purified Shk [1-35, Q16K ] fragment was ligated to the large vector fragment and transformed into OneShot Top10 bacteria. DNA from the transformed bacterial colonies was isolated and submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pDC324-16435-IgG2-HC-L10-ShK [1-35, Q16K ] construct encodes an IgG2-HC-L10-ShK [1-35, Q16K ] fusion polypeptide (SEQ ID: 377).

Purification of3742 initial purification of conditioned media was performed by: the Fc region was captured using affinity FPLC of protein A Sepharose (GE Healthcare), and then the column was washed with Dulbecco's PBS (Invitrogen) without divalent cations and eluted with a 100mM acetic acid (pH3.5) step. The protein containing fractions were pooled together and neutralized to ph5.0 with 10N NaOH and diluted 5 volumes with water. The material was filtered through a 0.45 μm cellulose acetate filter (Corning) and further purified by cation exchange FPLC (SP Sepharose high Performance; GE Healthcare). The sample was applied to a column equilibrated with 100% buffer a (50mM acetic acid, ph5.0) and eluted with a gradient of 0 to 800mM NaCl over 30 column volumes. Peaks containing monovalent species were pooled together and formulated into 10mM sodium acetate, 9% sucrose (ph 5.0).

Analysis ofThe 3742 pool was analyzed by reduced and non-reduced SDS-PAGE using 4-12% tris-glycine gel (Invitrogen) with 2. mu.g of protein, stained with QuickBlue (Boston biologicals). There were no significant differences between the pools based on SDS-PAGE (fig. 35). Analytical SEC was carried out using a Biosep SEC-S3000 column (Phenomenex) using 50mM sodium phosphate, 250mM NaCl (pH6.9) as mobile phase and 1 ml/min Isocratic elution was performed (FIGS. 36A-D). Based on SEC data, all four pools showed relatively low levels of aggregation; however, pool 1 showed a slightly higher level than the other pools.

The final 3742 samples were characterized by LC-MS analysis of the reduced heavy (FIGS. 38A-D) and light chains (FIGS. 37A-D). The product was chromatographed on a Waters ACQUITY UPLC system through a Waters MassREP mini desalting column. The column was set to 80 ℃ and the protein was eluted using a linear gradient of increasing acetonitrile concentration in 0.1% formic acid. The column effluent was directed to a Waters LCT Premier ESI-TOF mass spectrometer for mass analysis. The instrument was run in forward V mode. The capillary voltage was set to 3,200V and the cone voltage to 80V. Mass spectra were collected from 800 to 3000m/z and deconvoluted using MaxEnt1 software supplied by the instrument manufacturer. All four pools gave expected masses within the error range of the instrument, indicating that all pools produced the expected product (FIGS. 37A-D and 38A-D).

Whole blood assayIn vitro assays were used to examine the effect of the toxin peptide analogue Kv1.3 inhibitor on the secretion of IL-2 and IFN-. gamma.. The efficacy of ShK analogs and conjugates in blocking T cell inflammation in human whole blood was examined using an in vitro assay that has been described previously (see example 46 of WO2008/088422a2, which is incorporated herein by reference in its entirety). Briefly, 50% of human whole blood was stimulated with thapsigargin to cause reservoir depletion, calcium mobilization, and cytokine secretion. To evaluate the efficacy of the molecules in blocking T cell cytokine secretion, various concentrations of kv1.3 blocking peptides and peptide-conjugates were pre-incubated with human whole blood samples for 30-60 minutes, followed by the addition of the thapsigargin stimulus. At 37 ℃ and 5% CO ₂After the next 48 hours, the conditioned medium was collected and the level of cytokine secretion was determined using a 4-spot electrochemiluminescence immunoassay (4-spot electrochemiluminescence immunoassay) from MesoScale Discovery. The cytokines IL-2 and IFN-g were secreted vigorously from blood isolated from multiple donors using thapsigargin stimulators. IL-2 and IFN-g produced in human whole blood following thapsigargin stimulation is produced by T cells, e.g., by intracellular cytokine staining and fluorescence activationCell Sorting (FACS) analysis. Kv1.3 is the major voltage-gated potassium channel present on T cells. Allowing K⁺Flow, Kv1.3 provides for persistent Ca²⁺Driving force of flow, permanent Ca²⁺Mobilization is required for efficient T cell activation and sustained elevation of intracellular calcium required for cytokine secretion. It has been previously shown that Kv1.3 inhibitors inhibit this calcium flow caused by TCR linkage (G.C.Koo et al, 1999, cell. Immunol.197, 99-107). Thapsigargin-induced storage depletion and TCR ligation in isolated T cells caused similar Ca²⁺Mobilization model (E.Donnadieu et al, 1991, J.biol.chem.267, 25864-25872), but we have found thapsigargin produces a more robust response in whole blood. Therefore, we used a bioassay to assess the biological activity of kv1.3 inhibitors by examining their ability to block thapsigargin-induced cytokine secretion from T cells in human whole blood. Since whole blood is a complex fluid containing high protein levels, the activity of peptides and peptide conjugates in this whole blood assay has the additional advantage of assessing molecular stability in biologically relevant fluids over 48 hours. The whole blood assay provides an important confirmation of the Kv1.3 potency of the molecule as determined by electrophysiology (ePhys), since ePhys assays typically have a short duration (< 1-2 hours) and use protein-free saline. The longer duration of the whole blood assay may allow more efficient determination of equilibrium binding kinetics relative to ePhys studies with short durations. As can be seen in Table 7A (below), all four pools of 3742-ShK (1-35, Q16K) showed good potency in the human whole blood assay, indicating that the isolated molecules had obtained the correct tertiary structure and were able to remain fairly stable in serum for 48 hours. Table 7B (below) shows that the potency is comparable to other ShK-conjugated molecules.

TABLE 7A human whole blood ("WB") assays of 4 pools of 3742(SEQ ID NOS: 377; 109, 112; 109) of IL-2 and interferon- γ ("IFN γ") were performed as described in example 5 herein.

Table 7b. data showing the potency of multiple conjugates of [ Lys16] ShK in whole blood assays. As described in example 9 herein, the toxin peptides and toxin peptide analogs are pegylated. Immunoglobulin-containing compounds were recombinantly expressed and purified as described in example 8. Human whole blood ("WB") assays for IL-2 and interferon-gamma ("IFNg") were performed as described in example 5 herein.

Example 6

Ab 4341-ShK (1-35, Q16K), 4341-FGF21 and 16435-FGF21 fusion constructs were generated

Antibody 16435-huFGF21 fusion (Ab10162).The components of the 16435-huFGF21 fusion include:

(a)16435κLC(SEQ ID NO：109)；

(b)16435 HC (R118A; SEQ ID NO: 112); and

(c)16435 IgG2-HC-huFGF21[1-181](SEQ ID NO：384)：

QVQLVQSGAEVKKPGASVKVSCKAASGYTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAQLYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTTSKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLETREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLRGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS//SEQ ID NO：384。

the construct pTT5-aKLH120.6-IgG2-HC-L15-huFGF21[1-181] (SEQ ID NO: 130) was used as a template to generate a 16435huIgG2-HC-L15-huFGF21[1-181] fragment, which was digested with BsmBI and NotI and purified using a Qiagen gel purification kit. At the same time, pTT5-16435IgG2 HC was digested with BsmBI and NotI and run on a 1% agarose gel. The vector fragment containing 16435 heavy chain variable region was excised and gel purified by Qiagen gel purification kit. The purified huIgG2-HC-L15-huFGF21[1-181] fragment was ligated with a vector fragment containing the 16435 heavy chain variable region and transformed into DH10b bacteria. DNA from the transformed bacterial colonies was isolated and submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pTT5-16435-IgG2-HC-L15-huFGF21[1-181] construct encodes an IgG2-HC-L15-huFGF21[1-181] fusion polypeptide (SEQ ID: 384).

Antibody 4341-huFGF21 fusion (Ab10163).4341-ShK[1-35，Q16K]Components of the fusion (Ab10163) include

(a)4341κ LC(SEQ ID NO：115)；

(b)4341 HC (Y125A; SEQ ID NO: 118); and

(c)4341 IgG2-HC-huFGF21[1-181](SEQ ID NO：386)：

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS//SEQ ID NO：386。

the construct pTT5-aKLH120.6-IgG2-HC-L15-huFGF21[1-181] was used as template to generate a huIgG2-HC-L15-huFGF21[1-181] fragment, which was digested with BsmBI and NotI and purified with Qiagen gel purification kit. Meanwhile, pTT5-4341 TgG2HC was digested with BsmBI and NotI and run on a 1% agarose gel. The larger fragment containing the 4341 heavy chain variable region was excised and gel purified by Qiagen gel purification kit. Purified huIgG2-HC-L15-huFGF21[1-181] fragments were ligated into a large vector fragment containing 4341 heavy chain variable regions and transformed into DH10b bacteria. DNA from the transformed bacterial colonies was isolated and submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pTT5-4341-IgG2-HC-L15-huFGF21[1-181] construct encodes an IgG2-HC-L15-huFGF21[1-181] fusion polypeptide (SEQ ID: 386).

