Hybrid immunoglobulin containing non-peptidyl linkage

Abstract

To provide new protein compounds, incorporating nonprotein chains, that are both flexible and extendible, as well as processes for producing such compounds.SOLUTION: The present invention provides a compound having the following structure (In the formula, A is a biologically active structure of the compound; Z is a protein component of the compound, where the protein component comprises one or more polypeptides, where at least one of the one or more polypeptides comprises consecutive amino acids, where the consecutive amino acids (i) are identical to a stretch of consecutive amino acids present in a chain of an Fc domain of an antibody; (ii) bind to an Fc receptor; and (iii) have at their N-terminus a sequence selected from the group consisting of a cysteine or selenocysteine; the dashed line between B and Z represents a peptidyl linkage; and the solid line between A and B represents a nonpeptidyl linkage), as well as intermediates dimers thereof, and processes of producing the compounds of the invention.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 61 / 953,650, filed March 14, 2014, the entire contents of which are incorporated herein by reference.

[0002] This application incorporates by reference the nucleotide and / or amino acid sequences present in file name "150313_0893_86150_PCT_Sequence_Listing_REB.txt", which is 499 kilobytes in size and was created on Mar. 13, 2015 in IBM-PC format with MS-Windows operating system compatibility, and which are contained in a text file submitted as part of this application on Mar. 13, 2015.

[0003] Throughout this application, various publications are referenced. The disclosures of all references are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this invention pertains. BACKGROUND OF THEINVENTION

[0004] Proteins tend to form compact globular or fibrous structures that minimize their exposure to solvent. This tendency is inherent both to the polypeptide backbone, which tends to adopt secondary structures through hydrogen bonding, and to side-chain interactions that promote tertiary folding. Thus, previous efforts to introduce "flexibility" into antibodies using peptides have been largely inadequate. For example, a combination of amino acids that tend to interact with the solvent (e.g., serine) and those that break helical structure (e.g., glycine) is often used. This approach is useful for making fusion proteins, e.g., single-chain antibody fragments (scFv), but the resulting structures are indeed quite compact and have no evidence of extensibility (see, e.g., Robert et al, (2009) Engineered antibody intervention strategies for Alzheimer's disease and related dementias by targeting amyloid and toxic oligomers. Protein Eng. Des. Sel. 22, 199-208). Moreover, such sequences may pose additional problems due to intrinsic immunogenicity and susceptibility to proteolytic degradation.

[0005] There is a need for novel protein compounds that incorporate non-protein chains that are flexible and extensible, and methods for producing such compounds. Summary of the Invention

[0006] The present invention relates to a compound having the structure

[0007] [ka]

[0008] where A is a biologically active structure of the compound; Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of which comprises consecutive amino acids, the consecutive amino acids being (i) F of an antibody; c (ii) F c (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is a chemical structure that connects A and Z, The dotted line between B and Z represents a peptidyl bond. The solid line between A and B represents a non-peptidyl bond).

[0009] The present invention also provides a compound having the structure

[0010] [ka]

[0011] wherein Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of which comprises consecutive amino acids, the consecutive amino acids being (i) an F of an antibody; c (ii) F c (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is R a and C, The dotted line between B and Z represents a peptidyl bond. L is -N 3 , alkyne,

[0012] [ka]

[0013] Group (where R 5 is an alkyl group or an aryl group;

[0014] [ka]

[0015] is selected from the group consisting of a tetrazine group, a tetrazine group, and a trans-cyclooctene group; R a is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0016] [ka]

[0017] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0018] [ka]

[0019] [is].

[0020] The present invention also provides a compound having the structure

[0021] [ka]

[0022] where A is a biologically active structure of the compound; Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of which comprises consecutive amino acids, the consecutive amino acids being (i) F of an antibody; c (ii) F c (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is a chemical structure that connects A and Z, The dotted line between B and Z represents a peptidyl bond. The solid line between A and B represents a non-peptidyl bond. 1. A method for producing a) obtaining A' comprising A or a derivative of A and a first terminal reactive group; b) obtaining B' comprising B or a derivative of B, a second terminal reactive group, and a third terminal reactive group, the second terminal reactive group being capable of reacting with the first terminal reactive group to form a non-peptidyl bond; c) obtaining Z' comprising Z or a derivative of Z and a fourth terminal reactive group, the fourth terminal reactive group being capable of reacting with a third terminal reactive group to form a peptidyl bond; and d) reacting A', B' and Z' in any order to produce a compound. The present invention provides a method comprising: [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows the preparation of alkyne-modified TNR1B by cleavage of the TNR1B-intein fusion protein with cystyl-propargylamide. The intein by-product is removed by chitin chromatography. Azide-modified TNR1B and cycloalkyne-modified TNR1B are similarly prepared using cystyl-3-azidopropylamide and various cyclooctyne (e.g., DIBAC) derivatives of cysteine, respectively. [Diagram 2] Figure 2 shows the cleavage of TNR1B by (1) cysteine, (2) cysteine ​​+ mercaptoethanesulfonate (MESNA), (3) cystyl-propargylamide, (4) cystyl-propargylamide + MESNA, and (5) MESNA. All compounds were used at 50 mM concentration. [Diagram 3] FIG. 3 shows the preparation of azide-modified Fc6 by linkage (peptidyl) of Fc6 dimer and azide-DKTHT-thioester (Table 1). [Figure 4] Figure 4 shows the preparation of azide-modified Fc6 by linking (peptidyl) Fc6 dimer and azide-PEG-DKTHT-thioester (Table 1). Cycloalkyne-modified Fc is similarly prepared by using DIBAC-PEG12-thioester. [Diagram 5] FIG. 5 shows SDS-PAGE analysis (reducing conditions) of (1) unmodified Fc6, (2) the Az-DKTHT-Fc6 reaction product of FIG. 3, and (3) the Az-PEG4-DKTHT-Fc6 reaction product of FIG. [Figure 6]FIG. 6 shows the synthesis of TNR1B-alkyne-azide-Fc6 by conjugation (non-peptidyl) of alkyne-modified TNR1B and Az-DKTHT-Fc6. [Figure 7] 7 shows the synthesis of TNR1B-alkyne-azide-PEG-Fc6 by coupling (non-peptidyl) of alkyne-modified TNR1B and azide-PEG-DKTHT-Fc6. In this example, n=4. [Figure 8] FIG. 8 shows SDS-PAGE analysis (reducing conditions) of (1) alkyne modified TNR1B alone, (2) alkyne modified TNR1B+Az-DKTHT-Fc6 in the absence of catalyst, (3) alkyne modified TNR1B+Az-DKTHT-Fc6+catalyst resulting in the products of FIG. 6, and (4) dialyzed alkyne modified TNR1B+Az-DKTHT-Fc6+catalyst increasing the formation of the products of FIG. 6, (5) alkyne modified TNR1B+Az-PEG4-DKTHT-Fc6 in the absence of catalyst, (6) alkyne modified TNR1B+Az-PEG4-DKTHT-Fc6+catalyst resulting in the products of FIG. 7, and (7) dialyzed alkyne modified TNR1B+Az-PEG4-DKTHT-Fc6+catalyst increasing the formation of the products of FIG. 7. The arrows correspond to (a) Mr approx. 75,000, (b) Mr approx. 65,000, (c) Mr approx. 43,000, and (d) Mr approx. 28,000. [Figure 9] Figure 9 shows SDS-PAGE analysis (reducing conditions) of (1) TNF1B-Fc fusion protein (etanercept) alone, (2) alkyne-modified TNR1B+Az-DKTHT-Fc6+catalyst resulting in the product of Figure 6, (3) TNF1B-Fc fusion protein (etanercept), and (4) alkyne-modified TNR1B+Az-PEG4-DKTHT-Fc6 resulting in the product of Figure 7. Arrows correspond to (a) Mr approx. 75,000, (b) Mr approx. 65,000, (c) Mr approx. 43,000, and (d) Mr approx. 28,000. [Figure 10]Figure 10 shows SDS-PAGE analysis (reducing conditions) of (1) unmodified Fc6+ catalyst, (2) alkyne-modified TNR1B + unmodified Fc6+ catalyst (3) Az-DKTHT-Fc6+ catalyst, (4) alkyne-modified TNR1B + Az-DKTHT-Fc6+ catalyst resulting in the product of Figure 6, and (5) alkyne-modified TNR1B alone. Arrows correspond to (a) Mr approx. 75,000, (b) Mr approx. 65,000, (c) Mr approx. 43,000, (d) Mr approx. 28,000, and (e) Mr approx. 27,000. [Figure 11] Figure 11 shows the tryptic peptidation of the TNR1B-alkyne-azide-DKTHT-Fc6 product (Mr approx. 75,000) of Figure 10 by LC / MS. The underlined peptide sequences are derived from the parent TNR1B (top) and Fc6 (bottom) sequences and were identified by LC / MS. [Figure 12] Figure 12 shows SPR analysis of TNF- binding by the TNR1B-alkyne-azide-DKTHT-Fc6 (left panel) and TNR1B-alkyne-azide-PEG4-DKTHT-Fc6 (right panel) reaction products of Figure 9. Kinetic binding data are summarized in Table 2. [Figure 13] FIG. 13 shows the preparation of adalimumab Fab' in a three-step process: 1) IdeS cleavage to Fab'2+Fc' fragments, 2) Protein A chromatography to remove the Fc' fragment, and 3) mild reduction of the Fab'2 fragment to a Fab' fragment using 2-mercaptoethylamine (MEA). [Figure 14]14 shows SDS-PAGE analysis of (1) adalimumab, (2) adalimumab after IdeS cleavage, (3) adalimumab Fab'2 after Protein A purification, (4) adalimumab Fab' after MEA treatment of Fab'2 purified by Protein A, (5) adalimumab Fab'2 after Protein A purification, and (6) adalimumab Fab' after MEA treatment of Fab'2 purified by Protein A. Samples in lanes 1, 2, 5, and 6 were analyzed under reducing conditions; samples in lanes 3 and 4 were analyzed under non-reducing conditions. Arrows correspond to (a) heavy chain, (b) heavy chain Fc' fragment, (c) heavy chain Fd' (variable region-containing) fragment, and (d) light chain. [Figure 15] Figure 15 shows the preparation of cycloalkyne modified Fab' by reaction of Adalimumab Fab' with DIBAC-PEGy-Lys(Mal). In this example, PEGy=PEG12. [Figure 16] FIG. 16 shows SDS-PAGE analysis (non-reducing conditions) of the synthesis and purification of cycloalkyne-modified adalimumab Fab'. The top panels show reactions at (1-6) pH 7.4 and (7-12) pH 7.0. The DIBAC-PEGy-Lys(Mal) to Fab' rations were (1) 0, (2) 10:1, (3) 5:1, (4) 2.5:1, (5) 1.2:1, (6) 0.6:1, (7) 0, (8) 10, (9) 5, (10) 2.5, (11) 1.2, and (12) 0.6:1. The bottom panels show (1) unreacted Fab', (2) through (12) Protein L flow-through fractions containing only cycloalkyne-modified Fab'. [Figure 17] FIG. 17 shows SDS-PAGE analysis (reducing conditions) of (1) Fc6, (2) Az-DKTHT-Fc6, (3) Az-PEG12-DKTHT-Fc6, (4) Az-PEG24-DKTHT-Fc6, and (5) Az-PEG36-DKTHT-Fc6. [Figure 18]FIG. 18 shows size-exclusion chromatography of (a) Az-PEG36-DKTHT-Fc6, (b) Az-PEG24-DKTHT-Fc6, (c) Az-PEG12-DKTHT-Fc6, (d) Az-DKTHT-Fc6, and (e) Fc6. [Figure 19] FIG. 19 shows the synthesis of Fab′-PEGy-alkyne-azide-PEGx-Fc6 by conjugation (non-peptidyl) of cycloalkyne-modified adalimumab Fab′ and azide-modified Fc6. [Figure 20] FIG. 20 shows the Fab'-PEGy-alkyne-azide-PEGx-Fc6 product series. [Figure 21] FIG. 21 shows SDS-PAGE analysis of (1) adalimumab whole antibody, (2) adalimumab Fab', (3) Fab'-PEG12-alkyne, (4) Fab'-PEG12-alkyne+Az-DKTHT-Fc6, (5) Az-DKTHT-Fc6, (6) Fab'-PEG12-alkyne+Az-PEG12-DKTHT-Fc6, (7) Az-PEG12-DKTHT-Fc6, (8) Fab'-PEG12-alkyne+Az-PEG24-DKTHT-Fc6, (9) Az-PEG24-DKTHT-Fc6 alone, (10) Fab'-PEG12-alkyne+Az-PEG36-DKTHT-Fc6, (11) Az-PEG36-DKTHT-Fc6, and (12) Fc6. Samples were run under reducing (top panel) and non-reducing (bottom panel) conditions. In the top panel, the arrows indicate (a) Fab'-PEGy-alkyne-azide-PEGx-Fc6 heavy chain. In the bottom panel, the arrows indicate (a) two-handed Fab'-PEGy-alkyne-azide-PEGx-Fc6 molecule and (b) one-handed Fab'-PEGy-alkyne-azide-PEGx-Fc6 molecule. [Figure 22]FIG. 22 shows size exclusion chromatography (SEC) of both reaction products, namely, (a) Fab'-PEG12-alkyne-azide-PEG36-DKTHT-Fc6, (b) Fab'-PEG12-alkyne-azide-PEG24-DKTHT-Fc6, (c) Fab'-PEG12-alkyne-azide-PEG12-DKTHT-Fc6, (d) Fab'-PEG12-alkyne-azide-DKTHT-Fc6, and (e) total adalimumab. [Figure 23] Figure 23 shows the inhibition of TNF-α cytotoxicity in WEHI cells by the reaction products. The top panel shows (a) Fc6 control, (b) cycloalkyne modified Fab', (c) Fab'-PEG12-alkyne-azide-DKTHT-Fc6, and (d) Fab'-PEG12-alkyne-azide-PEG12-DKTHT-Fc6. The bottom panel shows (a) Fc6 control, (b) cycloalkyne modified Fab', (c) Fab'-PEG12-alkyne-azide-PEG24-DKTHT-Fc6, and (d) Fab'-PEG12-alkyne-azide-PEG36-DKTHT-Fc6. [Figure 24] FIG. 24 shows purified N3-Px-Fc proteins by SDS-PAGE under reducing (left) and non-reducing (right) conditions: Fc6 control (lane a), N3-P0-Fc (lane b), N3-P12-Fc (lane c), N3-P24-Fc (lane d), N3-P36-Fc (lane e), and N3-P48-Fc (lane f). [Diagram 25] FIG. 25 shows the structure and synthesis of a cyclooctyne-modified GLP-1 analog (GLP1-P7-DBCO). [Figure 26] FIG. 26 shows the reaction of GLP1-P7-DBCO with N3-Px-Fc protein. [Figure 27] FIG. 27 shows the structure of the GLP1-triazole-Fc hybrid immunoglobulin. [Figure 28]FIG. 28 shows purified GLP1-triazole-Fc hybrid immunoglobulins by SDS-PAGE under reducing (left) and non-reducing (right) conditions: Fc6 control (lane a), GLP1-P4-DN-P0-Fc (lane b), GLP1-P4-DN-P12-Fc (lane c), GLP1-P4-DN-P24-Fc (lane d), GLP1-P4-DN-P36-Fc (lane e), and GLP1-P4-DN-P48-Fc (lane f). [Figure 29] FIG. 29 directly compares GLP1-triazole-Fc hybrid immunoglobulin and N3-Px-Fc proteins by SDS-PAGE under reducing conditions: Fc6 control (lane a), N3-P0-Fc (lane b), GLP1-P4-DN-P0-Fc (lane c), N3-P12-Fc (lane d), GLP1-P4-DN-P12-Fc (lane e), N3-P24-Fc (lane f), GLP1-P4-DN-P24-Fc (lane g), N3-P36-Fc (lane h), GLP1-P4-DN-P36-Fc (lane i), N3-P48-Fc (lane j), and GLP1-P4-DN-P48-Fc (lane k). [Diagram 30] FIG. 30 compares the induction of cAMP synthesis in GLP-1 receptor expressing cells by GLP1-triazole-Fc hybrid immunoglobulin and GLP-1. [Diagram 31] FIG. 31 shows the reaction between DIBAC-PEG11-DIBAC and N3-Px-Fc protein. [Diagram 32] FIG. 32 shows the structure of the cyclooctyne-Px-Fc protein. [Diagram 33] FIG. 33 shows DIBAC-P11-DN-P0-Fc reaction products by SDS-PAGE under reducing conditions: Fc6 control (lane b), unpurified reaction product (lanes c-e), purified N3-P0-Fc protein (lane f), and purified DIBAC-P11-DN-P0-Fc protein (lane g). [Diagram 34] FIG. 34 shows the reaction of azide-modified DNA with cyclooctyne-Px-Fc protein. [Diagram 35]FIG. 35 shows the structure of a DNA-triazole-Fc hybrid immunoglobulin. [Diagram 36] Figure 36 shows the DNA-triazole-Fc hybrid immunoglobulin reaction products by SDS-PAGE under reducing conditions. The 5AzD-let7d oligonucleotide concentrations (mg / ml) were as follows: marker (lane a), 0 (lane b), 2.5 (lane c), 1.25 (lane d), 0.063 (lane e), 0.031 (lane f), 0.016 (lane g), 0.08 (lane h). [Figure 37] FIG. 37 shows the structures and synthesis of trastuzumab mutants, cys1H-IgG1, and azide-modified trastuzumab heavy chain (N3-Px-Hc). [Figure 38] FIG. 38 shows the structures and synthesis of trastuzumab variants, cys1H-IgG1, and azide-modified trastuzumab light chain (N3-Px-Lc). [Figure 39] FIG. 39 shows the structure and synthesis of cyclooctyne-modified DM-1 (DM1-P4-DBCO). [Diagram 40] FIG. 40 shows the reaction between cyclooctyne-modified DM-1 and N3-Px-Hc protein. [Diagram 41] FIG. 41 shows the structure of the DM1-P4-triazole-Px-Hc hybrid immunoglobulin. [Diagram 42] FIG. 42 shows the reaction between cyclooctyne-modified DM-1 and N3-Px-Lc protein. [Diagram 43] FIG. 43 shows the structure of the DM1-P4-triazole-Px-Lc hybrid immunoglobulin. [Diagram 44] FIG. 44 shows the reaction between tetrazine-DBCO and N3-Px-Fc protein. [Diagram 45] FIG. 45 shows the structure of tetrazine-modified Fc protein (Tet-Px-Fc). [Figure 46]FIG. 46 shows purified Tet-Px-Fc proteins by SDS-PAGE under reducing (left) and non-reducing (right) conditions: Fc6 control (lane a), Tet-P0-Fc (lane b), Tet-P12-Fc (lane c), Tet-P24-Fc (lane d), Tet-P36-Fc (lane e), and Tet-P48-Fc (lane f). [Figure 47] FIG. 47 shows the reaction between TCO-PEG12-DBCO and N3-Px-Fc protein. [Figure 48] FIG. 48 shows the structure of the trans-cyclooctene-modified Fc protein (Tco-Px-Fc). [Figure 49] Figure 49 shows Tco-P12-Px-Fc protein by SDS-PAGE under reducing conditions. Tco-P12-DMCO linker concentrations (mg / ml) were as follows: 32 (lane a), 16 (lane b), 8 (lane c), 4 (lane d), 2 (lane e), 1 (lane f), 0.5 (lane g), 0.25 (lane h), 0.125 (lane i), and 0 (lane j). [Figure 50] Figure 50 shows the reaction products of NH2-PEG23-N3 and DBCO-TT-P12-P36-Fc protein by SDS-PAGE under reducing conditions. The NH2-PEG23-N3 linker concentrations (mg / ml) were as follows: 0.12 (lane a), 0.06 (lane b), 0.03 (lane c), 0.015 (lane d), 0.0075 (lane e), 0.0038 (lane f), 0.002 (lane g), 0.001 (lane h), 0 (lane i). [Figure 51] FIG. 51 shows the structure and synthesis of a trans-cyclooctene modified GLP-1 analog (GLP1-P6-Tco). [Figure 52] FIG. 52 shows the reaction between the GLP1-P6-Tco peptide and the Tet-Px-Fc protein. [Figure 53] FIG. 53 shows the structure of the GLP1-dihydropyrididine-Fc hybrid immunoglobulin. [Figure 54]FIG. 54 shows the structure and synthesis of a tetrazine-modified GLP-1 analog (GLP1-P6-Tet). [Figure 55] FIG. 55 shows the reaction between the GLP1-P6-Tet peptide and the Tco-Px-Fc protein. [Figure 56] FIG. 56 shows the structure of the GLP1-P6-TT-Px-Fc hybrid immunoglobulin. [Figure 57] FIG. 57 shows purified GLP1-dihydropyridine-Fc hybrid immunoglobulins by SDS-PAGE under reducing (left) and non-reducing (right) conditions: Fc6 control (lane a), GLP1-P6-TT-P0-Fc (lane b), GLP1-P6-TT-P12-Fc (lane c), GLP1-P6-TT-P24-Fc (lane d), GLP1-P6-TT-P36-Fc (lane e), and GLP1-P6-TT-P048-Fc (lane f). [Figure 58] FIG. 58 directly compares N3-Px-Fc(I) protein, Tet-Px-Fc(II) protein, and GLP1-dihydropyrididine-Fc(III) hybrid immunoglobulin by SDS-PAGE under reducing conditions: Fc6 control (lane a), N3-P0-Fc (lane b), Tet-P0-Fc (lane c), GLP1-P6-TT-P0-Fc (lane d), N3-P12-Fc (lane e), Tet-P12-Fc (lane f). lane f), GLP1-P6-TT-P12-Fc (lane g), N3-P24-Fc (lane h), Tet-P24-Fc (lane i), GLP1-P6-TT-P24-Fc (lane j), N3-P36-Fc (lane k), Tet-P36-Fc (lane l), GLP1-P6-TT-P36-Fc (lane m), N3-P48-Fc (lane n), Tet-P48-Fc (lane o), and GLP1-P6-TT-P48-Fc (lane p). [Figure 59] FIG. 59 shows the time course of the reaction of GLP1-P7-DBCO with N3-P36-Fc and the time course of the reaction of GLP1-P6-Tco with Tet-P36-Fc. [Figure 60]FIG. 60 compares the induction of cAMP synthesis in GLP-1 receptor expressing cells by GLP1-dihydropyrididine-Fc hybrid immunoglobulin and GLP-1. [Figure 61] FIG. 61 shows the structure and synthesis of trans-cyclooctene modified adalimumab Fab (Fab-P3-Tco). [Figure 62] FIG. 62 shows the reaction between Fab-P3-Tco and Tet-Px-Fc proteins. [Figure 63] FIG. 63 shows the structure of a Fab-dihydropyridine-Fc hybrid immunoglobulin. [Figure 64] Figure 64 shows Fab-dihydropyridin-Fc hybrid immunoglobulins by SDS-PAGE under reducing conditions: marker (lane a), adalimumab (lane b), Fab-P3-TT-P0-Fc (lane c), Fab-P3-TT-P12-Fc (lane d), Fab-P3-TT-P24-Fc (lane e), Fab-P3-TT-P36-Fc (lane f), Fab-P3-TT-P48-Fc (lane g), Fab-P3-Tco (lane h), Tet-P0-Fc (lane i), Tet-P12-Fc (lane j), Tet-P24-Fc (lane k), Tet-P36-Fc (lane l), Tet-P48-Fc (lane m). [Figure 65] FIG. 65 shows the structure and synthesis of azide- and trans-cyclooctene-modified olanzapine (Ola-P12-Tco). [Figure 66] FIG. 66 shows the reaction between Ola-P12-Tco and Tet-Px-Fc proteins. [Figure 67] FIG. 67 shows the structure of an olanzapine-dihydropyrididine-Fc hybrid immunoglobulin. Detailed Description of the Invention