4341-ShK[1-35，Q16K]Fusions (antibodies 10164).4341-ShK[1-35，Q16K]The components of the fusion (Ab10164) include:

(a)4341κ LC(SEQ ID NO：115)；

(b)4341 HC (Y125A; SEQ ID NO: 118); and

(c)4341 IgG2-HC-ShK[1-35，Q16K](SEQ ID NO：388)：

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//SEQ ID NO：388。

The construct pDC324-16435-HC-L10-IgG2-ShK [1-35, Q16K ] (SEQ ID NO: 376) was used as a template to generate a huIgG2-HC-L10-ShK [1-35, Q16K ] fragment, which was digested with BsmBI and NotI and purified using Qiagen gel purification kit. At the same time, pTT5-4341 IgG2 HC was digested with BsmBI and NotI and run on a 1% agarose gel. The larger fragment containing the 4341 heavy chain variable region was excised and gel purified by Qiagen gel purification kit. Purified huIgG2-HC-L10-ShK [1-35, Q16K ] fragment was ligated to a large vector fragment containing the 4341 heavy chain variable region and transformed into DH10b bacteria. DNA from the transformed bacterial colonies was isolated and submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pTT5-4341-IgG2-HC-L10-ShK [1-35, Q16K ] construct encodes an IgG2-HC-L10-ShK [1-35, Q16K ] fusion polypeptide (SEQ ID NO: 388).

Example 7

Ab4341-ShK, 4341-FGF21 and 16435-FGF21 fusion expression, purification and analysis

Transient transfection was performed in HEK293-6E cells as follows. Human embryonic kidney 293 cell lines (293-6E cells) stably expressing Epstein Barr virus nuclear antigen-1 were obtained from the National Research Council (Montreal, Canada). Cells were maintained as serum-free suspension cultures using F17 medium (Invitrogen, Carlsbad, CA) supplemented with 6mM L-glutamine (Invitrogen, Carlsbad, CA), 1.1% F-68Pluronic (Invitrogen, Carlsbad, CA) and 250 μ g/ul geneticin (Invitrogen, Carlsbad, CA). Suspension cell cultures were maintained in Erlenmeyer flask cultures. In moist 5% CO ₂The flask was shaken under an atmosphere at 37 ℃ and 65 rpm. Stock solutions (1mg/ml) of 25-kDa linear PEI (Polysciences, Warrington, Pa.) were prepared with water, acidified to pH2.0 with HCl until dissolved, then neutralized with NaOH, sterilized by filtration (0.2 μm), aliquoted, andstored at-20 ℃ for further use. Tryptone N1 was obtained from OrganoTechni s.a. (tekni science, QC, Canada). Stock solutions (20%, w/v) were prepared from F17 medium, filter sterilized through a 0.2 μm filter, and stored at 4 ℃ until use. Transfection is usually performed on the 1L scale. Cells (293-6E) were cultured to a viable cell density of 1.1X10⁶Individual cells/ml, then transfection complexes were prepared at 1/10th volume of the final culture volume. For the 1-L transfection culture, transfection complexes were prepared in 100ml F17 basal medium, and 500. mu.g of plasmid DNA (heavy and light chain DNA, 1: 1 ratio) was first diluted with 100ml F17 medium. After 5 minutes incubation at room temperature, 1.5ml of PEI solution was added. The complex was gently vortexed and then incubated at room temperature for 15 minutes. Cells were transfected by adding the transfection complex mixture to cells in shake flask culture. 24 hours after transfection, tryptone N1 was added to the transfected culture to a final concentration of 0.5%, and the transfected culture was incubated in a humidified 5% CO ₂The atmosphere was kept on a shaker at 37 ℃ and 65rpm for an additional 5 days, after which the culture was harvested. Conditioned media was harvested by centrifugation at 4000rpm and then sterile filtered through a 0.2 μm filter (Corning Inc.).

Transiently expressed antibodies were purified using recombinant protein A agarose gel (GE Healthcare) and conditioned media was loaded directly onto the column at 5 ml/min at 7 ℃. The column was then washed with 10 column volumes of Dulbecco's PBS containing no divalent cations and then eluted with 100mM acetic acid (pH 3.5). Eluted antibody was collected based on the chromatogram and pH was adjusted to 5.0 using 2M Tris base. The pooled antibodies were then filtered through a 0.8/0.22 μm syringe filter and then dialyzed against 10mM acetic acid, 9% sucrose (pH 5.0). The buffer exchanged antibody was then concentrated using a Vivaspin30kDa centrifugal concentrator (Sartorius) and the concentrated product was filtered through a 0.22 μm cellulose acetate filter. All conditioned media, including mock transfections, were analyzed using a 1.0mm Tris-glycine 4-20% SDS-PAGE (FIG. 39A) loaded with 10. mu.l of conditioned media run at 35mA/1000V/250W for 55 minutes. Bands above the 250 molecular weight standard not observed in mock transfected samples are likely to be the expressed product. All three experimental transfections showed significant amounts of the desired product on SDS-PAGE.

The product quality of the antibody fusions was analyzed on a 1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) using reducing and non-reducing loading buffers (FIG. 39B). Electrophoresis of all candidates was as expected by non-reducing and reducing SDS-PAGE; however, 10162 and 10163 showed a slightly slower migration than the expected bands, which may indicate partial glycosylation. Antibody homogeneity was further analyzed using 1 size exclusion column (Phenomenex SEC3000, 7.8x300mM) with a mobile phase of 50mM sodium phosphate, 250mM NaCl (ph6.8) flowing at 1.0 mL/min. 10162 and 10163 fusions elute as expected and exhibit relatively low levels of high molecular weight species; however, 10164 fusion eluted earlier than expected, possibly indicating aggregation.

LC-MS analysis was performed on the reduced light chain (FIGS. 41A-C) and heavy chain (FIGS. 42A-C) of the final 4341-ShK, 4341-FGF21 and 16435-FGF21 samples, respectively. Prior to reduction, FGF21 fusion samples were deglycosylated using PNGase F technology as described by the manufacturer (QA Bio, LLC, Palm dester, CA) except that the substrate to enzyme ratio was 10 μ g substrate to 1 μ L enzyme. The product was chromatographed using an Agilent1100 capillary HPLC system through a zorbax sb 300C 850 x1mm3 mini column. The column was set at 75 ℃ and elution of protein was performed using a gradient of increasing n-propanol concentration in 0.1% trifluoroacetic acid. The column effluent was directed to an Agilent-TOF mass spectrometer for mass analysis. The capillary voltage was set to 3,200V and the fragmentation voltage was set to 225V. Mass spectra were collected from 800 to 3000m/z and deconvoluted using MassHunter software supplied by the instrument manufacturer. All samples had the expected mass within the error range of the instrument, indicating that all libraries contained the expected product.

Example 8

Expression and purification of monovalent or multivalent immunoglobulin-and/or Fc domain-toxin peptide analog fusions

Various monovalent, divalent, and trivalent structures were expressed and purified for comparison, including exemplary embodiments of the invention, as shown in table 7B in example 5. Those include antibody IgG 2-or IgG1-ShK fusion variants (see FIGS. 1F-L). For example, a bivalent Fc-L10-ShK [1-35], monovalent immunoglobulin heavy chain- [ Lys16] ShK fusion antibody; see fig. 1F). IgG2Fc/Fc-ShK variants (see FIG. 1A), bivalent Fc-L10-ShK [2-35], monovalent Fc/Fc-L10-ShK [2-35], were also prepared for comparison by recombinant methods as described in Sullivan et al, WO2008/088422, and specific examples 1, 2, and 56 therein, which are incorporated by reference in their entirety, or modified as described herein.