[0024] The present invention relates to a compound having the structure

[0025] [ka]

[0026] where A is a biologically active structure of the compound; Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of which comprises consecutive amino acids, the consecutive amino acids being (i) F of an antibody; c (ii) F c (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is a chemical structure that connects A and Z, The dotted line between B and Z represents a peptidyl bond. The solid line between A and B represents a non-peptidyl bond).

[0027] In some embodiments, cysteine ​​or selenocysteine ​​is naturally occurring in the series of consecutive amino acids. In some embodiments, cysteine ​​and selenocysteine ​​are not naturally occurring in the series of consecutive amino acids.

[0028] In some embodiments, the contiguous amino acids have a sequence at the N-terminus selected from the group consisting of cysteine, selenocysteine, CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP, and CDTPPPCPRCP.

[0029] In some embodiments, the F of the antibody c The domain is the naturally occurring F of an antibody. c It is a domain.

[0030] In some embodiments, the F of the antibody c The domain is the variant F of the antibody. c It is a domain.

[0031] In some embodiments, the variant F of the antibody c The domain is the mutated F of the antibody. c It is a domain.

[0032] In some embodiments, the mutated F c The domains are substitution mutants.

[0033] In some embodiments, the substitution variant is an F at the N-terminus, C-terminus, or other than the N-terminus or C-terminus. c It has an amino acid substitution at a certain position in the domain.

[0034] In some embodiments, a substitution variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 amino acid substitutions in its stretch of contiguous amino acids.

[0035] In some embodiments, the substitutions are conservative amino acid substitutions.

[0036] In some embodiments, the mutated F c The domains are amino acid addition variants.

[0037] In some embodiments, the amino acid addition variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 additional amino acids in its stretch of consecutive amino acids.

[0038] In some embodiments, the mutated F c The domains are amino acid deletion mutants.

[0039] In some embodiments, an amino acid deletion mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 deleted amino acids in its stretch of consecutive amino acids.

[0040] In some embodiments, the consecutive amino acids are cIt is identical to a stretch of at least 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 consecutive amino acids present in the domain chain.

[0041] In some embodiments, the contiguous amino acids are identical to a series of amino acids in the hinge region, CH2 region, or CH3 region of an Fc domain, or a portion thereof.

[0042] In some embodiments, the solid line between A and B represents the following structure:

[0043] [ka]

[0044] represents a non-peptidyl bond comprising Where:

[0045] [ka]

[0046] teeth,

[0047] [ka]

[0048] (where R 5 is an alkyl group or an aryl group. and R 1 is H or part of an additional structure which is a cyclic structure, where the additional cyclic structure is R 1 or R 1 Includes part of R 2 or R 2 Part of, and R 2 and the carbon between the alkene double bond, however,

[0049] [ka]

[0050] but,

[0051] [ka]

[0052] If R 3 is H,

[0053] [ka]

[0054] but,

[0055] [ka]

[0056] If

[0057] [ka]

[0058] teeth,

[0059] [ka]

[0060] is a triazole ring containing

[0061] [ka]

[0062] but,

[0063] [ka]

[0064] If

[0065] [ka]

[0066] teeth,

[0067] [ka]

[0068] is an N-alkyl or aryl substituted isoxazoline ring comprising R 2 represents an organic structure connecting to either A or B, and R 4 represents an organic structure connecting A or B to the other.

[0069] In some embodiments, the solid line between A and B represents the following structure:

[0070] [ka]

[0071] represents a non-peptidyl bond comprising Here, R 1 is H or part of an additional structure which is a cyclic structure, where the additional cyclic structure is R 1 or R 1 Includes part of R 2 or R 2 Part of, and R 2 and the alkene double bond may also be included.

[0072] In some embodiments, the solid line between A and B represents the following structure:

[0073] [ka]

[0074] represents a non-peptidyl bond comprising Here, R 1 is H or part of an additional structure which is a cyclic structure, where the additional cyclic structure is R 1 or R 1 Includes part of R 2 or R 2 Part of, and R 2 and the alkene double bond may also be included.

[0075] In some embodiments, the solid line between A and B represents the following structure:

[0076] [ka]

[0077] represents a non-peptidyl bond comprising Here, R 1 is part of an additional structure that is a cyclic structure, where the additional cyclic structure is R 1 or R 1 Includes part of R 2 or R 2 Part of, and R 2 and the alkene double bond may also be included.

[0078] In some embodiments, the solid line between A and B represents the following structure:

[0079] [ka]

[0080] represents a non-peptidyl bond comprising Here, R 1 is part of an additional structure that is a cyclic structure, where the additional cyclic structure is R 1 or R 1 Includes part of R 2 or R 2 Part of, and R2 and the alkene double bond may also be included.

[0081] In some embodiments, R 1 and R 2 are linked by at least one direct bond to i)R 1 Part of ii) R 2 Part of iii) R 2 and the carbon between the alkene double bond, iv) Alkene double bonds and A ring structure comprising:

[0082] In some embodiments, R 1 is optionally substituted at any position,

[0083] [ka]

[0084] is selected from the group consisting of:

[0085] In some embodiments, R 1 is optionally substituted at any position

[0086] [ka]

[0087] It is.

[0088] In some embodiments, R 1 is optionally substituted at any position

[0089] [ka]

[0090] It is.

[0091] In some embodiments, R 1 is optionally substituted at any position

[0092] [ka]

[0093] It is.

[0094] In some embodiments, R 2 The carbon between the alkene double bond and i) Single bond with R 2 and is substituted with two substituents independently selected from the group consisting of hydrogen, halogen, optionally substituted benzyl, optionally substituted alkyl, or optionally substituted alkoxy; ii) R by double bonds and single bonds 2 is directly bonded to

[0095] In some embodiments, R 2 The carbon between the and alkene double bond is replaced with two hydrogens, and the single bond is R 2 is directly bonded to

[0096] In some embodiments, R 2 The carbon between the alkene double bond and the R is bounded by a double bond and a single bond. 2 is directly bonded to

[0097] In some embodiments, R 2 The carbon between the alkene double bond and the R is bounded by a double bond and a single bond. 2 is directly attached to form a phenyl ring which may be optionally substituted at any position.

[0098] In some embodiments, R 2 teeth,

[0099] [ka]

[0100] and R 2 is linked to A or B by J, J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0101] [ka]

[0102] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0103] [ka]

[0104] It is.

[0105] In some embodiments, R 2 teeth,

[0106] [ka]

[0107] and R 2 is connected to A or B by J, and R 2 R by nitrogen atom 1 is bound to J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0108] [ka]

[0109] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0110] [ka]

[0111] It is.

[0112] In some embodiments, R 2 is optionally substituted at any position,

[0113] [ka]

[0114] and R 2 R depending on the nitrogen or carbon atom of 1 is bound to R 2is linked to A or B by J, J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0115] [ka]

[0116] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0117] [ka]

[0118] It is.

[0119] In some embodiments, R 2 is optionally substituted at any position

[0120] [ka]

[0121] It is.

[0122] In some embodiments, R 2 is optionally substituted at any position

[0123] [ka]

[0124] It is.

[0125] In some embodiments, R 2 is optionally substituted at any position

[0126] [ka]

[0127] It is.

[0128] In some embodiments, R 2 is optionally substituted at any position

[0129] [ka]

[0130] It is.

[0131] In some embodiments, R 2 is optionally substituted at any position

[0132] [ka]

[0133] It is.

[0134] In some embodiments, R 2 is optionally substituted at any position

[0135] [ka]

[0136] It is.

[0137] In some embodiments, the group R 1 and R 2 is optionally substituted at any position,

[0138] [ka]

[0139] and J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0140] [ka]

[0141] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0142] [ka]

[0143] It is.

[0144] In some embodiments, the group R 1 and R 2 is optionally substituted at any position

[0145] [ka]

[0146] It is.

[0147] In some embodiments, the group R 1 and R 2 is optionally substituted at any position

[0148] [ka]

[0149] It is.

[0150] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0151] [ka]

[0152] represents a non-peptidyl bond comprising J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0153] [ka]

[0154] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0155] [ka]

[0156] It is.

[0157] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0158] [ka]

[0159] represents a non-peptidyl bond including:

[0160] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0161] [ka]

[0162] represents a non-peptidyl bond including:

[0163] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0164] [ka]

[0165] represents a non-peptidyl bond comprising J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0166] [ka]

[0167] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0168] [ka]

[0169] It is.

[0170] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0171] [ka]

[0172] represents a non-peptidyl bond including:

[0173] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0174] [ka]

[0175] represents a non-peptidyl bond including:

[0176] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0177] [ka]

[0178] represents a non-peptidyl bond comprising J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0179] [ka]

[0180] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0181] [ka]

[0182] It is.

[0183] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0184] [ka]

[0185] represents a non-peptidyl bond including:

[0186] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0187] [ka]

[0188] represents a non-peptidyl bond including:

[0189] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0190] [ka]

[0191] represents a non-peptidyl bond comprising J is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0192] [ka]

[0193] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0194] [ka]

[0195] It is.

[0196] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0197] [ka]

[0198] represents a non-peptidyl bond including:

[0199] In some embodiments, the solid line between A and B represents the following structure, optionally substituted at any position:

[0200] [ka]

[0201] represents a non-peptidyl bond including:

[0202] In some embodiments, the solid line between A and B represents the following structure:

[0203] [ka]

[0204] represents a non-peptidyl bond including:

[0205] In some embodiments, R 1 is H.

[0206] In some embodiments, J is an organic structure comprising a [PEG(y)]z group.

[0207] In some embodiments, J is a polyalkylene glycol, a polyoxyalkylated polyol, a polyvinyl alcohol, a polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), or an organic structure comprising a polysaccharide group.

[0208] In some embodiments, J is C 1 ~C 4 It is an organic structure that contains an alkyl group.

[0209] In some embodiments, J is an organic structure that comprises a succinimide.

[0210] In some embodiments, J is an organic structure that comprises an amine.

[0211] In some embodiments, J is an organic structure comprising succinyl, malonyl, glutaryl, phthalyl, or adipoyl.

[0212] In some embodiments, J is an organic structure that comprises malonyl.

[0213] In some embodiments, J is an organic structure comprising an amino acid.

[0214] In some embodiments, J is an organic structure that includes a cysteine.

[0215] In some embodiments, J is an organic structure that includes a lysine.

[0216] In some embodiments, J is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0217] [ka]

[0218] It is an organic structure consisting of a chain of three moieties selected from the group consisting of:

[0219] In some embodiments, J is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0220] [ka]

[0221] It is an organic structure consisting of a chain of four moieties selected from the group consisting of:

[0222] In some embodiments, J is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0223] [ka]

[0224] It is an organic structure consisting of a chain of five moieties selected from the group consisting of:

[0225] In some embodiments, J comprises a [PEG(y)]z group attached to a lysine.

[0226] In some embodiments, J is a C bonded to a succinimide group. 1 ~C 4 Contains an acyl group.

[0227] In some embodiments, J is C 1 ~C 4 Contains lysine bound to acyl.

[0228] In some embodiments, J comprises a [PEG(y)]z group attached to the glutaryl.

[0229] In some embodiments, J is [PEG(y)], C 2 ~C 5 Acyl, succinyl, malonyl, glutaryl, amino acid, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0230] [ka]

[0231] It is an organic structure consisting of a chain of five moieties selected from the group consisting of:

[0232] In some embodiments, J is a bond.

[0233] In some embodiments, J is cysteine.

[0234] In some embodiments, J has the structure

[0235] [ka]

[0236] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). has.

[0237] In some embodiments, J has a linear structure.

[0238] In some embodiments, J has a branched structure.

[0239] In some embodiments, R 2 teeth,

[0240] [ka]

[0241] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). It is.

[0242] In some embodiments, R 2 teeth,

[0243] [ka]

[0244] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). It is.

[0245] In some embodiments, R 2 teeth,

[0246] [ka]

[0247] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). It is.

[0248] In some embodiments, the group R 1 and R 2 teeth,

[0249] [ka]

[0250] or

[0251] [ka]

[0252] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). It is.

[0253] In some embodiments, the group R 1 and R 2 teeth,

[0254] [ka]

[0255] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). It is.

[0256] In some embodiments, the group R 1 and R 2 teeth,

[0257] [ka]

[0258] (wherein n is 1 to 3, m is 1 to 4, y is 1 to 100, and z is 1 to 10). It is.

[0259] In some embodiments, the solid line between A and B represents the following structure:

[0260] [ka]

[0261] represents a non-peptidyl bond comprising [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0262] [ka]

[0263] It is.

[0264] In some embodiments, the solid line between A and B represents the following structure:

[0265] [ka]

[0266] represents a non-peptidyl bond including:

[0267] In some embodiments, the solid line between A and B represents the following structure:

[0268] [ka]

[0269] represents a non-peptidyl bond including:

[0270] In some embodiments, the solid line between A and B represents the following structure:

[0271] [ka]

[0272] represents a non-peptidyl bond comprising [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0273] [ka]

[0274] and [PEG(x)]w is x=1 to 100 and w=1 to 10.

[0275] [ka]

[0276] It is.

[0277] In some embodiments, y is 1 to 20. In some embodiments, y is 21 to 40. In some embodiments, y is 41 to 60. In some embodiments, y is 61 to 80. In some embodiments, y is 30 to 50. In some embodiments, y is 12, 24, 36, or 48. In some embodiments, z is 1. In some embodiments, z is 0.

[0278] In some embodiments, the terminal carbonyl of the [PEG(y)]z group is part of an amide bond.

[0279] In some embodiments, the terminal amine of the [PEG(y)]z group is part of an amide bond.

[0280] In some embodiments, R4 is x=1~100 and w=0~5

[0281] [ka]

[0282] It is.

[0283] In some embodiments, x is 1 to 20. In some embodiments, x is 21 to 40. In some embodiments, x is 41 to 60. In some embodiments, x is 61 to 80. In some embodiments, x is 30 to 50. In some embodiments, x is 12, 24, 36, or 48.

[0284] In some embodiments, w is 1. In some embodiments, w is 0.

[0285] In some embodiments, R 4 has the following structure:

[0286] [ka]

[0287] has.

[0288] In some embodiments, R 2 is connected to A by J and to R 4 is bonded to B.

[0289] In some embodiments, R 2 is connected to B by J and to R 4 is bonded to A.

[0290] In some embodiments, R 4 is attached to B through the terminal carbonyl carbon.

[0291] In some embodiments, the solid line between A and B represents the following structure:

[0292] [ka]

[0293] (where p=0~5, 0~10, 0~50, or 0~100) represents a non-peptidyl bond including:

[0294] In some embodiments, R 2 is bonded to A by a carbon-nitrogen bond or a carbon-sulfur bond.

[0295] In some embodiments, R 2 is attached to A by a carbon-nitrogen bond.

[0296] In some embodiments, the carbon-nitrogen bond is an amide bond.

[0297] In some embodiments, R 2 is attached to A by a biologically labile bond.

[0298] In some embodiments, R 2 is linked to A by an amide bond between the C-terminal amino acid of A and the amino group in B.

[0299] In some embodiments, the terminal amino acid is a cysteine.

[0300] In some embodiments, R 2 is attached to A by a carbon-sulfur bond.

[0301] In some embodiments, R 2 is R 2 is linked to A by a carbon-sulfur bond formed between and the free thiol.

[0302] In some embodiments, R 2 is attached to A by a succinimide-sulfur bond.

[0303] In some embodiments, J comprises a branching residue.

[0304] In some embodiments, J is linked to two or more A's by said branching residues.

[0305] In some embodiments, B comprises a branching residue.

[0306] In some embodiments, B is linked to two or more A's by non-peptidyl bonds, each through a branched residue.

[0307] In some embodiments, the solid line between A and B represents the following structure:

[0308] [ka]

[0309] represents a non-peptidyl bond comprising Where X a is a chemical structure containing cyclooctane fused to dihydropyridazine, R a represents an organic structure connecting to either A or B, and R b represents an organic structure connecting A or B to the other.

[0310] In some embodiments, X a has the following structure:

[0311] [ka]

[0312] (where R c is H, alkyl or aryl) or a tautomer thereof.

[0313] In some embodiments, X a has the following structure:

[0314] [ka]

[0315] (where R c is H, alkyl or aryl) or a tautomer thereof.

[0316] In some embodiments, R a is connected to cyclooctane, R b is connected to a dihydropyridazine.

[0317] In some embodiments, the solid line between A and B represents the following structure:

[0318] [ka]

[0319] (where R c is H, alkyl or aryl) or a non-peptidyl bond including a tautomer thereof.

[0320] In some embodiments, X a has the following structure:

[0321] [ka]

[0322] (where R c is H, alkyl or aryl) or a tautomer thereof.

[0323] In some embodiments, R c is methyl.

[0324] In some embodiments, R a and R b is independently a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0325] [ka]

[0326] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0327] [ka]

[0328] It is.

[0329] In some embodiments, R a and R b are independently a bond or each moiety is [PEG(y)]z, C 2 ~C 5 Acyl, succinyl, malonyl, glutaryl, amino acid, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0330] [ka]

[0331] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0332] [ka]

[0333] It is.

[0334] In some embodiments, Ra or Rb is attached to A by a carbon-nitrogen bond or a carbon-sulfur bond.

[0335] In some embodiments, Ra or Rb is attached to A by a carbon-nitrogen bond.

[0336] In some embodiments, the carbon-nitrogen bond is an amide bond.

[0337] In some embodiments, Ra or Rb is linked to A by a biologically labile bond.

[0338] In some embodiments, Ra or Rb is linked to A by an amide bond between the C-terminal amino acid of A and an amino group on B.

[0339] In some embodiments, the terminal amino acid is a cysteine.

[0340] In some embodiments, Ra or Rb is attached to A by a carbon-sulfur bond.

[0341] In some embodiments, Ra or Rb is R 2 is linked to A by a carbon-sulfur bond formed between and the free thiol.

[0342] In some embodiments, Ra or Rb is linked to A by a succinimide-sulfur bond.

[0343] In some embodiments, Ra or Rb comprises a branched residue.

[0344] In some embodiments, Ra or Rb is linked to two or more A by a branched residue.

[0345] In some embodiments, the biological activity of A is increased when A is part of a compound or dimer of the invention compared to the biological activity of A when A is not linked to any other structure.

[0346] In some embodiments, A comprises the structure of a compound that is a drug approved for the treatment of a subject suffering from a disease.

[0347] In some embodiments, the subject is a mammalian subject.

[0348] In some embodiments, the mammalian subject is a human subject.

[0349] In some embodiments, A comprises an organic compound structure having a molecular weight of less than 1000 Daltons, a DNA aptamer, an RNA aptamer, an oligonucleotide, or a biologically active protein.

[0350] In some embodiments, the oligonucleotide is an antisense oligonucleotide.

[0351] In some embodiments, the oligonucleotide is an RNA interference-inducing molecule.

[0352] In some embodiments, the oligonucleotide encodes an RNA interference-inducing molecule.

[0353] In some embodiments, A comprises a primary or secondary amine.

[0354] In some embodiments, A is linked to B by a primary or secondary amine.

[0355] In some embodiments, A comprises a primary amine.

[0356] In some embodiments, A comprises aripiprazole or oseltamivir.

[0357] In some embodiments, A comprises a secondary amine.

[0358] In some embodiments, A is a respiratory medication, an antiasthma medication, an analgesic, an antidepressant, an antianginal medication, an antiarrhythmic medication, an antihypertensive medication, an antidiabetic medication, an antihistamine, an anti-infective medication, an antibiotic, an anti-inflammatory medication, an anti-Parkinson's medication, an antipsychotic medication, an antipyretic medication, an antiulcer medication, an attention deficit hyperactivity disorder (ADHD) medication, a central nervous system stimulant, a decongestant, or a psychostimulant.

[0359] In some embodiments, A is selected from the group consisting of alprenolol, acebutolol, amidefrin, amineptine, amosulalol, amoxapine, amphetaminil, atenolol, atomoxetine, balofloxacin, bametan, befunolol, benazepril, benfluorex, benzoctamine, betahistine, betaxolol, bevantolol, bifemelane, bisoprolol, brinzolamide, bufeniode, butethamine, camilofine, carazolol, carticaine, carvedilol, cephaeline, ciprofloxacin, clozapine, cephalosporin ... pine, clobenzorex, clorprenaline, cyclopentamine, delapril, demexiptyline, denopamine, desipramine, desloratadine, diclofenac, dimetofrine, dioxadrol, dobutamine, dopexamine, doripenem, dorzolamide, droprenylamine, duloxetine, eltoprazine, enalapril, enoxacin, epinephrine, ertapenem, esaprazole, esmolol, etoxadrol, fasudil, fendiline, fenetylline, fenfluramine, fenoldopam, fenoterol, fenproporex, Flecainide, fluoxetine, formoterol, frovatriptan, gaboxadol, garenoxacin, gatifloxacin, grepafloxacin, hexoprenaline, imidapril, indalpine, indecainide, indeloxazine hydrochloride, isoxsuprine, ispronicline, labetalol, landiolol, lapatinib, levophacetoperane, lisinopril, lomefloxacin, lotrafiban, maprotiline, mecamylamine, mefloquine, mepindolol, meropenem, metapramine, metaproterenol, methoxyphenamine, dextromethyl Phenidate, methylphenidate, metipranolol, metoprolol, mitoxantrone, mivazerol, moexipril, moprolol, moxifloxacin, nebivolol, nifenalol, nipradilol, norfloxacin, nortriptyline, nylidrine, olanzapine, oxamniquine, oxprenolol, oxyfedrine, paroxetine, perhexiline, phenmetrazine, phenylephrine, phenylpropylmethylamine, phoredrine, picirolex, pimethylline, pindolol, pipemidic acid,Pyridocaine, practolol, pradofloxacin, pramipexole, pramiverine, prenalterol, prenylamine, prilocaine, procaterol, pronethalol, propafenone, propranolol, propylhexedrine, protokylol, protriptyline, pseudoephedrine, reboxetine, rasagiline, (r)-rasagiline, repinotan, reproterol, rimiterol, ritodrine, safinamide, sa lubutamol / albuterol, salmeterol, sarizotan, sertraline, silodosin, sotalol, soterenol, sparfloxacin, spirapril, sulfinalol, synephrine, tamsulosin, tebanicline, tianeptine, tirofiban, tretoquinol, trimetazidine, troxipide, varenicline, vildagliptin, viloxazine, biquidil, or xamoterol.