A transient expression system is used to produce toxin peptide analog-Fc fusions ("peptide antibodies") or other immunoglobulin fusion embodiments. HEK293-6E cells at 37 ℃ in 5% CO₂F17 medium supplemented with L-glutamine (6mM) and Geneticin (Geneticin) (25. mu.g/ml) in 3L Fernbacherrenmeyer flasks was maintained at a density of between 2e5 and 1.2e6 cells/ml and shaken at 65 RPM. Upon transfection, cells were diluted to 1.1x10 with 90% of the final culture volume of the above-mentioned F17 medium ⁶Individual cells/mL. The DNA complexes were prepared in Freestyle293 medium at 10% of the final culture volume. The DNA complex contained 500ug total DNA per liter of culture and 1.5ml of PEhnax per liter of culture. Once the ingredients are added, the DNA complex is shaken briefly and incubated at room temperature for 10 to 20 minutes before being added to the cell culture and returned to the incubator. The day after transfection, tryptone N1(5g/L) was added to the culture from a liquid 20% stock solution. Six days after transfection, cultures were centrifuged at 4,000RPM for 40 minutes to pellet the cells and the cultured media was harvested through a 0.45um filter.

In preparing the DNA complex, the ratio of plasmids is proportional to the desired molar ratio of peptides required to produce the desired product. The IgG2Fc/Fc-ShK composition included a 1: 1 ratio of IgG2Fc and IgG2 Fc-ShK. During expression, these assembled into IgG2Fc homodimers, IgG2Fc/Fc-ShK heterodimers, and IgG2Fc-ShK homodimers. The IgG2Fc/Fc-ShK heterodimer (monovalent form) was isolated during purification using cation exchange chromatography.

IgG2Fc-ShK [2-35 ]; IgG2Fc Shk [2-35, Q16K ]; IgG2Fc-Shk [1-35 ]; IgG2Fc-ShK [1-35, Q16K ] mammalian cell expression. The DNA sequence encoding the immunoglobulin Fc domain of human IgG2 fused in frame to a monomer or mutated ShK [2-35, Q16K ] of the Kv1.3 inhibitor peptide ShK [2-35] was constructed using standard PCR techniques:

MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ IDNO：1，

ShK [2-35] or ShK [2-35, Q16K ] and a 10 amino acid linker moiety were used as templates in pcDNA3.1(+) CMVi to generate molecules in PCR reactions (see Sullivan et al, WO2008/088422A2, example 2, FIGS. 15A-B). The initial Fc-2xL-ShK [1-35] used in pcDNA3.1(+) CMVi was used as a template to generate ShK [1-35] in a PCR reaction (Sullivan et al, WO2008/088422A2, example 1, FIGS. 14A-B, herein). These ShK constructs have the following modified VH21 signal peptide amino acid sequence MEWSWVFLFFLSVTTGVHS// SEQ id no: 2, the sequence was generated from the pseleexis-Vh 21-hIgG2-Fc template with the following oligonucleotides:

5'-CAT GAA TTC CCC ACC ATG GAA TGG AGC TGG-3' (SEQ ID NO: 3); and

5’-CA CGG TGG GCA CTC GAC TTT GCG CTC GGA GTG GACACC-3’(SEQ ID NO：4)。

wild type ShK [2-35] (amino acid sequence GGGGSGGGGSSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC// SEQ ID NO: 6) with an N-terminal linker extension is encoded by the following DNA sequence: GGAGGAGGAGGATCCGGAGGAGGAGGAAGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCGCCTTCCAGTGCAAGCACAGCATGA AGTACCGCCTGAGCTTCTGCCGCA AGACCTGCGGCACCTGC// SEQ ID NO: 5. a fragment comprising this coding sequence (SEQ ID NO: 5) was generated using the following oligonucleotides (SEQ ID NO: 7 and SEQ ID NO: 8) and the initial Fc-L10-ShK [2-35] in pcDNA3.1(+) CMVi as template (Sullivan et al, WO2008/088422A2, example 2, FIGS. 15A-B, incorporated by reference):

5'-GTC CAC TCC GAG CGC AAA GTC GAG TGC CCA CCGTGC C-3' (SEQ ID NO: 7); and

5’-TCC TCC TCC TTT ACC CGG AGA CAG GGA GAG-3’//(SEQ ID NO：8)。

mutant ShK was generated using site-directed mutagenesis and following the manufacturer's instructions using Stratagene's QuikChange multiple site-directed mutagenesis kit catalog No. 200531 [2-35, Q16K ]. The oligonucleotides used to generate the mutagenesis were:

5 '-GCT GCA CCG CCT TCA AGT GCA AGC ACA GC 3' (SEQ ID NO: 9); and

5'-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3' (SEQ ID NO: 10); and the initial Fc-L10-ShK [2-35] used in pcDNA3.1(+) CMVi as a template (Sullivan et al, WO2008/088422A2, example 2 therein, FIGS. 15A-B), to obtain the DNA coding sequence

Ggaggaggaggatccggaggaggaggaagcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgc// (SEQ ID NO: 11) which encodes the amino acid sequence Shk (2-35, K16) with an N-terminal linker extension:

ggggsggggsscidtipksrctafkckhsmkyrlsfcrktcgtc//SEQ ID NO：12)。

the initial Fc-2xL-ShK [1-35] used in pcDNA3.1(+) CMVi as a template (Sullivan et al, WO2008/088422A2, example 1 therein, FIGS. 14A-B) and the following oligonucleotides produced ShK [1-35] WT fragments:

5'-GTC CAC TCC GAG CGC AAA GTC GAG TGC CCA CCGTGC C-3' (SEQ ID NO: 7); and

5’-TCC TCC TCC TTT ACC CGG AGA CAG GGA GAG-3’(SEQID NO：8)。

The IgG2Fc region was generated using the following oligonucleotides and pSelexis Vh21-hIgG2-Fc template:

5'-CCG GGT AAA GGA GGA GGA GGA TCC GGA G-3' (SEQ ID NO: 13); and

5'-CAT GCG GCC GCT CAT TAG CAG GTG-3' (SEQ ID NO: 14), resulting in a fragment comprising the following DNA coding sequence:

gcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa// SEQ ID NO: 15 encoding the amino acid sequence appvagpsvffppkpkddmistmirrtrtpepvtcpvvvdvshdvspeevprenkfywyvddvdvvegvehnaktkpreqrnfnsfrvsvltpvvsltvqwdlgpkykkckvsnkgrappektiktktktkkqgpvqpplvpnryemprenckppvqvslcglvplvfgfrvpryvpryvphlvqmlhneqkslstpvqpgk SEQ o: 16).

PCR fragments were generated and the products run on a gel. After gel purification, the DNA fragments were put together in a PCR tube and stitched together with the following outer primers:

5'-CAT GAA TTC CCC ACC ATG GAA TGG AGC TGG-3' (SEQ ID NO: 3); and

5’-CAT GCG GCC GCT CAT TAG CAG GTG-3’(SEQ IDNO：14)。

the PCR product was digested with EcoRI and noti (roche) restriction enzymes and agarose gel purified by gel purification kit. Meanwhile, the pTT14 vector (Amgen vector containing CMV promoter, poly-A tail and puromycin resistance gene) was digested with EcoRI and NotI restriction enzymes and the large fragment was purified by gel purification kit. Each purified PCR product was ligated to large fragments and transformed into OneShot Top10 bacteria. DNA from the transformed bacterial colonies was isolated and subjected to EcoRI and NotI restriction enzyme digestions, and then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone of each construct was selected for large scale plasmid purification. The final pTT14-VH1SP-IgG2-Fc construct encodes an IgG2-Fc-L10-ShK (2-35) fusion polypeptide having the following sequence:

Mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykekvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfsosvmhealhnhytqkslslspgkggggsggggsscidtipksrctafqckhsmkyrlsfcrktcgtc//(SEQID NO：17)。

the pTT14-VH21SP-IgG2-Fc-L10-ShK (2-35, Q16K) construct encodes an IgG2-Fc L10-ShK (2-35, Q16K) fusion polypeptide sequence:

MewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsscidtipksrctafKckhsmkyrlsfcrktcgtc//SEQID NO：18；

And the pTT14-VH21SP-IgG2-Fc ShK1-35 construct comprises the coding sequence of an IgG2Fc-L10-ShK (1-35) fusion polypeptide having the sequence:

mewswvflfflsvttgvhserkveeppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykekvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltelvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsrscidtipksrctafqckhsmkyrlsfcrktcgtc//(SEQID NO：19)。

the generation of a VH21SP-IgG2-Fc only construct in pYDl6 (Amgen vector containing CMV promoter, poly a tail and hygromycin resistance gene) was performed as follows: the VH21 signal peptide was generated using the following oligonucleotides:

5'-CAT AAG CTT CCC ACC ATG GAA TGG AGC TGG-3' (SEQ ID NO: 20); and

5'-CA CGG TGG GCA CTC GAC TTT GCG CTC GGA GTG GACACC-3' (SEQ ID NO: 4), and uses the pSelexis template indicated above.