[0360] In some embodiments, A comprises a biologically active protein.

[0361] In some embodiments, A comprises a secreted protein.

[0362] In some embodiments, A comprises an extracellular domain of a protein.

[0363] In some embodiments, A is biologically active to the extent that it has target binding activity.

[0364] In some embodiments, A is an independently folding protein or portion thereof.

[0365] In some embodiments, A is a glycosylated protein.

[0366] In some embodiments, A comprises an intrachain disulfide bond.

[0367] In some embodiments, A is bound to a cytokine.

[0368] In some embodiments, the cytokine is TNFα.

[0369] In some embodiments, A comprises atrial natriuretic peptide (ANP), calcitonin, corticotropin releasing hormone (CRH), endothelin, gastric inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), an analog of GLP-1 or GLP-2, glucagon vasoactive intestinal peptide (GVIP), ghrelin, peptide YY, or secretin, or a portion thereof.

[0370] In some embodiments, A comprises a stretch of consecutive amino acids in the sequence HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG.

[0371] In some embodiments, A comprises at least one stretch of contiguous amino acids that is identical to a stretch of contiguous amino acids present in a Fab or Fab' heavy chain of the antibody.

[0372] In some embodiments, A comprises at least one stretch of contiguous amino acids that is identical to a stretch of contiguous amino acids present in a Fab or Fab' light chain of the antibody.

[0373] In some embodiments, A comprises at least one Fab or Fab' or a portion of at least one Fab or Fab' of an antibody.

[0374] In some embodiments, A comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a Fab or Fab' or portion thereof.

[0375] In some embodiments, A comprises Fab-1 or Fab'1 of an antibody, or a portion thereof.

[0376] In some embodiments, A comprises Fab-2 or Fab'2 of an antibody, or a portion thereof.

[0377] In some embodiments, A comprises two Fab or Fab' arms of an antibody.

[0378] In some embodiments, the Fab and Fab' are present in adalimumab.

[0379] In some embodiments, A comprises at least one stretch of contiguous amino acids that is identical to a stretch of contiguous amino acids present in a single chain antibody.

[0380] In some embodiments, A comprises at least one stretch of contiguous amino acids that is identical to a stretch of contiguous amino acids present in a TNFα receptor.

[0381] In some embodiments, the TNFα receptor is TNR1B.

[0382] In some embodiments, the compounds of the invention form part of a homodimer.

[0383] In some embodiments, the compounds of the invention form part of a heterodimer.

[0384] The present invention provides homodimers comprising the compounds of the present invention.

[0385] The present invention provides heterodimers comprising the compounds of the present invention.

[0386] In some embodiments, each compound of the dimer can be bound to the other by at least one disulfide bond.

[0387] In some embodiments, each compound of the dimer can be linked to the other by at least one disulfide bond between the Z of each compound.

[0388] In some embodiments, each compound of the dimer is linked to the other by at least one disulfide bond.

[0389] In some embodiments, each compound of the dimer is linked to the other by at least one disulfide bond between the Z of each compound.

[0390] In some embodiments, each compound of the dimer is non-covalently bound to the other.

[0391] The present invention relates to a compound having the structure

[0392] [ka]

[0393] wherein Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of which comprises consecutive amino acids, the consecutive amino acids being (i) an F of an antibody; c (ii) F c (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is R a and C, The dotted line between B and Z represents a peptidyl bond. L is -N 3 , alkyne,

[0394] [ka]

[0395] Group (where R 5 is an alkyl group or an aryl group;

[0396] [ka]

[0397] is selected from the group consisting of a tetrazine group, a tetrazine group, and a trans-cyclooctene group; R a is a bond or each moiety is [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0398] [ka]

[0399] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties independently selected from the group consisting of: [PEG(y)]z is y=1 to 100 and z=1 to 10.

[0400] [ka]

[0401] [is].

[0402] In some embodiments, cysteine ​​or selenocysteine ​​is naturally occurring in the series of consecutive amino acids. In some embodiments, cysteine ​​and selenocysteine ​​are not naturally occurring in the series of consecutive amino acids.

[0403] In some embodiments, the contiguous amino acids have a sequence at the N-terminus selected from the group consisting of cysteine, selenocysteine, CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP, and CDTPPPCPRCP.

[0404] In some embodiments, the F of the antibody c The domain is the naturally occurring F of an antibody. c It is a domain.

[0405] In some embodiments, the F of the antibody c The domain is the variant F of the antibody. c It is a domain.

[0406] In some embodiments, the variant F of the antibody c The domain is the mutated F of the antibody. c It is a domain.

[0407] In some embodiments, the mutated F c The domains are substitution mutants.

[0408] In some embodiments, the substitution variant is an F at the N-terminus, C-terminus, or other than the N-terminus or C-terminus. c It has an amino acid substitution at a certain position in the domain.

[0409] In some embodiments, a substitution variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 amino acid substitutions in its stretch of contiguous amino acids.

[0410] In some embodiments, the substitutions are conservative amino acid substitutions.

[0411] In some embodiments, the mutated F c The domains are amino acid addition variants.

[0412] In some embodiments, the amino acid addition variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 additional amino acids in its stretch of consecutive amino acids.

[0413] In some embodiments, the mutated F c The domains are amino acid deletion mutants.

[0414] In some embodiments, an amino acid deletion mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 deleted amino acids in its stretch of consecutive amino acids.

[0415] In some embodiments, the consecutive amino acids are c It is identical to a stretch of at least 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 consecutive amino acids present in the domain chain.

[0416] In some embodiments, the contiguous amino acids are identical to a series of amino acids in the hinge region, CH2 region, or CH3 region of an Fc domain, or a portion thereof.

[0417] In some embodiments, L is -N 3 It is.

[0418] In some embodiments, L is

[0419] [ka]

[0420] It is.

[0421] In some embodiments, L is an alkyne.

[0422] In some embodiments, the alkyne is a propargyl group.

[0423] In some embodiments, the alkyne is a cyclooctyne group.

[0424] In some embodiments, the alkyne has the structure

[0425] [ka]

[0426] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, and R5 is an aryl group or an alkyl group. has.

[0427] In some embodiments, the alkyne has the structure

[0428] [ka]

[0429] has.

[0430] In some embodiments, L is tetrazine.

[0431] In some embodiments, the tetrazine has the structure

[0432] [ka]

[0433] (wherein Rc is H, alkyl or aryl). has.

[0434] In some embodiments, the tetrazine has the structure

[0435] [ka]

[0436] (wherein Rc is H, alkyl or aryl). has.

[0437] In some embodiments, the tetrazine has the structure

[0438] [ka]

[0439] has.

[0440] In some embodiments, the tetrazine has the structure

[0441] [ka]

[0442] has.

[0443] In some embodiments, L is trans-cyclooctene.

[0444] In some embodiments, the trans-cyclooctene has the following structure:

[0445] [ka]

[0446] has.

[0447] In some embodiments, the trans-cyclooctene has the following structure:

[0448] [ka]

[0449] has.

[0450] In some embodiments, R a or R b is an organic structure comprising a [PEG(y)] z group.

[0451] In some embodiments, Ra or Rb is a polyalkylene glycol, a polyoxyalkylated polyol, a polyvinyl alcohol, a polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), or an organic structure comprising a polysaccharide group.

[0452] In some embodiments, Ra or Rb is C 1 ~C 4 It is an organic structure that contains an alkyl group.

[0453] In some embodiments, Ra or Rb is an organic structure that comprises a succinimide.

[0454] In some embodiments, Ra or Rb is an organic structure that contains an amine.

[0455] In some embodiments, Ra or Rb is an organic structure including succinyl, malonyl, glutaryl, phthalyl, adipoyl.

[0456] In some embodiments, Ra or Rb is an organic structure that contains malonyl.

[0457] In some embodiments, Ra or Rb is an organic structure comprising an amino acid.

[0458] In some embodiments, Ra or Rb is an organic structure that includes a cysteine.

[0459] In some embodiments, Ra or Rb is an organic structure that includes lysine.

[0460] In some embodiments, Ra or Rb is selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharides, branched residues, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0461] [ka]

[0462] It is an organic structure consisting of a chain of three moieties selected from the group consisting of:

[0463] In some embodiments, Ra or Rb is selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharides, branched residues, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C 10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0464] [ka]

[0465] It is an organic structure consisting of a chain of four moieties selected from the group consisting of:

[0466] In some embodiments, Ra or Rb is selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharides, branched residues, C 1 ~C 10 Alkyl, C 3 ~C 10 Cycloalkane, C 2 ~C10 Alkenes, C 5 ~C 10 Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C 2 ~C 5 Achill, C 2 ~C 5 Acylamino, C 2 ~C 5 acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acids, aryl groups, heteroaryl groups, carbamates, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0467] [ka]

[0468] It is an organic structure consisting of a chain of five moieties selected from the group consisting of:

[0469] In some embodiments, Ra or Rb comprises a [PEG(y)]z group attached to a lysine.

[0470] In some embodiments, Ra or Rb is a C bonded to a succinimide group. 1 ~C 4 Contains an acyl group.

[0471] In some embodiments, Ra or Rb is C 1 ~C 4 Contains lysine bound to acyl.

[0472] In some embodiments, R a or R b comprises a [PEG(y)] z group attached to a glutaryl.

[0473] In some embodiments, Ra or Rb is [PEG(y)]z, C 2~C 5 Acyl, succinyl, malonyl, glutaryl, amino acid, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene,

[0474] [ka]

[0475] (where X 1 is CH or N, and X 2 is CH 2 or a carbonyl group, R 5 is an aryl group or an alkyl group. and [PEG(y)]z is an organic structure consisting of a chain of 3, 4 or 5 moieties selected from the group consisting of y=1 to 100 and z=1 to 10.

[0476] [ka]

[0477] It is.

[0478] In some embodiments, Ra or Rb is a bond.

[0479] In some embodiments, Ra or Rb is cysteine.

[0480] In some embodiments, Ra or Rb has a linear structure.

[0481] In some embodiments, Ra or Rb has a branched structure.

[0482] In some embodiments, y is 1 to 20. In some embodiments, y is 21 to 40. In some embodiments, y is 41 to 60. In some embodiments, y is 61 to 80. In some embodiments, y is 30 to 50. In some embodiments, y is 12, 24, 36, or 48.

[0483] In some embodiments, z is 1.

[0484] In some embodiments, the terminal carbonyl of the [PEG(y)]z group is part of an amide bond.

[0485] In some embodiments, the terminal amine of the [PEG(y)]z group is part of an amide bond.

[0486] In some embodiments, Ra or Rb has x of 1 to 100 and w of 0 to 5.

[0487] [ka]

[0488] It is.

[0489] In some embodiments, x is 1 to 20. In some embodiments, x is 21 to 40. In some embodiments, x is 41 to 60. In some embodiments, x is 61 to 80. In some embodiments, x is 30 to 50. In some embodiments, x is 12, 24, 36, or 48.

[0490] In some embodiments, w is 1. In some embodiments, w is 0.

[0491] In some embodiments, Ra or Rb has the structure

[0492] [ka]

[0493] has.

[0494] In some embodiments, Ra or Rb is

[0495] [ka]

[0496] (wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50).

[0497] In some embodiments, Ra or Rb is

[0498] [ka]

[0499] (wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-30, 1-40, or 1-50; x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-30, 1-40, or 1-50; and z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-30, 1-40, or 1-50). It is.

[0500] In some embodiments, Ra or Rb is

[0501] [ka]

[0502] (wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-30, 1-40, or 1-50; and z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-30, 1-40, or 1-50). It is.

[0503] In some embodiments, Ra or Rb is

[0504] [ka]

[0505] (wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50).

[0506] In some embodiments, the solid line between A and B represents the following structure:

[0507] [ka]

[0508] represents a non-peptidyl bond comprising Here, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50; x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50; and z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50.

[0509] In some embodiments, the solid line between A and B represents the following structure:

[0510] [ka]

[0511] represents a non-peptidyl bond comprising Here, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50; x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50; and x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 30, 1 to 40, or 1 to 50.

[0512] The present invention relates to a compound having the structure

[0513] [ka]

[0514] where A is a biologically active structure of the compound; Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of which comprises consecutive amino acids, the consecutive amino acids being (i) F of an antibody; c (ii) F c (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is a chemical structure that connects A and Z, The dotted line between B and Z represents a peptidyl bond. The solid line between A and B represents a non-peptidyl bond. 1. A method for producing a) obtaining A' comprising A or a derivative of A and a first terminal reactive group; b) obtaining B' comprising B or a derivative of B, a second terminal reactive group, and a third terminal reactive group, the second terminal reactive group being capable of reacting with the first terminal reactive group to form a non-peptidyl bond; c) obtaining Z' comprising Z or a derivative of Z and a fourth terminal reactive group, the fourth terminal reactive group being capable of reacting with a third terminal reactive group to form a peptidyl bond; and d) reacting A', B' and Z' in any order to produce a compound. The present invention provides a method comprising:

[0515] In some embodiments, cysteine ​​or selenocysteine ​​is naturally occurring in the series of consecutive amino acids. In some embodiments, cysteine ​​and selenocysteine ​​are not naturally occurring in the series of consecutive amino acids.

[0516] In some embodiments, the contiguous amino acids have a sequence at the N-terminus selected from the group consisting of cysteine, selenocysteine, CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP, and CDTPPPCPRCP.

[0517] In some embodiments, the F of the antibody c The domain is the naturally occurring F of an antibody. c It is a domain.

[0518] In some embodiments, the F of the antibody c The domain is the variant F of the antibody. c It is a domain.

[0519] In some embodiments, the variant F of the antibody c The domain is the mutated F of the antibody. c It is a domain.

[0520] In some embodiments, the mutated F c The domains are substitution mutants.

[0521] In some embodiments, the substitution variant is an F at the N-terminus, C-terminus, or other than the N-terminus or C-terminus. c It has an amino acid substitution at a certain position in the domain.

[0522] In some embodiments, a substitution variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 amino acid substitutions in its stretch of contiguous amino acids.

[0523] In some embodiments, the substitutions are conservative amino acid substitutions.

[0524] In some embodiments, the mutated F c The domains are amino acid addition variants.

[0525] In some embodiments, the amino acid addition variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 additional amino acids in its stretch of consecutive amino acids.

[0526] In some embodiments, the mutated F c The domains are amino acid deletion mutants.

[0527] In some embodiments, an amino acid deletion mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 deleted amino acids in its stretch of consecutive amino acids.