The Fc region was generated using the pSelexis template described above and the following oligonucleotides:

5'-GTC CAC TCC GAG CGC AAA GTC GAG TGC CCA CCGTGC C-3' (SEQ ID NO: 7); and

5’-CAT GGA TCC TCA TTT ACC CGG AGA CAG GGA G-3’(SEQ ID NO：21)。

the PCR fragments were gel purified and used in a single PCR reaction with the outer primers GGT TGA GAG GTG CCA GAT GTC AGG GCT GCA GCA GCG GC// SEQ ID NO: 391 and CAG CTG CAC CTG ACC ACC ACC TCC ACCGCT ATG CTG AGC GCG// SEQ ID NO: 392 are sewn together. The resulting PCR fragment was gel purified and digested with HindIII and BamHI. At the same time, the pYD16 vector (Amgen vector containing CMV promoter, poly a tail and hygromycin resistance gene) was also cut with HindIII and BamHI and the large vector fragment was purified by Qiagen gel purification kit. The purified PCR product was ligated to large fragments and transformed into OneShotTop10 bacteria. DNA from transformed bacterial colonies was isolated and subjected to HindIII and BamHI restriction enzyme digestions and then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pYD16-VH21SP-IgG2-Fc construct encodes human IgG2-Fc (SEQ ID NO: 1 above).

IgG2-Fc ShK[1-35，Q16K]Mammalian expressionThe use of DNAPTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK [1-35, Q16K ]]A construct comprising a fragment of the following DNA coding sequence

ggatccggaggaggaggaagccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgctaatgagcggccgctcgaggccggcaaggccggatcc//(SEQ ID NO：22)

Cleavage was performed with BamHI/BamHI. The coding sequence (SEQ ID NO: 23) encodes a polypeptide having an N-terminal linker sequence: GSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC// (SEQ ID NO: 23) ShK (1-35, Q16K).

Meanwhile, the pTT14-hIgG2-Fc-ShK [1-35] WT construct was also digested with BamHI/BamHI to remove the Shk [1-35] coding region to give a coding sequence

Attggaatgggagcttccaggccctaccaggccaggccctgccaccaggccctgcaggcaccgcgcgctcgcacccaaggccctgccaaggcaccaggcaccaggcaccaggcaccaggcaccaggcgcaccaggcaccaggctcgcaccaggcaccaggcaccaggctcgctcgctcgctcgctccaggctcgctccaggctcaaggctcaaggctcagggcatcgctacgggcatcgctacaggctacgggcatcgctacgggcatagctcagggctacaggctacgctacgggctacaggctcagggctacgctcagggctacaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggcaccaggccggcaccaggcaccaggcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgc: 24) the coding sequence encodes the following amino acid sequence mewswfflffllsvtttgvvhsherkvfppkpkpkpkpktllmmertpirtpevtcvvvdhvshedpvpfedqfnvwdvveaktkpreqppreqfnsvsvltlvsltvhdqwlckvkekvkekvsnksnkglvpacktpacktpqqpvqplypprefemksweckuskvslcktfssfpidvsldvsfflstdvsldvsldvsffvsldvstwqgnqgnvsvgnvslhyqkslhstpgggg// (SFQ ID NO: 25).

The pTT14-hIgG2-Fc vector from which ShK has been removed was treated with Calf Intestinal Phosphatase (CIP) to remove the 5' phosphate group, and phenol/chloroform extraction was performed to prevent the vector from religation itself. The fragment inserted into ShK [1-35, Q16K ] was gel purified for isolation from its vector and purified using a Qiagen gel purification kit. The purified insert was ligated to a large vector fragment and transformed into OneShot Top10 bacteria. DNA from transformed bacterial colonies was isolated and subjected to BamHI restriction enzyme digestion and then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pTT14-IgG2-Fc-ShK [1-35, Q16K ] construct encodes the following IgG2Fc-L10-ShK (1-35, Q16K) fusion protein sequence:

mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdlmisrtpevtcvvvdvshedpcvqfnwyvdgvevhnakktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsrscidtipksrctafkckhsmkyrlsfcrktcgtc//(SEQID NO：26)。

the amino acid sequence of IgG2Fc-L10-ShK (1-35) is:

mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpcnnykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsrscidtipksrctafqckhsmkyrlsfcrktcgtC//(SEQ ID NO：30)。

the desired aKLH IgG2/Fc-ShK product contained one copy of each of the immediately above components (a) - (c), as constructed in FIG. 1E. For this reason, the ratio is 1: 1. This product can be described as half an antibody and half an Fc fusion ("half antibody") coupled together at the Fc domain. Other peptide components that must be removed from the culture are the aKLH Ab and Fc-ShK homodimers.

The initial Fc-L10-ShK [1-35] used in pcDNA3.1(+) CMVi was used as a template (described in example 1, FIGS. 14A-14B of Sullivan et al, Toxin Peptide Therapeutic Agents, PCT/US2007/022831 (published as WO 2008/088422), which is incorporated herein by reference in its entirety) and the following oligonucleotides to form the ShK [1-35] WT fragment:

5'-TCC CTG TCT CCG GGT GGA GGA GGA GGA TCC GGAG-3' (SEQ ID NO: 47); and 5'-CAT GCG GCC GCT CAT TAG CAG GTG-3' (SEQ ID NO: 14).

The PCR product was run on a 1% agarose gel. The band was punched out as an agarose wedge (agarose plug) and the wedge was placed in a new PCR reaction tube. The agarose wedge was then primed using the outer primers SEQ ID NO: 357 and SEQ ID NO: 330 is amplified by PCR. The PCR product was then digested with XbaI and NotI and purified with a PCR clean-up kit (Qiagen). At the same time, the pTT5 vector (Amgen vector containing CMV promoter and poly A tail) was cut by XbaI and NotI. The pTT5 vector was run on a 1% agarose gel and the larger fragment was excised and gel purified by Qiagen gel purification kit. The purified PCR products were ligated to large vector fragments and transformed into OneShot Top10 bacteria. DNA from the transformed bacterial colonies was isolated and subjected to XbaI and NotI restriction enzyme digestions, then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final pTT5-aKLH120.6-VKL SP-IgG2-HC-L10-ShK [1-35] construct encodes an IgG2-HC-L10-ShK [1-35] fusion polypeptide having the following amino acid sequence:

Mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggggsggggsrscidtipksrctafqckhsmkyrlsfcrktcgtc//(SEQ IDNO：48)。

To generate ShK [1-35, Q16K ] mutant forms of this construct, site-directed mutagenesis was performed using the Stratagene Quikchange multiple site-directed mutagenesis kit (Cat. No. 200531) and the following oligonucleotides according to the manufacturer's instructions:

5 '-GCT GCA CCG CCT TCAAGT GCAAGC ACA GC 3' (SEQ ID NO: 9); and

5'-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3' (SEQ ID NO: 10). The final construct pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK [1-35, Q16K ] encodes an IgG2-HC-L10-ShK [1-35, Q16K ] fusion polypeptide having the following amino acid sequence:

Mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykekvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggggsggggsrscidtipksrctafkckhsmkyrlsfcrktcgtc//(SEQ IDNO：49)。

aKLH-IgG2 heavy chain-L10-ShK [2-35, Q16K]Mammalian expressionThe DNA construct pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK [1-35 ]]As a carrier, ShK [1-35 ]]Cleavage was performed with BamHI/BamHI. From the plant without ShK [1-35 ]]The vector fragment of pTT5-aKLH120.6-VKLSP-IgG2-HC contained the coding sequence:

atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgccagatgtcaggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctaccacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagagatcgtgggagctactactggttcgacccctggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggagga//(SEQ ID NO：50)，

the coding sequence encodes the following amino acid sequence:

mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktvcrkccvccppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshcdpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvlvvhqdwlngkeykekvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggg//(SEQ ID NO：51)。

the vector fragments were then treated with Calf Intestinal Phosphatase (CIP) to remove the 5' phosphate group and phenol/chloroform extraction was performed to prevent the vector from re-ligating itself. The insertion sequence comes from the code IgG2Fc-L10-ShK (2-35, Q16K)

mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsscidtipksretafkckhsmkyrlsfcrktcgtc//(SEQID NO：18)，

pTTl4-VH21SP-IgG2-Fc-ShK [2-35, Q16K ], and the insert was also digested with BamHI/BamHI. The fragment inserted into ShK [2-35, Q16K ] was gel purified for isolation from its vector and purified using Qiagen gel purification kits. Will comprise a coding sequence encoding amino acid sequence GSGGGGS SCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 53)

gga tcc gga gga gga gga agc agc tgc atc gac acc atc ccc aag agc cgc tgc accgcc ttc aag tgc aag cac agc atg aag tac cgc ctg agc ttc tgc cgc aag acc tgc ggc acc tgctaa tga//(SEQ ID NO：52)，

The purified DNA insert of (a) was ligated to the large vector fragment and transformed into OneShotTop10 bacteria. DNA from transformed bacterial colonies was isolated and subjected to BamHI restriction enzyme digestion and then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final construct pTT5-aKLH-IgG2HC-L10-ShK [2-35, Q16K ] encodes an IgG2HC-L10-ShK [2-35, Q16K ] fusion polypeptide:

Mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggggsggggsscidtipksrctafkckhsmkyrlsfcrktcgtc//(SEQ ID NO：54)。

VH21 SP-N-terminal ShK [1-35 ]]Wild type-IgG 1-Fc mammalian expressionThe peptide ShK [1-35 ] encoding the Kv1.3 inhibitor fused in frame to the N-terminal Fc region of human IgG1 was constructed as follows]The DNA sequence of the monomer(s).

To construct the VH21SP-ShK (1-35) -L10-IgG1Fc expression vector, a PCR strategy was used to generate the VH21 signal peptide ShK (1-35) gene linked to the amino acids of 4 glycines and 1 serine, which were flanked by HindIII and BamHI restriction sites, and Fc-L10-OSK1, used in pcDNA3.1(+) CMVi, was used as a template to generate the amino acids of 4 glycines and 1 serine in a PCR reaction linked to an IgG1Fc fragment, which is flanked by BamHI and NotI restriction sites (described in Suvallin et al, example 41 of WO2008/088422A2 and FIGS. 42A-B, which are incorporated by reference).

To generate VH21SP-ShK (1-35) -G₄S, two oligonucleotides having the sequences shown below were used in a PCR reaction using Pfuturbo HotStart DNA polymerase (Stratagene), in which 35 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 75 ℃ for 45 seconds were performed; HindIII (aagctt) and BamHI (ggatcc) restriction sites are underlined:

a forward primer:

tgcagaagcttctagaccaccatggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttccagt// (SEQ ID NO: 55); and

reverse primer:

Ctccggatcctcctcctccgcaggtgccgcaggtcttgcggcagaagctcaggcggtacttcatgctgtgcttgcactggaaggcggtgcagcggctcttggggatggtgtcgat//(SEQ IDNO：56)。

the resulting PCR product was resolved on a 2% agarose gel into a 202bp band. The 202bpPCR product was purified using a PCR purification kit (Qiagen), then digested with HindIII and BamHI (Roche) restriction enzymes, and the agarose gel was purified by a gel recovery kit (Qiagen).

To generate G₄S-IgG1Fc, two oligonucleotides having the sequences shown below were used in a PCR reaction with Pfuturbo HotStart DNA polymerase (Stratagene) with 30 cycles at 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 75 ℃ for 1 minute; BamHI (ggatcc) and NotI (gcggccgc) restriction sites are underlined:

a forward primer:

gtaggatccggaggaggaggaagcgacaaaactcacac// (SEQ ID NO: 57); and

Reverse primer:

Cgagcggccgcttactatttacccggagacaggga//(SEQ ID NO：58)。

the resulting PCR product was resolved on a 1% agarose gel into a 721-bp band. The 721-bp PCR product was purified using a PCR purification kit (Qiagen), then digested with BamHI and NotI (Roche) restriction enzymes, and the agarose gel was purified by a gel recovery kit (Qiagen).

The pcDNA3.1(+) CMVi-Fc-L10-OSK1 vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by gel recovery kit. Gel purified 4GS-IgG1Fc fragment was ligated to purified large fragment and transformed to OneTop10(Invitrogen) to generate the pCMVi-Fc-L10-IgG1Fc vector. Subsequently, the pCMVi-Fc-L10-IgG1Fc vector was digested with HindIII and BamHI restriction enzymes and the large fragment was purified by gel recovery kit. The gel-purified VH21SP-ShK (1-35) -4GS fragment was ligated to the purified large fragment and transformedTo OneTop10(Invitrogen), thereby generating the pCMVi-VH21SP-ShK (1-35) -L10-IgG1Fc construct. DNA from transformed bacterial colonies was isolated and digested with BamHI and NotI restriction enzymes, and then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone per gene was selected for large scale plasmid purification. DNA from VH21SP-ShK (1-35) -L10-IgG1Fc in the pCMVi vector was re-sequenced to confirm the Fc and linker regions and the sequence was 100% identical to the sequence described above. The fragment VH21SP-ShK (1-35) -L10-IgG1Fc comprises a coding sequence

atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttccagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagcgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatagtaa//(SEQ ID NO：59)，

The coding sequence encodes VH21SP-ShK (1-35) -L10-IgG1Fc amino acid sequence mewswvfflfflsvttvgsvhrsgrcidtipksrctafckhsmkyrlfcrkttcgtcgggsggsggthtcpppapellggsvlfflfpkpfpldtftlmtcpirtpirtpirtpirtpirtpirtpvvphvshpknwvgefvgvevhntprovskintprevkvsnvkvsnvkvsnvkvsnvpkvsvqpstvporqpvsvqptpvytlpvppelpfplvplmvsvqpglvpslvvsldvsglvpglvplsglvplskqvssdvqvssdvkswesngpyvcsvmssglvksyk (SEQ ID: 60)

N-terminal ShK [1-35, Q16K]-aKLH HC and N-terminus ShK[1-35Q16K]Mammalian expression of-aKLH LCUse of a coding N-terminal ShK [1-35 ]]wild-type-L10-IgGl-Fc construct, site-directed mutagenesis was performed to generate the Q16K mutation in the ShK region using the following oligonucleotides:

5'-GCT GCA CCG CCT TCA AGT GCA AGC ACA GC-3'// (SEQ ID NO: 9); and

5’-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3’(SEQID NO：10)。

stratagene QuikChange multiple site-directed mutagenesis kit was used according to the manufacturer's instructions. The final construct of pCMVi-N-terminal-ShK [1-35Q16K ] -L10-IgGl-Fc encodes the following signal peptide (VH21SP) -ShK [1-35, Q16K ] -L10-IgGl-Fc fusion polypeptide:

Mewswvflfflsvttgvhsrscidfipksrctafkckhsmkyrlsfcrktcgtcggggsggggsdkthtcppcpapellggpsvflfppkpkdtlmisrtpcvtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk//(SEQID NO：61)。

to generate the N-terminal ShK [1-35, Q16K ] -aKLH HC construct, the following oligonucleotides were used:

5’-CAT TCT AGA CCA CCA TGG AAT GG-3’(SEQ ID NO：62)；

5’-CAG CTG CAC CTG GCT TCC TCC TCC TCC GG-3’(SEQ IDNO：63)；

and template pCMVi-N-terminal-ShK [1-35, Q16K ] -L10-IgG1-Fc to generate a PCR product comprising a signal peptide-ShK [1-35Q16K ] -L10 linker, resulting in a fragment comprising the following coding sequence:

atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgeggaggaggaggatccggaggaggaggaagc//(SEQ ID NO：64)，

The coding sequence encodes the VH21SP-ShK (1-35, Q16K) -L10 amino acid sequence MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTCGGGGSGGGGS// (SEQ ID NO: 65).

To generate the aKLH-HC fragment, oligonucleotides were used:

5’-GGA GGA GGA AGC CAG GTG CAG CTG GTG CAG-3’(SEQ ID NO：66)；

5’-CAT GCG GCC GCT CAT TTA CCC-3’(SEQ ID NO：67)；

and the template pTT5-aKLH120.6-HC to generate PCR products, thereby obtaining DNA fragments comprising the following coding sequences:

caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctaccacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagagatcgtgggagctactactggttcgacccctggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaactagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga//(SEQ ID NO：68)，

the coding sequence encodes an amino acid sequence

qvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcarstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltelvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk//(SEQ IDNO：69)，

Both PCR products were run on a gel and the appropriate size band was punched out as an agarose wedge. The agarose wedge was placed in a single new PCR reaction and the fragments were stitched together using the outermost primers (SEQ ID NO: 62) and (SEQ ID NO: 67). The PCR fragment was digested with XbaI and NotI and purified with Qiagen PCR clean-up kit. Meanwhile, the pTT5 vector was also cleaved with XbaI and NotI and gel-purified. The purified insert was ligated to a large vector fragment and transformed into OneShot Top10 bacteria. DNA from the transformed bacterial colonies was isolated and subjected to XbaI and NotI restriction enzyme digestions, then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. Although analysis of several clone sequences resulted in a 100% percent match to the above sequences, only one clone was selected for large scale plasmid purification. The final construct pTT 5-N-terminal ShK [1-35Q16K ] -L10-aKLH120.6-HC encodes VH21SP-ShK [1-35, Q16K ] -L10-aKLH120.6-HC fusion polypeptide:

Mcwswvflfflsvttgvhsrscidtipksrctafkckhsmkyrlsfcrktcgtcggggsggggsqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnggtnyaqkfqgrvimtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk//(SEQ ID NO：70)。

Finally, an N-terminal-ShK [1-35, Q16K ] -L10-aKLH120.6 Light Chain (LC) was produced in the same manner as described above. The following oligonucleotides were used:

5'-CAT TCT AGA CCA CCA TGG AAT GG-3' (SEQ ID NO: 62); and

5’-CAT CTG GAT GTC GCT TCC TCC TCC TCC GG-3’(SEQ IDNO：71)；

and template pCMVi-N-terminal-ShK [1-35Q16K ] -L10-IgG1-Fc to generate a PCR product comprising signal peptide-ShK [1-35, Q16K ] -L10, thereby obtaining a DNA fragment comprising the following coding sequence:

atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagc//(SEQ ID NO：64)，

the coding sequence encodes the amino acid sequence of the signal peptide (VH21SP) -ShK (1-35, Q16K) -L10 linker:

mewswvflfflsvttgvhsrscidtipksretafkckhsmkyrlsfcrktcgtcggggsggggs//(SEQ ID NO：65)。

using templates and oligonucleotides:

5'-GGA GGA GGA AGC GAC ATC CAG ATG ACC CAG TC-3' (SEQ ID NO: 72); and

5’-CAT CTC GAG CGG CCG CTC AAC-3’(SEQ ID NO：73)，

generating a vector comprising a coding sequence

atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacac catccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagcgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaagcccctaaacgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtctacagcataatagttacccgctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga// (SEQ ID NO: 74),

The coding sequence encodes the amino acid sequence of an N-terminal VH21SP-ShK [1-35, Q16K ] -L10-aKLH120.6 Light Chain (LC) with an N-terminal signal peptide:

mewswvflfflsvttgvhsrscidtipksrctafkckhsmkyrlsfcrktcgtcggggsggggsdiqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvpsrfsgsgsgteftltisslqpedfatyyclqhnsypltfgggtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec//(SEQ ID NO：75)。

two PCR fragments (DNA fragment containing the coding sequence (SEQ ID NO: 64) and aKLH120.6 light chain LC fragment containing the coding sequence (SEQ ID NO: 74)) were run on a gel and the appropriate size band was punched out as an agarose wedge. The agarose wedge was placed in a single new PCR reaction and the fragments were stitched together using the outermost primers (SEQ ID NO: 62) and (SEQ ID NO: 73). The resulting PCR fragment was cut with XbaI and NotI and purified with Qiagen PCR clean-up kit.

Meanwhile, the pTT14 vector (Amgen vector containing CMV promoter, poly A tail and puromycin resistance gene) was also cut with XbaI and NotI and gel-purified. The purified insert was ligated to a large vector fragment and transformed into OneShot Top10 bacteria. DNA from the transformed bacterial colonies was isolated and subjected to XbaI and NotI restriction enzyme digestions, then resolved on a 1% agarose gel. The DNA that produced the expected map was submitted for sequencing. The final construct pTT 14-N-terminal ShK [1-35Q16K ] -L10-aKLH120.6-LC encodes a signal peptide-ShK [1-35, Q16K ] -L10-aKLH120.6-LC fusion polypeptide sequence (i.e., SEQ ID NO: 75).

Example 9

Purification and evaluation of the comparative molecules: monovalent Fc/Fc-L10-ShK [2-35] heterodimers and monovalent or divalent Fc/Fc-ShK (1-35Q16K) (IgG2) heterodimers and other polypeptide molecules

Monovalent or divalent Fc-L10-ShK [2-35], monovalent or divalent Fc-L10-ShK [1-35], monovalent or divalent Fc-L10-ShK (1-35, Q16K), and other ShK-related polypeptide molecules listed in Table 7B (in example 5 herein) were expressed, isolated, and purified by the methods described herein. The pegylated and non-pegylated toxin peptide comparisons in table 7B were prepared synthetically as follows:

peptide synthesis N^αFmoc, side chain protected amino acids and H-Cys (Trt) -2C1-Trt resin were purchased from Novabiochem, Bachem or Sigma Aldrich. The following side chain protection strategy was employed: asp (OtBu), Arg (Pbf), Cys (Trt), Glu (OtBu), His (Trt), Lys (N)^εBoc), Ser (OtBu), Thr (OtBu) and Tyr (OtBu). ShK (RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC// SEQ ID NO: 378), [ Lys16]ShK (RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC// SEQ ID NO: 76) or other toxin peptide analog amino acid sequences were synthesized by SPPS on a CS Bio peptide synthesizer in a stepwise manner using DIC/HOBt coupling chemistry on a 0.2mmol equivalent scale and using H-Cys (Trt) -2Cl-Trt resin (0.2mmol, 0.32mmol/g loading). For each coupling cycle, 1mmol N ^αthe-Fmoc-amino acid was dissolved in 2.5mL of 0.4M 1-hydroxybenzotriazole (HOBt) in N, N-Dimethylformamide (DMF). To this solution was added 1.0mL of 1.0MN, N' -Diisopropylcarbodiimide (DIC) in DMF. The solution was stirred with bubbling nitrogen for 15 minutes to complete the pre-activation and then added to the resin. The mixture was shaken for 2 h. The resin was filtered and washed 3 times with DMF, 2 times with Dichloromethane (DCM) and then 3 times with DMF. Fmoc deprotection was performed by treatment with 20% piperidine in DMF (5mL, 2 × 15 min). The first 23 residues were mono-coupled by repeating the Fmoc-amino acid coupling and Fmoc removal steps described above. The remaining residues were double coupled by performing coupling steps twice followed by Fmoc-removal.

After synthesis, the resin was then drained and washed sequentially with DCM, DMF, DCM and then dried in vacuo. The peptide-resin was transferred to a 250-mL plastic round bottom flask. The peptide was deprotected and released from the resin by treatment with triisopropylsilane (1.5mL), 3, 6-dioxa-1, 8-octane-dithiol (DODT, 1.5mL), water (1.5mL), trifluoroacetic acid (TFA, 20mL), and stir bar and the mixture was stirred for 3 h. The mixture was filtered through a 150-mL sintered glass funnel into a 250-mL plastic round bottom flask. The mixture was filtered through a 150-mL sintered glass funnel into a 250-mL plastic round bottom flask and the filtrate was concentrated in vacuo. The crude peptide was precipitated by addition of cold diethyl ether, collected by centrifugation and then dried under vacuum.

Peptide foldingThe crude linear peptide (about 600mg) is dried, for example [ Lys16 ]]ShK peptide (SEQ ID NO: 76) or [ Lys16]ShK-Ala (also known as [ Lys16, Ala 36)]-ShK; SEQ ID NO: 379) the peptide was dissolved in 16mL of acetic acid, 64mL of water, and 40mL of acetonitrile. The mixture was stirred rapidly for 15 minutes to complete the dissolution. The peptide solution was added to a 2-L plastic bottle containing 1700mL of water and a large stir bar. To the so diluted solution was added 20mL of concentrated ammonium hydroxide to raise the pH of the solution to 9.5. Using small amount of acetic acid or NH as required₄OH adjusts the pH. The solution was stirred at 80rpm overnight and monitored by LC-MS. Folding was generally judged to be complete in 24 to 48h, and the solution was then quenched by the addition of acetic acid and TFA (pH 2.5). The aqueous solution was filtered (0.45 μm cellulose membrane).

Purification by reverse phase HPLCReversed-phase high performance liquid chromatography was performed on analytical columns (C18, 5 μm, 0.46 cm. times.25 cm) or preparative columns (C18, 10 μm, 2.2 cm. times.25 cm). Chromatographic separation was achieved using a linear gradient of buffer B in a (a ═ 0.1% aqueous TFA; B ═ 90% aqueous ACN containing 0.09% TFA), typically from 5% to 95% over 35 minutes at a flow rate of 1 mL/min for analytical analysis and from 5% to 65% over 90 minutes at a flow rate of 20 mL/min for preparative separation. Analytical and preparative HPLC fractions were characterized by ESMS and photodiode array (PDA) HPLC, pooled and lyophilized.