[0528] In some embodiments, the consecutive amino acids are c It is identical to a stretch of at least 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 consecutive amino acids present in the domain chain.

[0529] In some embodiments, the contiguous amino acids are identical to a series of amino acids in the hinge region, CH2 region, or CH3 region of an Fc domain, or a portion thereof.

[0530] In some embodiments, the solid line between A and B represents the following structure:

[0531] [ka]

[0532] represents a non-peptidyl bond comprising Where:

[0533] [ka]

[0534] teeth,

[0535] [ka]

[0536] (where R 5 is an alkyl group or an aryl group. and R 1 is H or part of an additional structure which is a cyclic structure, where the additional cyclic structure is R 1 or R 1 Includes part of R 2 or R 2 Part of, and R 2 and the carbon between the alkene double bond, however,

[0537] [ka]

[0538] but,

[0539] [ka]

[0540] If R 3 is H,

[0541] [ka]

[0542] but,

[0543] [ka]

[0544] If

[0545] [ka]

[0546] teeth,

[0547] [ka]

[0548] is a triazole ring containing

[0549] [ka]

[0550] but,

[0551] [ka]

[0552] If

[0553] [ka]

[0554] teeth,

[0555] [ka]

[0556] is an N-alkyl or aryl substituted isoxazoline ring comprising R 2 represents an organic structure connecting to either A or B, and R 4 represents an organic structure connecting A or B to the other, it is, a) obtaining A' comprising A or a derivative of A and a first terminal reactive group; b) obtaining B' comprising B or a derivative of B, a second terminal reactive group, and a third terminal reactive group, the second terminal reactive group being capable of reacting with the first terminal reactive group to form a non-peptidyl bond; c) obtaining C' comprising C or a derivative of C and a fourth terminal reactive group, the fourth terminal reactive group being capable of reacting with the third terminal reactive group to form a peptidyl bond; and d) reacting A', B' and C' in any order to produce a compound. Includes.

[0557] In some embodiments, step d) is carried out by first reacting A' with B',

[0558] [ka]

[0559] (where B″ comprises B and a third terminal reactive group, and the solid line between B″A represents a non-peptidyl bond), and then

[0560] [ka]

[0561] is reacted with C' to give the compound.

[0562] In some embodiments, step d) is carried out by first reacting C' with B',

[0563] [ka]

[0564] (where B″ comprises B and a second terminal reactive group, and the dotted line between B″C represents a peptidyl bond), and then

[0565] [ka]

[0566] is reacted with A' to obtain the compound.

[0567] In some embodiments, the first terminal reactive group is an azide, a thiol, a nitrone, or an alkyne.

[0568] In some embodiments, the first terminal reactive group is an alkyne.

[0569] In some embodiments, the alkyne is a cycloalkyne.

[0570] In some embodiments, the alkyne is an eight-membered ring.

[0571] In some embodiments, the alkyne is an azacyclooctyne.

[0572] In some embodiments, the cycloalkyne is a biarylazacyclooctyne.

[0573] In some embodiments, the cycloalkyne is cyclooctyne.

[0574] In some embodiments, the alkyne is a terminal alkyne.

[0575] In some embodiments, the first terminal reactive group is an azide, a thiol, or a nitrone.

[0576] In some embodiments, the first terminal reactive group is an azide.

[0577] In some embodiments, the first terminal reactive group is a thiol.

[0578] In some embodiments, the first terminal reactive group is a nitrone.

[0579] In some embodiments, the first terminal reactive group is an N-alkyl nitrone.

[0580] In some embodiments, the first terminal reactive group is an N-aryl nitrone.

[0581] In some embodiments, the second terminal reactive group is an azide, thiol, nitrone, or alkyne.

[0582] In some embodiments, the second terminal reactive group is an alkyne.

[0583] In some embodiments, the alkyne is a cycloalkyne.

[0584] In some embodiments, the alkyne is an eight-membered ring.

[0585] In some embodiments, the alkyne is an azacyclooctyne.

[0586] In some embodiments, the cycloalkyne is a biarylazacyclooctyne.

[0587] In some embodiments, the cycloalkyne is cyclooctyne.

[0588] In some embodiments, the alkyne is a terminal alkyne.

[0589] In some embodiments, the second terminal reactive group is an azide, thiol, or nitrone.

[0590] In some embodiments, the second terminal reactive group is an azide.

[0591] In some embodiments, the second terminal reactive group is a thiol.

[0592] In some embodiments, the second terminal reactive group is a nitrone.

[0593] In some embodiments, the second terminal reactive group is an N-alkyl nitrone.

[0594] In some embodiments, the second terminal reactive group is an N-aryl nitrone.

[0595] In some embodiments, the first terminal reactive group is a terminal alkyne and the second terminal reactive group is an azide, thiol, or nitrone.

[0596] In some embodiments, the second terminal reactive group is an azide.

[0597] In some embodiments, the second terminal reactive group is a thiol.

[0598] In some embodiments, the second terminal reactive group is a nitrone.

[0599] In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

[0600] In some embodiments, the first terminal reactive group is an azide, thiol, or nitrone and the second terminal reactive group is a terminal alkyne.

[0601] In some embodiments, the first terminal reactive group is an azide.

[0602] In some embodiments, the first terminal reactive group is a thiol.

[0603] In some embodiments, the first terminal reactive group is a nitrone.

[0604] In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

[0605] In some embodiments, the first terminal reactive group is a cycloalkyne and the second terminal reactive group is an azide, thiol, or nitrone.

[0606] In some embodiments, the first terminal reactive group is an azide.

[0607] In some embodiments, the first terminal reactive group is a thiol.

[0608] In some embodiments, the first terminal reactive group is a nitrone.

[0609] In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

[0610] In some embodiments, the first terminal reactive group is an azide, thiol, or nitrone and the second terminal reactive group is a cycloalkyne.

[0611] In some embodiments, the first terminal reactive group is an azide.

[0612] In some embodiments, the first terminal reactive group is a thiol.

[0613] In some embodiments, the first terminal reactive group is a nitrone.

[0614] In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

[0615] In some embodiments, the cycloalkyne is an eight-membered ring.

[0616] In some embodiments, the cycloalkyne is an azacyclooctyne.

[0617] In some embodiments, the cycloalkyne is a biarylazacyclooctyne.

[0618] In some embodiments, the cycloalkyne is cyclooctyne.

[0619] In some embodiments, the first terminal reactive group is an azide and the second terminal reactive group is a terminal alkyne, or the first terminal reactive group is an azide and the second terminal reactive group is a cycloalkyne, or the first terminal reactive group is a thiol and the second terminal reactive group is a cycloalkyne, or the first terminal reactive group is an N-alkyl nitrone or N-aryl nitrone and the second terminal reactive group is a cyclooctyne.

[0620] In some embodiments, the second terminal reactive group is an azide and the first terminal reactive group is a terminal alkyne, or the second terminal reactive group is an azide and the first terminal reactive group is a cycloalkyne, or the second terminal reactive group is a thiol and the first terminal reactive group is a cycloalkyne, or the second terminal reactive group is an N-alkyl nitrone or N-aryl nitrone and the first terminal reactive group is a cyclooctyne.

[0621] In some embodiments, the first terminal reactive group and the second terminal reactive group react to form a triazole, a thiolene, an N-alkylisoxazoline, or an N-arylisoxazoline.

[0622] In some embodiments, the first terminal reactive group and the second terminal reactive group react to form a triazole.

[0623] In some embodiments, the first terminal reactive group and the second terminal reactive group react to form a thiolene.

[0624] In some embodiments, the first terminal reactive group and the second terminal reactive group react to produce an N-alkylisoxazoline or an N-arylisoxazoline.

[0625] In some embodiments, reacting the first reactive group with the second reactive group results in a yield of reaction of at least 80%, 85%, or 90% in less than 3, 6, 12, 18, 24, 30, 36, 42, 48, or 72 hours.

[0626] In some embodiments, the solid line between A and B represents the following structure:

[0627] [ka]

[0628] represents a non-peptidyl bond comprising Where X a is a chemical structure containing cyclooctane fused to dihydropyridazine, R a represents an organic structure connecting to either A or B, and R b represents an organic structure connecting A or B to the other.

[0629] In some embodiments, the first terminal reactive group is trans-cyclooctene, or tetrazine.

[0630] In some embodiments, the first terminal reactive group is trans-cyclooctene.

[0631] In some embodiments, the alkyne is a tetrazine.

[0632] In some embodiments, the second terminal reactive group is trans-cyclooctene, or tetrazine.

[0633] In some embodiments, the second terminal reactive group is trans-cyclooctene.

[0634] In some embodiments, the second terminal reactive group has the structure:

[0635] [ka]

[0636] has.

[0637] In some embodiments, the second terminal reactive group has the structure:

[0638] [ka]

[0639] has.

[0640] In some embodiments, the second terminal reactive group is a tetrazine.

[0641] In some embodiments, the second terminal reactive group has the structure:

[0642] [ka]

[0643] (where R c is N, alkyl or aryl) has.

[0644] In some embodiments, the second terminal reactive group has the structure:

[0645] [ka]

[0646] has.

[0647] In some embodiments, the first terminal reactive group is trans-cyclooctene and the second terminal reactive group is tetrazine.

[0648] In some embodiments, the first terminal reactive group is tetrazine and the second terminal reactive group is trans-cyclooctene.

[0649] In some embodiments, the first terminal reactive group and the second terminal reactive group react to produce a chemical structure comprising a cyclooctane fused to a dihydropyridazine.

[0650] In some embodiments, the first terminal reactive group and the second terminal reactive group react to form the following chemical structure:

[0651] [ka]

[0652] (where R c is N, alkyl or aryl) Or it produces a tautomer thereof.

[0653] In some embodiments, the third terminal reactive group and the fourth terminal reactive group are each independently an amino acid or an amino acid derivative.

[0654] In some embodiments, the third terminal reactive group is threonine or a threonine derivative.

[0655] In some embodiments, the third terminal reactive group is a thioester derivative of an amino acid.

[0656] In some embodiments, the fourth terminal reactive group is cysteine, selenocysteine, homocysteine, or homoselenocysteine, or a derivative of cysteine, selenocysteine, homocysteine, or homoselenocysteine.

[0657] In some embodiments, the fourth terminal reactive group is cysteine ​​or a derivative of cysteine.

[0658] In some embodiments, the fourth terminal reactive group is cysteine.

[0659] In some embodiments, A' is i) obtaining A″ comprising A or a derivative of A and a series of consecutive amino acids including an intein; ii) obtaining a cysteine, selenocysteine, homocysteine, or homoselenocysteine ​​residue, or a substituted derivative of a cysteine, selenocysteine, homocysteine, or homoselenocysteine ​​residue, substituted at the C-terminus with an organic structure comprising an alkyne, an azide, a thiol, or a nitrone; and iii) reacting A″ with a substituted cysteine ​​residue to produce A′.

[0660] In some embodiments, the alkyne-containing organic structure is an N-propargylamine.

[0661] In some embodiments, A' is i) obtaining A″ comprising A or a derivative of A and comprising at least one free thio group; ii) obtaining a compound comprising a first terminal reactive group and a terminal maleimide; and iii) reacting A″ with the compound of step ii) to produce A′. It is prepared by:

[0662] In some embodiments, A″ is a) obtaining a polypeptide A''', which comprises A or a derivative of A and contains at least one disulfide bond; and b) by treating A''' with mercaptoethylamine (MEA) to yield A''.

[0663] In some embodiments, A''' is a) obtaining a monoclonal antibody comprising A or a derivative of A and comprising at least one disulfide bond; and b) treating the polypeptide of step a) with IdeS to produce A''' It is prepared by:

[0664] In some embodiments, the monoclonal antibody binds to TNFα.

[0665] In some embodiments, the monoclonal antibody is adalimumab.

[0666] In some embodiments, when R1 is hydrogen and the first terminal reactive group is an alkyne, in step d), B' is reacted in the presence of a metal catalyst.

[0667] In some embodiments, when R1 is hydrogen and the second terminal reactive group is an alkyne, in step d), B' is reacted in the presence of a metal catalyst.

[0668] In some embodiments, the metal catalyst is Ag(I) or Cu(I).

[0669] In some embodiments, A' comprises one or more branching residues, each branching residue comprising an additional first terminal reactive group.

[0670] In some embodiments, B' comprises one or more branching residues, each branching residue comprising an additional second terminal reactive group.

[0671] In some embodiments, B' comprises one or more branching residues, each branching residue comprising an additional third terminal reactive group.

[0672] In some embodiments, the branching residues are amino acid residues.

[0673] In some embodiments, the amino acid residue is lysine or a lysine derivative, arginine or an arginine derivative, aspartic acid or an aspartic acid derivative, glutamic acid or a glutamic acid derivative, asparagine or an asparagine derivative, glutamine or a glutamine derivative, tyrosine or a tyrosine derivative, cysteine ​​or a cysteine ​​derivative, ornithine or an ornithine derivative.

[0674] In some embodiments, the amino acid residue is substituted at the N-position with a residue that contains a terminal amino or carbonyl reactive group.

[0675] In some embodiments, the branched residue is an organic residue that includes two or more terminal amino groups or two or more terminal carbonyl groups.

[0676] In some embodiments, the organic residue is iminodipropionic acid, iminodiacetic acid, 4-amino-pimelic acid, 4-amino-heptanedioic acid, 3-aminohexanedioic acid, 3-aminoadipic acid, 2-aminooctanedioic acid, or 2-amino-6-carbonyl-heptanedioic acid.

[0677] In some embodiments, the method is performed in the absence of a non-thiol reducing agent.

[0678] In some embodiments, the method is carried out in the absence of a thiol reducing agent.

[0679] In some embodiments, the method is carried out in the presence of a thiol reducing agent.

[0680] In some embodiments, the method is performed with an overall yield of 80% or greater.

[0681] In some embodiments, the method is performed with an overall yield of 90% or greater.

[0682] In some embodiments, reacting the first reactive group with the second reactive group results in a reaction yield of at least 50%, 55%, 60%, 65%, 70%, 80%, 85% or 90% in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 60 minutes.

[0683] In some embodiments, B is an organic acid residue.

[0684] In some embodiments, B is a series of 1 to 50 amino acid residues and optionally an organic acid residue.

[0685] In some embodiments, B is a stretch of 1 to 10 contiguous amino acids.

[0686] In some embodiments, B comprises a stretch of consecutive amino acids in the sequence EPKSCDKTHTCPPCP, ERKCCVECPPCP, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3, ESKYGPPCPSCP, or a portion thereof.

[0687] In some embodiments, B has a threonine at the C-terminus.

[0688] In some embodiments, Z comprises one C, and C is a first polypeptide of the compound, the first polypeptide comprising consecutive amino acids, the consecutive amino acids being (i) an F of an antibody. c (ii) F c binds to a receptor, and (iii) has a sequence at its N-terminus selected from the group consisting of cysteine, selenocysteine, CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP, and CDTPPPCPRCP.

[0689] In some embodiments, C comprises consecutive amino acids, the consecutive amino acids being (i) an F of the antibody. c (ii) F c binds to a receptor, and (iii) has a sequence at its N-terminus that contains a naturally occurring cysteine ​​selected from the group consisting of CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP, and CDTPPPCPRCP.