Mass spectrometric analysisMass spectra were acquired on a single quadrupole mass spectrometer equipped with an electrospray atmospheric pressure current source. Samples (25 μ L) were injected into a flowing solvent (10 μ L/min; 30: 50: 20ACN/MeOH containing 0.05% TFA) coupled directly to the ionization source through a fused silica capillary interface (50 μm i.d.). The sample droplets were ionized at a positive potential of 5kV and passed through the interface plate into the analyzer and then through the orifice (100-. Full scan mass spectra were acquired over the mass range 400-2200Da with a scan step size of 0.1 Da. Molecular masses were deduced from the observed m/z values.

Pegylation, purification and analysisPeptides such as [ Lys16 ]]ShK (SEQ ID NO: 76) or [ Lys16]ShK-Ala (SEQ ID NO: 379) is selectively pegylated by reductive alkylation at its N-terminus using activated linear or branched PEG. In 50mM NaH containing 20mM sodium cyanoborohydride and 2 molar excess of 20kDa monomethoxy-PEG-aldehyde (NOF, Japan)₂PO₄(pH4.5) conjugation was performed at 2mg/ml in reaction buffer. The conjugation reactions were stirred at room temperature for approximately 5 hours and their progress was monitored by RP-HPLC. The completed reaction was quenched by 4-fold dilution with 20mM NaOAc (pH4) and cooled to 4 ℃. The PEG-peptide was then purified by chromatography at 40C using a SP Sepharose HP column (GE Healthcare, Piscataway, N.J.) and eluting the column with a linear 0-1M NaCl gradient in 20mM NaOAc (pH 4.0). Eluted peak fractions were analyzed by SDS-PAGE and RP-HPLC and the purity of pooled pools was determined to be > 97%. The major contaminants observed were di-pegylated toxin peptide analogs. The selected pool was concentrated to 2-5mg/ml by centrifugation through a 3kDaMWCO membrane and dialyzed into 10mM NaOAc (pH4) with 5% sorbitol. The dialyzed pool was then filter sterilized through a 0.2 micron filter and was > 97% pure as determined by SDS-PAGE (data not shown). Reverse phase HPLC on Agilent1100 model HPLC with 0.1% TFA/H₂O flows through at 1 ml/minA5 μm300 SB-C84.6x50mm column (Agilent) was used and the column temperature was maintained at 40 ℃. PEG-peptide samples (20 μ g) were injected and eluted with a linear 6-60% gradient while monitoring the 215nm wavelength.

A fusion protein.Generally, fig. 1A and 1B show schematic representations of monovalent and divalent Fc-toxin peptide (or toxin peptide analog) fusion proteins (or "peptide antibodies"), respectively. The bivalent Fc-ShK molecule is a homodimer comprising two Fc-ShK chains. The monovalent Fc-ShK toxin peptide (or toxin peptide analog) molecule is a heterodimer comprising one Fc chain and one Fc-ShK (or analog) chain. A monovalent Fc-ShK molecule is considered monovalent since it contains only a single ShK peptide per dimer. The construct or chain, referred to as an Fc- (toxin peptide analog), comprises an N-terminal Fc region and an optional flexible linker sequence (e.g., L10 peptidyl linker GGSGGGGS; SEQ ID NO: 153) covalently attached to the toxin peptide or toxin peptide analog such that the orientation from N-terminus to C-terminus should be: an Fc-linker-toxin peptide or toxin peptide analog.

In examples 1 and 2 of Sullivan et al, WO2008/088422A2, the activity of bivalent Fc-ShK peptide antibodies, namely Fc-L10-ShK (1-35) and Fc-L10-ShK (2-35), expressed by mammalian cells is described. In example 1 of WO2008/088422a2, the isolation of monovalent Fc-L10-ShK (1-35) molecules is also described, which are formed as small by-products during expression. The monovalent antibody #3742-ShK (1-35, Q16K) conjugate provides an effective block of T cell cytokine secretion in human whole blood (see tables 7A-B in example 5 herein).

Example 10

Pharmacokinetic (PK) studies in rats and cynomolgus monkeys

Rat PKThe pharmacokinetic profile of 16435 and 4341 antibodies was determined in adult Sprague-dawley (sd) rats (n ═ 3 per group) by: 5mg/kg was injected subcutaneously and approximately 250. mu.L of blood was collected from the tail vein at 0, 0.25, 1, 4, 24, 48, 72, 168, 336, 504, 672, 840 and 1008 hours post-administrationIn a serum separation tube. After collection, each sample was maintained at room temperature and after 30-40 minutes of clotting time, the samples were centrifuged at 11,500rpm for about 10 minutes at 2-8 ℃ using a calibrated Eppendorf5417R centrifuge system (brinkmann instruments, inc., Westbury, NY). The collected sera were then transferred to pre-labeled (for each rat) cryo-storage tubes and stored at-60 ℃ to-80 ℃ for future analysis. To measure serum sample concentrations from PK study samples, the following methods were used: an 1/2 area black plate (Corning3694) was coated with 2. mu.g/ml of anti-hu Fc, antibody 1.35.1, in PBS and incubated overnight at 4 ℃. The plates were then washed and washed with I-Block at 4 deg.C ^TM(Applied Biosystems) was blocked overnight. If samples need to be diluted, they are diluted with rat SD serum. Standards and samples were then diluted 1: 20 with 1 XPBS +1M NaCl + 0.5% Tween 20 and 1% BSA buffer (5% serum). The plates were washed and 50- μ l samples of the diluted standards and samples were transferred to the antibody 1.35.1 coated plates and incubated at room temperature for 1.5 h. The plate was washed and 50. mu.l of 100ng/ml solution in I-Block was added^TMAnti-hu Fc antibody 21.1-HRP conjugate in + 5% BSA and incubated for 1.5 h. The plates were washed and then 50. mu.l of Pico substrate was added, immediately after which the plates were analysed photometrically. Methods and apparatus for using non-compartmental(enterprise version 5.1.1, 2006,corona View, CA) analyzed the time concentration data (fig. 34.0). The pharmacokinetic profiles of these two antibodies in Sprague-Dawley rats are shown in figure 43. PK parameters for 16435 and 4341 antibodies in SD rats are summarized in table 8 (below). Both molecules had good PK profiles in rats with a half-life of more than 10 days.

TABLE 8 PK parameters of antibodies 16435 and 4341 in SD rats.

Macaca fascicularis PKThe pharmacokinetic profile of the 16435 and 4341 antibodies was also determined in cynomolgus monkeys (n ═ 2 per group) by injecting two consecutive subcutaneous doses of 1mg/kg at day 0 and 5mg/kg at day 57, respectively. Serum samples were collected prior to dosing, at 0.5, 2, 4, 8, 12, 24, 48, 96, 168, 336, 504, 672, 840, 1008, 1176, 1344 (before the second dose) hours after the first dose of 1mg/kg, and at 0.5, 2, 4, 8, 12, 24, 48, 96, 168, 336, 360, 384, 432, 504, 672, 840, 1008, 1176, 1344 hours after the 2 nd dose of 5 mg/kg. Samples were assayed for 16435 and 4341 antibody levels by an anti-IgG sandwich ELISA as described above. Methods and apparatus for using non-compartmental Time concentration data was analyzed. The pharmacokinetic profile of these two antibodies in cynomolgus monkeys is shown in figure 44. PK parameters for 16435 and 4341 antibodies in cynomolgus monkeys are summarized in table 9 (below). Both molecules showed good PK profile in cynomolgus monkeys, whichThe half-lives of meso16435 and 4341 are about 12 and 21 days, respectively. 4341 antibody has a better PK profile than 16435, and has shown normal hu IgG clearance in monkeys based on FcRn binding and in the absence of any target-mediated drug disposition (TMDD) clearance mechanism. Furthermore, the results in figure 44 show that even with multiple dosing in cynomolgus monkeys, neither antibody 16435 nor 4341 indicates a significant change in immune response-mediated clearance in cynomolgus monkeys. If there is a significant immune response resulting in abnormal antibody clearance, it is expected after the second dose because the first dose stimulates the immune system.

Table 9 PK parameters of antibodies 16435 and 4341 in cynomolgus monkeys.

Abbreviations

Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific instances.