[0690] In some embodiments, C is a polypeptide component of the compound, the polypeptide component comprising consecutive amino acids, the consecutive amino acids being (i) an F of an antibody. c (ii) F c binds to a receptor, and (iii) has a sequence at its N-terminus that contains a non-naturally occurring cysteine ​​or selenocysteine.

[0691] In some embodiments, C comprises consecutive amino acids, which are identical to a stretch of consecutive amino acids present in an Fc domain chain of an antibody selected from the group consisting of IgG, IgM, IgA, IgD, and IgE.

[0692] In some embodiments, C comprises consecutive amino acids, which are identical to a certain stretch of consecutive amino acids present in an Fc6 domain chain of an antibody.

[0693] In some embodiments, C further comprises contiguous amino acids that are identical to a stretch of contiguous amino acids present in a chain of the antibody other than the Fc domain chain of the antibody.

[0694] In some embodiments, C comprises consecutive amino acids that are identical to a stretch of consecutive amino acids present in the Fab or Fab' heavy chain of the antibody.

[0695] In some embodiments, C comprises consecutive amino acids that are identical to a stretch of consecutive amino acids present in the Fab or Fab' light chain of the antibody.

[0696] In some embodiments, Z further comprises a second polypeptide, which second polypeptide comprises consecutive amino acids that are identical to a stretch of consecutive amino acids present in a chain of the antibody other than the Fc domain chain of the antibody.

[0697] In some embodiments, the second polypeptide comprises contiguous amino acids that are identical to a stretch of contiguous amino acids present in a Fab or Fab' heavy chain of the antibody.

[0698] In some embodiments, the second polypeptide comprises contiguous amino acids that are identical to a stretch of contiguous amino acids present in a Fab or Fab' light chain of the antibody.

[0699] In some embodiments, Z comprises an antibody or a portion thereof.

[0700] In some embodiments, Z comprises at least one Fab or Fab', or a portion of at least one Fab or Fab', of an antibody.

[0701] In some embodiments, Z comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a Fab or Fab', or a portion thereof.

[0702] In some embodiments, Z comprises Fab-1 or Fab'1 of an antibody, or a portion thereof.

[0703] In some embodiments, Z comprises Fab-2 or Fab'2 of an antibody, or a portion thereof.

[0704] In some embodiments, Z comprises the two Fab or Fab' arms of an antibody.

[0705] In some embodiments, the antibody is an IgG, IgM, IgA, IgD, or IgE antibody.

[0706] In some embodiments, the Fab or Fab' is present in adalimumab.

[0707] In some embodiments, Z comprises at least one stretch of contiguous amino acids that is identical to a stretch of contiguous amino acids present in a single chain antibody.

[0708] In some embodiments, the C-terminus of the C is F of the modified antibody. c It includes a certain series of consecutive amino acids present in a domain chain.

[0709] In some embodiments, the C-terminus of C is cysteine, selenocysteine, homocysteine ​​or homoselenocysteine, or a derivative of cysteine, selenocysteine, homocysteine ​​or homoselenocysteine.

[0710] In some embodiments, B is linked to Z by a peptidyl bond between the N-terminal cysteine ​​or selenocysteine ​​of the Z polypeptide and an amino acid or organic acid residue of B.

[0711] In some embodiments, Z comprises a second polypeptide and B is linked to Z by a peptidyl bond between the N-terminal cysteine ​​or selenocysteine ​​of the second polypeptide of Z and an amino acid or organic acid residue of B.

[0712] In some embodiments, B is linked to C by a peptidyl bond between the N-terminal cysteine ​​or selenocysteine ​​of C and an amino acid or organic acid residue of B.

[0713] In some embodiments, Z comprises one polypeptide, which is C.

[0714] In some embodiments, Z comprises two polypeptides, C and a second polypeptide.

[0715] The present invention provides homodimers and heterodimers comprising the compounds of the invention.

[0716] In some embodiments, each compound of the dimer can be bound to the other by at least one disulfide bond.

[0717] In some embodiments, each compound of the dimer can be linked to the other by at least one disulfide bond between the C or second polypeptide of each compound.

[0718] In some embodiments, each compound of the dimer is linked to the other by at least one disulfide bond.

[0719] In some embodiments, each compound of the dimer is linked to the other by at least one disulfide bond between the C or second polypeptide of each compound.

[0720] In some embodiments, each compound of the dimer is non-covalently bound to the other.

[0721] In some embodiments, the dimer is

[0722] [ka]

[0723] [ka]

[0724] [ka]

[0725] [ka]

[0726] [ka]

[0727] [ka]

[0728] [ka]

[0729] It is.

[0730] Additional non-limiting examples of dimers of the compounds of the present invention are shown in the Figures.

[0731] In some embodiments, the branching residues are lysine or a lysine derivative, arginine or an arginine derivative, aspartic acid or an aspartic acid derivative, glutamic acid or a glutamic acid derivative, asparagine or an asparagine derivative, glutamine or a glutamine derivative, tyrosine or a tyrosine derivative, cysteine ​​or a cysteine ​​derivative, ornithine or an ornithine derivative.

[0732] In some embodiments, the branching residue is an amino acid substituted at the N-position with a residue that contains a terminal amino or carbonyl reactive group, hi some embodiments, the branching residue is an organic residue that contains two or more terminal amino groups or two or more terminal carbonyl groups.

[0733] In some embodiments, the branched residue is an organic residue that includes two or more terminal amino groups. In some embodiments, the branched residue is an organic residue that includes two or more terminal carbonyl groups. In some embodiments, the branched residue is diaminopropionic acid. In some embodiments, the branched residue is a diaminopropionic carbonyl compound.

[0734] In some embodiments, the branching residue is 4-(carbonylmethoxy)phenylalanine, 2-amino-6-(carbonylmethylamino)hexanoic acid, S-(carbonylpropyl)cysteine, S-(carbonylethyl)cysteine, S-(carbonylmethyl)cysteine, N-(carbonylethyl)glycine, N-(carbonylmethyl)glycine, iminodipropionic acid, iminodiacetic acid, 4-amino-pimelic acid, 4-amino-heptanedioic acid, 3-aminohexanedioic acid, 3-aminoadipic acid, 2-aminooctanedioic acid, or 2-amino-6-carbonyl-heptanedioic acid.

[0735] In some embodiments, the branching residue is Fmoc-L-Asp-AMC, Fmoc-L-Asp-pNA, Fmoc-L-Glu-AMC, Fmoc-L-Glu-pNA, Fmoc-L-Glu(Edans)-OH, Fmoc-L-Glu(PEG-biotinyl)-OH, (S)-Fmoc-2-amino-hexanedioic acid-6-tert-butyl ester, (S)-Fmoc-2-amino-adipic acid-6-tert-butyl ester, (S)-Fmoc-Aad(OtBu)-OH, (S)-Fmoc-2-amino-5 ... -butoxycarbonyl-hexanedioic acid-6-tert-butyl ester, (S)-Fmoc-2-amino-heptanedioic acid-7-tert-butyl ester, (S)-Fmoc-2-amino-pimelic acid-7-tert-butyl ester, (S)-Fmoc-2-amino-6-tert-butoxycarbonyl-heptanedioic acid-7-tert-butyl ester, (S)-Fmoc-2-amino-octanedioic acid-8-tert-butyl ester, (S)-Fmoc-2-amino-suberic acid-8-tert-butyl ester, (S)-Fmoc -Asu(OtBu)-OH, (R)-Fmoc-3-amino-hexanedioic acid-1-tert-butyl ester, (R)-Fmoc-3-amino-adipic acid-1-tert-butyl ester, (R)-Fmoc-4-amino-heptanedioic acid-1-tert-butyl ester, (R)-Fmoc-4-amino-pimelic acid-1-tert-butyl ester, Boc-iminodiacetic acid, Fmoc-iminodiacetic acid, Boc-iminodipropionic acid, Fmoc-iminodipropionic acid, Fmoc-N-(tert-butoxycarbonylmethyl) -glycine, Fmoc-N-(tert-butoxycarbonylethyl)-glycine, Fmoc-L-Cys(tert-butoxycarbonylmethyl)-OH, (R)-Fmoc-2-amino-3-(tert-butoxycarbonylmethylsulfanyl)-propionic acid, Fmoc-L-Cys(tert-butoxycarbonylpropyl)-OH, (R)-Fmoc-2-amino-3-(3-tert-butoxycarbonylpropylsulfanyl)-propionic acid, Fmoc-L-Cys(tert-butoxycarbonylethyl)-OH,It is prepared from (R)-Fmoc-2-amino-3-(2-tert-butoxycarbonylethylsulfanyl)-propionic acid, Fmoc-4-(tert-butoxycarbonylmethoxy)-L-phenylalanine, or (S)-Fmoc-2-amino-6-(Boc-tert-butoxycarbonylmethylamino)-hexanoic acid.

[0736] In some embodiments, the branching residue is N-α-Boc-DL-diaminopropionic acid, N-α-Boc-D-diaminopropionic acid, N-α-Boc-L-diaminopropionic acid, N-α-Fmoc-L-diaminopropionic acid, N-α-Boc-N-β-Alloc-D-diaminopropionic acid, N-α-Boc-N-β-Alloc-L-diaminopropionic acid, N-α-Fmoc-N-β-alloc-L-diaminopropionic acid, N-α-N-β -Bis-Boc-L-diaminopropionic acid, N-α-Fmoc-N-β-Boc-D-diaminopropionic acid, N-α-Fmoc-N-β-Boc-L-diaminopropionic acid, N-α-ZN-β-Boc-L-diaminopropionic acid, N-α-Boc-N-β-Fmoc-D-diaminopropionic acid, N-α-Boc-N-β-Fmoc-L-diaminopropionic acid, N-α-Boc-N-β-Fmoc-L-diaminopropionic acid, N-α-N-β-Bis-Fmoc-L-diaminopropionic acid, N-α-ZN-β-F moc-L-diaminopropionic acid, N-α-Boc-N-β-ZL-diaminopropionic acid, N-α-Fmoc-N-β-ZL-diaminopropionic acid, N-α-Fmoc-N-β-(Boc-aminooxyacetyl)-L-diaminopropionic acid, N-α-Boc-N-γ-Fmoc-D-diaminobutyric acid, N-α-Boc-N-γ-Fmoc-L-diaminobutyric acid, N-α-Boc-N-γ-Fmoc-L-diaminobutyric acid, N-α-Fmoc-N-γ-Bo c-D-Diaminobutyric acid, N-α-Fmoc-N-γ-Boc-L-diaminobutyric acid, N-α-Fmoc-N-γ-Alloc-L-diaminobutyric acid, (S)-Nb-Fmoc-N-γ-Boc-3,4-diaminobutyric acid, HL-ornithine, Na-Boc-N-δ-Alloc-L-ornithine, Na-Fmoc-N-δ-Alloc-L-ornithine, Na-Fmoc-N-δ-Boc-L-ornithine, (S)-Boc-2-amino-5-azido-pentanoic acid.DCHA, (S)-Fmoc-2-amino-5-azido-pentanoic acid, NaN-δ-bis-Boc-NaN-δ-bis(3-Boc-aminopropyl)-L-ornithine, N-α-Boc-N-β-N-δ-N-δ-tris(3-Boc-aminopropyl)-L-ornithine, Fmoc-L-Lys(biotin)-OH, Fmoc-L-Lys(Dabcyl)-OH, Fmoc-L-Lys(Boc)(Me)-OH, Fmoc-L-Lys(Boc)(iPr)-OH, (2S,5R)-Fmoc-2-amino-4-(3-B oc-2,2-Dimethyl-oxazolidin-5-yl)-butyric acid, (S)-Fmoc-2-amino-6-(Boc-tert-butoxycarbonylmethyl-amino)-hexanoic acid, (S)-Fmoc-2-amino-7-(Boc-amino)-heptanoic acid, Fmoc-L-Arg(Me)(Pbf)-OH, Fmoc-L-Arg(Me)2(Pbf)-OH, Fmoc-L-Arg(Me)2-OH, (S)-Fmoc-3-amino-5-[(N'-Pbf-pyrrolidine-1-carboximidoyl)-amino]-pentanoic acid, Fmo c-L-Homoarg(Et)2-OH, Boc-3-amino-5-(Fmoc-amino)-benzoic acid, 3,5-bis[2-(Boc-amino)ethoxy]-benzoic acid, Fmoc-4-[2-(Boc-amino)ethoxy]-L-phenylalanine, N,N-bis(N'-Fmoc-3-aminopropyl)-glycine potassium hemisulfate, N,N-bis(N'-Fmoc-3-aminopropyl)-glycine potassium hemisulfate, Fmoc-N-(2-Boc-aminoethyl)-glycine, Fmoc-N-(3-Bo c-aminopropyl)-glycine, Fmoc-N-(4-Boc-aminobutyl)-glycine, (R,S)-N-α-Fmoc-N-a'-Boc-diaminoacetic acid, N,N'-bis-Fmoc-diaminoacetic acid, (S)-N-4-Fmoc-N-8-Boc-diaminooctanoic acid, (R,S)-N-Fmoc-N'-Boc-imidazolidine-2-carboxylic acid, Fmoc-p(NH-Boc)-L-Phe-OH, Boc-p(NH-Fmoc)-L-Phe-OH, or Boc-p(NH-Z)-L-Phe-OH.

[0737] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments, and thus all combinations of the various elements described herein are within the scope of the invention.

[0738] When a range for a parameter is given, it is understood that the invention also refers to all integers and tenths of that range. For example, "0.2-5 mg / kg / day" is a disclosure of 0.2 mg / kg / day, 0.3 mg / kg / day, 0.4 mg / kg / day, 0.5 mg / kg / day, 0.6 mg / kg / day, etc., up to 5.0 mg / kg / day. term As used herein, unless otherwise stated, each of the following terms shall have the definition given below.

[0739] Peptidyl bond: structure

[0740] [ka]

[0741] The peptidyl bond may be a peptide bond.

[0742] Series of Consecutive Amino Acids: A plurality of amino acids arranged in a chain, each connected to the previous amino acid by a peptide bond, except that the first amino acid in the chain may, optionally, not be connected to the previous amino acid. The amino acids in the chain may be naturally occurring or non-naturally occurring, or may include mixtures thereof. The amino acids may be genetically encoded, naturally occurring but not genetically encoded, non-naturally occurring, or any selection thereof, unless otherwise indicated.

[0743] N-terminal amino acid residue: Free α-amino (NH 2 ) functional group or α-amino (NH 2 ) The terminal residue of a string of two or more consecutive amino acids having a functional group derivative.

[0744] N-terminus: the free α-amino (NH 2 ) group (or a derivative thereof).

[0745] C-terminal amino acid residue: the terminal residue of a series of two or more contiguous amino acids having a free α-carboxyl (COOH) functional group or a derivative of an α-carboxyl (COOH) functional group.

[0746] C-terminus: the free α-carboxyl (COOH) group (or a derivative thereof) of the C-terminal amino acid residue.

[0747] "Biologically active structure," as used herein, refers to a structure of a molecule or fragment thereof that is capable of performing a function or action, or stimulating or responding to a function, action or response in a biological context (e.g., within an organism, cell, or in vitro model thereof), thereby treating a disease or condition, or localizing or targeting a compound of the invention to a site of a disease or condition in the body. Biologically active structures may include at least one structure of a small molecule, such as a polypeptide, a nucleic acid, or an organic or inorganic small molecule.

[0748] "Bond", unless otherwise specified or contrary to context, is understood to include covalent bonds, dipole-dipole interactions such as hydrogen bonding, and intermolecular interactions such as van der Waals forces.

[0749] A "signal sequence" is a short (3-60 amino acids long) peptide chain that directs post-translational transport of a polypeptide.

[0750] As used herein, "amino acid" refers, in one aspect, to the L- or D-isomers of the genetically encoded amino acids, namely, isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, arginine, serine, histidine, tyrosine, selenocysteine, and pyrrolysine, and also includes homocysteine ​​and homoselenocysteine.

[0751] Other examples of amino acids include the L or D isomers of taurine, GABA, dopamine, lanthionine, 2-aminoisobutyric acid, dehydroalanine, ornithine and citrulline, as well as non-naturally occurring homologues and synthetically modified forms thereof, such as amino acids having alkylene chains shortened or lengthened by up to two carbon atoms, amino acids containing optionally substituted aryl groups, amino acids containing halogenated groups, including halogenated alkyl and aryl groups, and beta or gamma amino acids, and cyclic analogs.

[0752] Depending on the presence of ionizable amino and carboxyl groups, the amino acids in these embodiments may be in the form of acidic or basic salts or in neutral form. Individual amino acid residues may be modified by oxidation or reduction. Other contemplated modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, and methylation of the α-amino groups of lysine, arginine, and histidine side chains.

[0753] Covalent derivatives may be made by linking particular functional groups to amino acid side chains or at the N- or C-terminus.

[0754] Compounds containing amino acids with R group substitutions are within the scope of the present invention. It is understood that the substituents and substitution patterns in the compounds of the present invention can be selected by one of ordinary skill in the art to yield chemically stable compounds from readily available starting materials.

[0755] As used herein, "naturally occurring amino acid" refers to the genetically encoded amino acids, i.e., isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, arginine, serine, histidine, tyrosine, selenocysteine, pyrrolysine, and the L- or D-isomers of homocysteine ​​and homoselenocysteine.

[0756] As used herein, an "unnatural amino acid" is an amino acid that has a C between the α carbon and S or Se. 3 ~C 10 It refers to chemically modified L or D isomers of isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, arginine, serine, histidine, tyrosine, selenocysteine, pyrrolysine, homocysteine, homoselenocysteine, taurine, GABA, dopamine, lanthionine, 2-aminoisobutyric acid, dehydroalanine, ornithine, or citrulline, including cysteine ​​and selenocysteine ​​derivatives with aliphatic side chains.In one embodiment, the aliphatic side chain is alkylene.In another embodiment, the aliphatic side chain is alkylene or alkynylene.

[0757] In addition to the contiguous amino acid sequences described herein, it is contemplated that variants thereof can be made by introducing appropriate nucleotide changes into the coding DNA and / or by synthesis of the desired contiguous amino acid sequence. One of skill in the art will appreciate that if expression is by a synthetic method selected (e.g., rather than chemical synthesis), the amino acid changes may alter post-translational processing of the contiguous amino acid sequences described herein, for example, by altering the number and position of glycosylation sites or altering the membrane anchoring properties.

[0758] Modifications of the sequences described herein can be made using any of the techniques and guidelines for conservative and non-conservative mutations described, for example, in U.S. Pat. No. 5,364,934. Modifications can be substitutions, deletions, or insertions of one or more codons encoding the contiguous amino acid sequence of interest, resulting in an amino acid sequence change compared to the original sequence. Optionally, the modification is by substituting at least one amino acid with any other amino acid in one or more of the domains. Guidance in determining which amino acid residues to insert, substitute, or delete without adversely affecting the desired activity can be found by comparing the sequence to sequences of known homologous protein molecules and minimizing the number of amino acid sequence changes made in areas of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, e.g., replacing leucine with serine, i.e., conservative amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. Permitted variations may be determined by synthetically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the original full-length or mature sequence, with any terminal variations being understood to be made within the context of the invention disclosed herein.

[0759] Amino acid sequence variants of binding partners are generated with a variety of objectives in mind, including increasing the affinity of the binding partner for its ligand, facilitating the stability, purification, and production of the binding partner, altering its plasma half-life, improving therapeutic efficacy, and reducing the severity or occurrence of side effects during therapeutic use of the binding partner.

[0760] It is contemplated herein that amino acid sequence variants of these sequences include insertional, substitutional or deletion variants. Such variants can be made by site-directed mutagenesis of nucleotides in DNA encoding the target-binding monomer, thereby obtaining DNA encoding the variant, which is then expressed in recombinant cell culture. Fragments having up to about 100-150 amino acid residues can also be conveniently made by in vitro synthesis. Such amino acid sequence variants are predetermined variants and do not occur in nature. The variants exhibit the qualitative biological activity (including target binding) of the unmutated form, although not necessarily the same quantitative values ​​as the unmutated form. The site at which the amino acid sequence variation is introduced is predetermined, but the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random or saturation mutagenesis (where all 20 possible residues are inserted) is performed at the target codon, and the expressed variants are screened for the optimal combination of desired activity. Such screening is within the ordinary skill in the art.

[0761] Amino acid insertions are usually on the order of about 1-10 amino acid residues, substitutions are typically introduced for single residues, and deletions range from about 1-30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletion of two residues or insertion of two residues. It will be abundantly clear from the following discussion that substitutions, deletions, insertions, or any combination thereof may be introduced or combined to arrive at a final structure.

[0762] In one aspect, the invention provides a nucleic acid sequence that has at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, to the amino acid sequences, figures, SEQ ID NOs. disclosed herein, or to the sequence listing of this application. and more preferably, at least about 90% sequence identity, more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity, even more preferably at least about 98% sequence identity, and even more preferably at least about 99% sequence identity.

[0763] Percent amino acid sequence identity values ​​can be readily obtained, for example, using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)).

[0764] Fragments of the original sequence are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared to the full-length original protein. Any terminal modifications are understood to be within the context of the invention disclosed herein.

[0765] Certain fragments lack amino acid residues that are not essential for a desired biological activity of the sequence of interest.

[0766] Any of several conventional techniques may be used. The desired peptide fragment, or fragment of a series of contiguous amino acids, may be chemically synthesized. Alternative approaches include enzymatic digestion, e.g., generating fragments by treating the protein with an enzyme known to cleave the protein at sites defined by specific amino acid residues, or by digesting the DNA with appropriate restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding the desired polypeptide / sequence fragment by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are used at the 5' and 3' primers in the PCR.

[0767] In certain embodiments, conservative substitutions of interest are shown under the heading of preferred substitutions in Table 1. If such substitutions result in a change in biological activity, then more substantial changes, such as those shown as exemplary substitutions in Table 1 or further described below in connection with classes of amino acids, are introduced and the products screened.

[0768] [Table 1]

[0769] Substantial alterations in the function or immunological identity of the sequence are achieved by selecting substitutions that are significantly different in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, e.g., a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into the following groups based on common properties of their side chains: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) Basic: asn, gln, his, lys, arg; (5) Residues that affect chain orientation: gly, pro; (6) Aromatic; trp, tyr, phe.

[0770] Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0771] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)), or other known techniques can be performed on cloned DNA to generate mutant DNA.

[0772] Scanning amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is generally the preferred scanning amino acid within this group because it is less likely to remove the side chain other than the β carbon and alter the main-chain conformation of the mutant (Cunningham and Wells, Science, 244:1081-1085 (1989)). Alanine is also generally preferred because it is the most common amino acid. Furthermore, alanine is commonly found in both buried and exposed positions (Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not result in a sufficient amount of mutants, an isosteric amino acid can be used.

[0773] Covalent modification: A series of consecutive amino acids may be covalently modified. One type of covalent modification involves reacting the targeted amino acids with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues not involved in the -xx-bond. Derivatization with bifunctional agents is useful, for example, for crosslinking with water-soluble support matrices or surfaces for use in methods for purifying the antisequence of the antibody of interest, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters including 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), bifunctional maleimides, such as bis-N-maleimido-1,8-octane, and methyl-3-((p-azidophenyl)dithio)propioimidate, and other agents.

[0774] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0775] Another type of covalent modification involves altering the original glycosylation pattern of a series of contiguous amino acids. "Altering the original glycosylation pattern," for purposes herein, is intended to mean deleting one or more glycans found in the amino acid sequence (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and / or enzymatic means) and / or adding one or more glycosylation sites that are not present in the original sequence. In addition, the phrase includes qualitative changes in the glycosylation of the original protein, including changes in the nature and proportion of the various glycans present.

[0776] Addition of glycosylation sites to an amino acid sequence may be achieved by altering the amino acid sequence, either by adding one or more serine or threonine residues to the original sequence or by substituting one or more serine or threonine residues (for O-linked glycosylation sites). The amino acid sequence may also be optionally altered through changes at the DNA level, in particular by mutating the DNA encoding the amino acid sequence at preselected bases to produce a codon that translates into the desired amino acid.

[0777] Another means of increasing the number of carbohydrate moieties on an amino acid sequence is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87 / 05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0778] Removal of carbohydrate moieties present in the amino acid sequence may be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues targeted for glycosylation. Techniques for chemical glycosylation are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987), and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be accomplished by the use of a variety of endo- and exoglycosidases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0779] Another type of covalent modification involves linking the amino acid sequence to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, by the methods described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0780] The terms "substituted," "substituted," and "substituent" refer to a functional group in which one or more bonds to a hydrogen atom are replaced with a bond to a non-hydrogen atom, provided that the normal valences are maintained and the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon or hydrogen atom are replaced with one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituents include halogen (i.e., F, Cl, Br, and I); alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, and trifluoromethyl; aryl groups such as phenyl; heteroaryl groups such as triazole, dihydropyridazine, and tetrazole; hydroxyl; alkoxy groups such as methoxy, ethoxy, n-propyl, and isopropoxy; aryloxy groups such as phenoxy; arylalkyloxy groups such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups such as sulfonate, trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; sulfnitro, nitrosyl; mercapto; sulfanyl groups such as methylsulfanyl, ethylsulfanyl, and propylsulfanyl; cyano; amino groups such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. In the case where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted, singly or multiply, by one or more of the disclosed or claimed substituent moieties. "Independently substituted" means that the (two or more) substituents can be the same or different. In the compounds used in the methods of the present invention, alkyl, heteroalkyl, monocyclic, bicyclic, aryl, heteroaryl, and heterocyclic groups can be substituted by replacing one or more hydrogen atoms with alternative non-hydrogen atoms. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, and carbamoyl.

[0781] It is understood that the substituents and substitution patterns in the compounds used in the methods of the present invention can be selected by one of ordinary skill in the art to result in compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. It is understood that when a substituent is itself substituted with more than one group, these multiple groups can be on the same carbon or on different carbons, so long as a stable structure results.

[0782] In selecting compounds for use in the methods of the present invention, one of skill in the art will appreciate the various substituents, i.e., R 1 , R 2 It will be appreciated that the etc. should be selected in accordance with well-known principles of chemical structure connectivity.

[0783] As used herein, "alkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups, which may be unsubstituted or substituted, having the specified number of carbon atoms. 1 ~C n C as in "alkyl" 1 ~C n is defined to include groups having 1, 2, ..., n-1 or n carbons in a linear or branched arrangement. For example, "C 1 ~C 6 C as in "alkyl" 1 ~C 6 is defined to include groups having 1, 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement, specifically including methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl and hexyl. Unless otherwise specified, contains 1 to 12 carbon atoms. Alkyl groups can be unsubstituted or substituted with one or more substituents, including but not limited to halogen, alkoxy, alkylthio, trifluoromethyl, difluoromethyl, methoxy and hydroxyl. One embodiment is C 1 ~C 12 Alkyl, C 2 ~C 12Alkyl, C 3 ~C 12 Alkyl, C 4 ~C 12 In one embodiment, the alkyl group is C 1 ~C 8 Alkyl, C 2 ~C 8 Alkyl, C 3 ~C 8 Alkyl, C 4 ~C 8 Alkyl, etc. Alkyl is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0784] As used here, "C 1 ~C 4 "Alkyl" is a branched C 1 ~C 4 Alkyl and Linear C 1 ~C 4 The term "alkyl" includes both aryl and alkyl.

[0785] As used herein, the term "cycloalkane" refers to a monocyclic or bicyclic ring system that may be unsaturated or partially unsaturated, i.e., has one or more double bonds. Monocyclic ring systems include saturated cyclic hydrogen groups containing 3 to 8 carbon atoms. Examples of monocyclic ring systems include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic fused ring systems include a cycloalkyl ring fused to another cycloalkyl ring. Examples of bicyclic fused ring systems include, but are not limited to, decalin, 1,2,3,7,8,8a-hexahydronaphthalene, and the like. Thus, C 3 ~C 10Cycloalkanes contain cyclic rings of alkanes of 3 to 8 total carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, etc.). Cycloalkane groups can be unsubstituted or substituted with one or more substituents, including, but not limited to, halogen, alkoxy, alkylthio, trifluoromethyl, difluoromethyl, methoxy, and hydroxyl. Cycloalkanes are intended to include moieties that are monovalent, divalent, trivalent, etc.

[0786] As used herein, the term "cycloalkene" refers to a cycloalkane having one or more double bonds. 5 ~C 10 Cycloalkene includes cyclic rings of alkanes having 5 to 10 total carbon atoms (e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, cyclooctenyl, or cyclooctadienyl, etc.). Cycloalkene is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0787] As used herein, "alkene" includes branched and straight-chain aliphatic hydrocarbon groups having one or more double bonds and the specified number of carbon atoms, which may be unsubstituted or substituted. 2 ~C n Alkenes such as C 2 ~C n is defined to include groups having 2, 3, ..., n-1 or n carbons in a linear or branched arrangement. For example, C 2 ~C 10 C as in alkenes 2 ~C 10is defined as a group having 2, 3, 4, 5...10 carbons in a linear or branched arrangement, and specifically includes vinyl, allyl, 1-butene, 2-butene, isobutene, 1-pentene, 2-pentene, etc. The alkene group may be unsubstituted or substituted with one or more substituents, including, but not limited to, halogen, alkoxy, alkylthio, trifluoromethyl, difluoromethyl, methoxy, and hydroxyl. One aspect is C 2 ~C 3 Alkenes, C 2 ~C 4 Alkenes, C 2 ~C 5 Alkene, etc. Alkene is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0788] As used herein, "acyl" refers to an alkyl group having a ketone at the 1-position. For example, an embodiment of "acyl" can be acetyl, propionyl, butyryl, and valeryl. As another example, an embodiment of "acyl" is

[0789] [ka]

[0790] (where n is 1 to 10) In another embodiment, n is 1-4.

[0791] Therefore, "C 2 ~C 5 "Acyl" can be acetyl, propionyl, butyryl, or valeryl. Acyl is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0792] C 2 ~C 5Acylamino is an acyl group, as defined above, further substituted with an amine. The amine may be linked to the carbonyl portion of the acyl group to form an amide, or may be linked to the non-carbonyl portion of the acyl group. For example, the amino group may be at the α-position, β-position, γ-position, δ-position, etc. As a further example, acylamino includes both α-aminoacetyl and acetamido groups. Acylamino includes β-aminopropionyl.