Ac acetyl group (for acetylated residue)

AcBpa acetylated p-benzoyl-L-phenylalanine

ACN nitriles

AcOH acetic acid

ADCC antibody-dependent cellular cytotoxicity

Aib Aminoisobutyric acid

bA beta-alanine

Bpa p-benzoyl-L-phenylalanine

BrAc Bromoacetyl (BrCH)₂C(O)

BSA bovine serum albumin

Bzl benzyl radical

Cap hexanoic acid

CBC complete blood cell count

COPD chronic obstructive pulmonary disease

CTL cytotoxic T lymphocytes

DCC dicyclohexylcarbodiimide

Dde 1- (4, 4-dimethyl-2, 6-dioxy-cyclohexylidene) ethyl

DNP 2, 4-dinitrophenol

DOPC 1, 2-dioleoyl-sn-glycero-3-phosphocholine

DOPE 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine

DPPC 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine

DSPC 1, 2-distearoyl-sn-glycero-3-phosphocholine

DTT dithiothreitol

EAE Experimental autoimmune encephalomyelitis

ECL enhanced chemiluminescence

ESI-MS electrospray ionization mass spectrometry

FACS fluorescence activated cell sorting

Fmoc fluorenylmethyloxycarbonyl

GHT Glycine, hypoxanthine, and thymidine

HOBt 1-hydroxybenzotriazole

HPLC high performance liquid chromatography

HSL homoserine lactone

IB Inclusion bodies

KCa calcium activated potassium channels (including IKCa, BKCa, SKCa)

KLH keyhole limpet hemocyanin

Kv Voltage-gated Potassium channel

Lau lauric acid

LPS lipopolysaccharide

LYMPH lymphocytes

MALDI-MS matrix assisted laser desorption ionization mass spectrometry

Me methyl group

MeO methoxy group

MeOH methanol

MHC major histocompatibility complex

MMP matrix metalloprotease

MW molecular weight

MWCO molecular weight cut-off

1-Nap 1-naphthylalanine

NEUT neutrophils

Nle leucine

NMP N-methyl-2-pyrrolidone

OAc acetate

PAGE Polyacrylamide gel electrophoresis

PBMC peripheral blood mononuclear cells

PBS phosphate buffered saline

Pbf 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl

PCR polymerase chain reaction

Pharmacodynamics of PD

Pec pipecolic acid

PEG poly (ethylene glycol)

pGlu pyroglutamic acid

Pic picolinic acid

PK pharmacokinetics

pY phosphotyrosine

RBS ribosome binding site

RT Room temperature (about 25 ℃ C.)

Sar sarcosine

SDS sodium dodecyl sulfate

STK serine-threonine kinases

t-Boc tert-butoxycarbonyl

tBu tert-butyl

TCR T cell receptor

TFA trifluoroacetic acid

THF thymic humoral factor

Trt trityl radical

Claims (37)

1. An isolated immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

(a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:323 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:188 or SEQ ID NO: 190; or

(b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO 321 and the light chain variable region comprises the amino acid sequence of SEQ ID NO 188 or SEQ ID NO 190; or

(c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO 325 and the light chain variable region comprises the amino acid sequence of SEQ ID NO 182, SEQ ID NO 188 or SEQ ID NO 190.

2. An isolated immunoglobulin comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein:

(a) the light chain variable region comprises the amino acid sequence of SEQ ID NO 196 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 335, SEQ ID NO 349, SEQ ID NO 351, SEQ ID NO 353, SEQ ID NO 355 or SEQ ID NO 359; or

(b) The light chain variable region comprises the amino acid sequence of SEQ ID NO 204 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 349 or SEQ ID NO 355; or

(c) The light chain variable region comprises the amino acid sequence of SEQ ID NO 202 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 349; or

(d) The light chain variable region comprises the amino acid sequence of SEQ ID NO 192 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 357, SEQ ID NO 359, or SEQ ID NO 369; or

(e) The light chain variable region comprises the amino acid sequence of SEQ ID NO 194 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 335, SEQ ID NO 349 or SEQ ID NO 351.

3. The isolated immunoglobulin of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 323; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188.

4. The isolated immunoglobulin of claim 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO 196; and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 353.

5. The isolated immunoglobulin of claim 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO 202; and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 349.

6. The isolated immunoglobulin of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 325; and the light chain variable region comprises the amino acid sequence of SEQ ID NO 190.

7. The isolated immunoglobulin of claim 1 or claim 2, wherein the isolated immunoglobulin comprises an antibody or antibody fragment.

8. The isolated immunoglobulin of claim 7 comprising IgG1, IgG2, IgG3, or IgG 4.

9. The isolated immunoglobulin of claim 7 comprising a monoclonal antibody.

10. The isolated immunoglobulin of claim 9 comprising a human antibody.

11. The isolated immunoglobulin of claim 10, comprising:

(a) 113, or an immunoglobulin heavy chain comprising the aforementioned sequence lacking one, two, three, four or five amino acid residues at the N-terminus or C-terminus or both; and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO 110, or comprising the aforementioned sequences lacking one, two, three, four or five amino acid residues at the N-terminus or the C-terminus or both; or

(b) 125, or the aforementioned sequences lacking one, two, three, four or five amino acid residues at the N-terminus or C-terminus or both; and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO 122, or the foregoing sequences lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; or

(c) 101, or an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 101, or the aforementioned sequences lacking one, two, three, four, or five amino acid residues at the N-terminus or C-terminus, or both; and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO 98, or comprising the aforementioned sequences lacking one, two, three, four or five amino acid residues at the N-terminus or C-terminus or both; or

(d) An immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO 119, or comprising the aforementioned sequences lacking one, two, three, four or five amino acid residues at the N-terminus or C-terminus or both; and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO 116, or comprising the aforementioned sequences lacking one, two, three, four or five amino acid residues at the N-terminus or the C-terminus or both.

12. The isolated immunoglobulin of any of claims 1-6 or 8-11, further comprising 1-24 pharmacologically active chemical moieties conjugated thereto.

13. The isolated immunoglobulin of claim 12, wherein the pharmacologically active chemical moiety is a pharmacologically active polypeptide.

14. The isolated immunoglobulin of claim 13, wherein the immunoglobulin is recombinantly produced.

15. The isolated immunoglobulin of claim 14, wherein the immunoglobulin comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is inserted into the primary amino acid sequence of the immunoglobulin heavy chain within an internal loop of the immunoglobulin heavy chain Fc domain.

16. The isolated immunoglobulin of claim 13, wherein the immunoglobulin comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is conjugated at the N-terminus or C-terminus of the immunoglobulin heavy chain.

17. The isolated immunoglobulin of claim 13, wherein the immunoglobulin comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is conjugated at the N-terminus or C-terminus of the immunoglobulin light chain.

18. The isolated immunoglobulin of claim 13, wherein the pharmacologically active polypeptide is a toxin peptide, an IL-6 binding peptide, a CGRP peptide antagonist, a bradykinin B1 receptor peptide antagonist, a PTH agonist peptide, a PTH antagonist peptide, an ang-1 binding peptide, an ang-2 binding peptide, a myostatin binding peptide, an EPO-mimetic peptide, a FGF21 peptide, a TPO-mimetic peptide, an NGF binding peptide, a BAFF antagonist peptide, GLP-1 or a peptidomimetic thereof, or GLP-2 or a peptidomimetic thereof.

19. The isolated immunoglobulin of claim 18, wherein the toxin peptide is ShK or an ShK peptide analog.

20. A pharmaceutical composition comprising an immunoglobulin according to any one of claims 1 to 19; and a pharmaceutically acceptable diluent, excipient or carrier.

21. An isolated nucleic acid encoding the immunoglobulin of any one of claims 1-11.

22. An isolated nucleic acid encoding the immunoglobulin of claim 3.

23. An isolated nucleic acid encoding the immunoglobulin of claim 4.

24. An isolated nucleic acid encoding the immunoglobulin of claim 5.

25. An isolated nucleic acid encoding the immunoglobulin of claim 6.

26. An isolated nucleic acid encoding the immunoglobulin of claim 11.

27. An isolated nucleic acid encoding the immunoglobulin of any one of claims 13-19.

28. A vector comprising the isolated nucleic acid of claim 21.

29. A vector comprising the isolated nucleic acid of any one of claims 22 to 26.

30. A vector comprising the isolated nucleic acid of claim 27.

31. The vector of claim 28, comprising an expression vector.

32. The vector of claim 29, comprising an expression vector.

33. The vector of claim 30, comprising an expression vector.

34. An isolated host cell comprising the expression vector of any one of claims 31-33.

35. A method, comprising:

(a) culturing the host cell of claim 34 in a culture medium under conditions that allow expression of the immunoglobulin encoded by the expression vector; and

(b) recovering the immunoglobulin from the culture medium.

36. The immunoglobulin of claim 1, wherein 30 micromolar of the immunoglobulin does not significantly bind 30 nanomolar of soluble human IL-17R (SEQ ID NO:89) in an aqueous solution incubated under physiological conditions as determined by a surface plasmon resonance binding assay.

37. The immunoglobulin of claim 2, wherein 10 micromolar of the immunoglobulin does not significantly bind 10 nanomolar of soluble human TR2 (SEQ ID NO:82) in an aqueous solution incubated under physiological conditions as determined by a surface plasmon resonance binding assay.