[0793] C 2 ~C 5 Acyloxy is an acyl group, as defined above, further substituted with oxygen. The oxygen may be linked to the carbonyl portion of the acyl group to form an amide, or may be linked to the non-carbonyl portion of the acyl group. For example, the oxygen group may be at the α-position, β-position, γ-position, δ-position, etc. As a further example, acyloxy includes both α-oxyacetyl and acetate groups. Acyloxy includes β-oxypropionyl.

[0794] As used herein, "amino" includes primary, secondary, tertiary, and quaternary amines. Amino thus includes the -NH- group, -NH 2 group, -NR- group, -NR 2 + -group, -NRH + -group, -NH 2 + -group, -NH 3 + group, and -NR 3 + groups where R is alkyl or aryl. Amino is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0795] As used herein, "sulfur" includes -S- and -SH groups. The term sulfur is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0796] As used herein, "oxygen" includes -O- and -OH groups. The term sulfur is intended to refer to moieties that are monovalent and divalent.

[0797] As used herein, "succinyl" is derived from succinic acid by removal of one or both hydroxyl groups. One embodiment is -C(O)-CH 2 -CH 2 It can be -C(O)-. Succinyl is intended to include monovalent, divalent, trivalent, etc. moieties.

[0798] As used herein, "malonyl" is derived from malonic acid by removal of one or both hydroxyl groups. One embodiment is -C(O)-CH 2 It can be -C(O)-. Malonyl is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0799] As used herein, "glutaryl" is derived from glutaric acid by removal of one or both hydroxyl groups. One embodiment is -C(O)-CH 2 -CH 2 -CH 2 It can be -C(O)-. Glutaryl is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0800] As used herein, "adipoyl" is derived from adipic acid by removal of one or both hydroxyl groups. One embodiment is -C(O)-CH 2 -CH 2 -CH 2 -CH 2 It can be -(O)-. Adipoyl is intended to include moieties that are monovalent, divalent, trivalent, etc.

[0801] A "polyalkylene glycol" is derived from a polyalkylene glycol by removal of both hydrogens from a hydroxyl group. An embodiment may be derived from a polyethylene glycol, a polypropylene glycol, or a polybutylene glycol. An embodiment of a "polyalkylene glycol" is:

[0802] [ka]

[0803] (where n is 1 to 10) It is possible.

[0804] As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic, or polycyclic carbocyclic ring of up to 10 atoms in each ring, in which at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include, but are not limited to, phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, phenanthryl, anthryl, or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

[0805] As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or polycyclic carbocyclic ring of up to 10 atoms in each ring, in which at least one ring is aromatic and may be unsubstituted or substituted. Examples of such aryl elements include, but are not limited to, phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydronaphthyl, indanyl, phenanthryl, anthryl, or acenaphthyl. When the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

[0806] The term "heteroaryl," as used herein, refers to any stable monocyclic, bicyclic, or polycyclic ring of up to 10 atoms in each ring, in which at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine, or pyrizidine rings: (a) fused to a 6-membered aromatic (unsaturated) heterocycle having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocycle having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocycle having one nitrogen atom with one oxygen atom or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocycle having one heteroatom selected from O, N, or S.Heteroaryl groups within the scope of this definition include benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, dihydropyrididine, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthapyridinyl, oxadiazolyl, oxazolyl, oxazoline, and the like. , isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydro Examples of heteroaryls include, but are not limited to, furanyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. In cases where a heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, it is understood that attachment is via the aromatic ring or via the heteroatom-containing ring, respectively.If the heteroaryl contains a nitrogen atom, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

[0807] The term "phenyl" is intended to mean an aromatic six-membered ring containing six carbons, and any substituted derivatives thereof.

[0808] The term "benzyl" is intended to mean a methylene directly attached to a benzene ring. A benzyl group is a methyl group in which the hydrogen has been replaced by a phenyl group, and any of the substituted derivatives thereof.

[0809] The term "triazole" is intended to mean a heteroaryl having a 5-membered ring containing 2 carbon atoms and 3 nitrogen atoms, and any substituted derivatives thereof.

[0810] Dihydropyridazine may be optionally substituted, such as 1,2-dihydropyridazine.

[0811] [ka]

[0812] 1,4-Dihydropyridazine

[0813] [ka]

[0814] 1,6-Dihydropyridazine

[0815] [ka]

[0816] and 4,5-dihydropyridazine

[0817] [ka]

[0818] Includes.

[0819] Chemical structures containing cyclooctane fused to dihydropyridazine include, but are not limited to, chemical structures containing cyclooctane fused to the 3- and 4-positions of dihydropyridazine, or chemical structures containing saturated cycloocta[d]pyridazine, both of which may be optionally substituted. For example, chemical structures containing cyclooctane fused to dihydropyridazine include, but are not limited to, 2,4a,5,6,7,8,9,10-octahydrocycloocta[d]pyridazine, each of which may be optionally substituted.

[0820] [ka]

[0821] 4a,5,6,7,8,9,10,10a-Octahydrocycloocta[d]pyridazine

[0822] [ka]

[0823] 2,3,5,6,7,8,9,10-Octahydrocycloocta[d]pyridazine

[0824] [ka]

[0825] or 1,2,5,6,7,8,9,10-octahydrocycloocta[d]pyridazine

[0826] [ka]

[0827] Examples of the chemical structure include:

[0828] [ka]

[0829] Examples of tautomers of include, but are not limited to,

[0830] [ka]

[0831] Examples include:

[0832] In some embodiments, the dihydropyridazine is oxidized to pyridazine.

[0833] In some embodiments, the dihydropyridazine is reduced to provide an open ring structure having a 1,4-dicarbonyl compound.

[0834] The compounds used in the methods of the present invention may be made by techniques that are well known in organic synthesis and familiar to those of skill in the art, but these may not be the only means to synthesize or obtain the desired compounds.

[0835] The compounds of the subject invention can be converted into prodrugs to optimize absorption and bioavailability. Formation of prodrugs includes, but is not limited to, reaction of the free hydroxyl group with a carboxylic acid to form an ester, reaction of the free hydroxyl group with phosphoryl chloride to form a phosphate ester followed by hydrolysis, or reaction of the free hydroxyl group with an amino acid to form an amino acid ester, methods of which have been previously described by Chandran in WO2005 / 046575. Substituents are selected and the resulting analogs are evaluated according to principles well known in the art of medicinal and pharmaceutical chemistry, such as quantification of structure-activity relationships, optimization of biological activity, and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties.

[0836] The various R groups attached to the aromatic rings of the compounds disclosed herein may be attached to the ring by standard procedures, such as those described in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the contents of which are incorporated herein by reference.

[0837] The compounds of the invention may be made by the techniques described in Vogel's Textbook of Practical Organic Chemistry, AI Vogel, AR Tatchell, BS Furnis, AJ Hannaford, PWG Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated herein by reference, however, these may not be the only means to synthesize or obtain the desired compounds.

[0838] Those of skill in the art will immediately understand that the definitions of substituents and moieties (e.g., the moieties J, Ra, and Rb) shown herein are intended to conform to standard rules of chemical valency. For example, where a structure shown herein requires that a particular substituent or moiety (e.g., a moiety in a linear chain of moieties) be divalent, those of skill in the art will immediately understand that the definition of that substituent or moiety is divalent in a manner that conforms to standard rules of chemical valency.

[0839] One of skill in the art will readily appreciate that some divalent moieties depicted in the present invention may be linked to other chemical structures in more than one way, for example, the depicted structure may be linked to other chemical structures when rotated or inverted.

[0840] In some embodiments of the invention, the compound comprises a non-proteinaceous polymer. In some embodiments, the non-proteinaceous polymer may be a hydrophilic synthetic polymer, i.e., a polymer that does not otherwise occur in nature. However, naturally occurring polymers produced by recombinant or in vitro methods are useful, as are polymers isolated from nature. Hydrophilic polyvinyl polymers, such as polyvinyl alcohol and polyvinylpyrrolidone, are within the scope of the invention. Particularly useful are polyalkylene ethers, such as polyethylene glycol, polypropylene glycol, polyoxyethylene esters, or methoxypolyethylene glycol; polyoxyalkylenes, such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronic); polymethacrylates; carbomers; and the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid (galacturonate). branched or unbranched polysaccharides, such as homopolysaccharides and heteropolysaccharides, including lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrin, glycogen, including polysaccharide subunits of hyaluronic acid, D-mannuronic acid (e.g., polymannuronic acid or alginic acid), D-glucosamine, D-galactosamine, D-glucose, and neuraminic acid; polymers of sugar alcohols, such as polysorbitol and polymannitol; and heparin or heparon. salt Salts of the compounds disclosed herein are within the scope of the present invention. As used herein, a "salt" is a salt of a compound of the present invention, which has been modified by making an acid or base salt of the compound. FC Domain The term "Fc domain" as used herein generally refers to a monomeric or dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain. The Fc domain may comprise a native or mutated Fc sequence. Although the boundaries of an immunoglobulin heavy chain Fc domain may vary, the Fc domain of a human IgG heavy chain is generally defined to extend from the amino acid residues of the hinge region to the carboxyl terminus of the Fc sequence. An immunoglobulin Fc sequence comprises two constant regions, namely a CH2 region and a CH3 region, and optionally a CH4 region. The human Fc domain may be derived from any suitable immunoglobulin, e.g., IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD, or IgM.

[0841] Suitable Fc domains are produced by recombinant DNA expression of a pre-Fc chimeric polypeptide comprising 1) an Fc domain polypeptide having an N-terminal cysteine ​​residue adjacent to a signal peptide obtained from a secreted or transmembrane protein, which is cleaved before the mature peptide having an N-terminal cysteine ​​residue.

[0842] Suitable examples of signal peptides are sonic hedgehog (SHH) (GenBank Acc. No. NM000193), IFNα-2 (IFN) (GenBank Acc. No. NP000596), and cholesterol ester transferase (CETP) (GenBank Accession No. NM000078). Other suitable examples include Indian hedgehog (GenBank Acc. No. NM002181), desert hedgehog (GenBank Acc. No. NM021044), IFNα-1 (GenBank Acc. No. NP076918), IFNα-4 (GenBank Acc. No. NM021068), IFNα-5 (GenBank Acc. No. NM002169), IFNα-6 (GenBank Acc. No. NM021002), IFNα-7 (GenBank Acc. No. NM021057), IFNα-8 (GenBank Acc. No. NM002170), IFNα-10 (GenBank Acc. No. NM002171), IFNα-13 (GenBank Acc. No. NM006900), IFNα-14 (GenBank Acc. No. NM002172), IFNα-15 (GenBank Acc. No. NM002173), IFNα-16 (GenBank Acc. No. NM002174), IFNα-17 (GenBank Acc. No. NM002175), IFNα-18 (GenBank Acc. No. NM002176), IFNα-19 (GenBank Acc. No. NM002187), IFNα-20 (GenBank Acc. No. NM002189), IFNα-21 (GenBank Acc. No. NM002188), IFNα-22 (GenBank Acc. No. NM002189), IFNα-30 (GenBank Acc. No. NM002189), IFNα-40 (GenBank Acc. No. NM021068), IFNα-5 (GenBank Acc. No. NM002169), IFNα-6 (GenBank Acc. No Acc. No. NM002172), IFNα-16 (GenBank Acc. No. NM002173), IFNα-17 (GenBank Acc. No. NM021268), and IFNα-21 (GenBank Acc. No. NM002175).

[0843] Suitable examples of Fc domains and their pre-Fc chimeric polypeptides are shown in SEQ ID NO: 1 to SEQ ID NO: 96. The Fc domains are obtained by expressing the pre-Fc chimeric polypeptides in cells under conditions that cause secretion and cleavage of the signal peptide. The pre-Fc chimeric polypeptides may be expressed in either prokaryotic or eukaryotic host cells. Preferably, mammalian host cells are transfected with an expression vector encoding the pre-Fc chimeric polypeptide.

[0844] The Fc domain of human IgG1 with N-terminal sequences CDKTHTCPPCPAPE, CPPCPAPE and CPAPE are shown in SEQ ID NO:1, SEQ ID NO:9 and SEQ ID NO:17, respectively, and the DNA sequences encoding them are shown in SEQ ID NO:2, SEQ ID NO:10 and SEQ ID NO:18, respectively. The IgG1 domain of SEQ ID NO:1 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO:3 (SHH signal peptide), SEQ ID NO:5 (IFN signal peptide) and SEQ ID NO:7 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8, respectively. The IgG1 domain of SEQ ID NO:9 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO:11 (SHH signal peptide), SEQ ID NO:13 (IFN signal peptide) and SEQ ID NO:15 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16, respectively. The IgG1 domain of SEQ ID NO: 17 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO: 19 (SHH signal peptide), SEQ ID NO: 21 (IFN signal peptide) and SEQ ID NO: 23 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, respectively.

[0845] The Fc domains of human IgG2 with N-terminal sequences CCVECPPCPAPE, CVECPPCPAPE, CPPCPAPE and CPAPE are shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41 and SEQ ID NO:49, respectively, and the DNA sequences encoding them are shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42 and SEQ ID NO:50, respectively. The IgG2 domain of SEQ ID NO:25 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO:27 (SHH signal peptide), SEQ ID NO:29 (IFN signal peptide) and SEQ ID NO:31 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO:28, SEQ ID NO:30 and SEQ ID NO:32, respectively. The IgG2 domain of SEQ ID NO:33 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO:35 (SHH signal peptide), SEQ ID NO:37 (IFN signal peptide) and SEQ ID NO:39 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:40, respectively. The IgG2 domain of SEQ ID NO: 41 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO: 43 (SHH signal peptide), SEQ ID NO: 45 (IFN signal peptide) and SEQ ID NO: 47 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48, respectively. The IgG2 domain of SEQ ID NO: 49 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO: 51 (SHH signal peptide), SEQ ID NO: 53 (IFN signal peptide) and SEQ ID NO: 55 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, respectively.

[0846] N-terminal sequence (CPRCPEPKSDTPPP) 3The Fc domains of human IgG3 with -CPRCPAPE, CPRCPAPE and CPAPE are shown in SEQ ID NO:57, SEQ ID NO:65 and SEQ ID NO:73, respectively, and the DNA sequences encoding them are shown in SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:42 and SEQ ID NO:74, respectively. The IgG3 domain of SEQ ID NO:57 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO:59 (SHH signal peptide), SEQ ID NO:61 (IFN signal peptide) and SEQ ID NO:63 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:64, respectively. The IgG3 domain of SEQ ID NO:65 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO:67 (SHH signal peptide), SEQ ID NO:69 (IFN signal peptide) and SEQ ID NO:71 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO:68, SEQ ID NO:70 and SEQ ID NO:72, respectively. The IgG3 domain of SEQ ID NO: 73 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO: 75 (SHH signal peptide), SEQ ID NO: 77 (IFN signal peptide) and SEQ ID NO: 79 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO: 76, SEQ ID NO: 78 and SEQ ID NO: 80, respectively.

[0847] The sequences of the Fc domain of human IgG4 with the N-terminal sequences CPSCPAPE and CPAPE are shown in SEQ ID NO: 81 and SEQ ID NO: 89, respectively, and the DNA sequences encoding them are shown in SEQ ID NO: 82 and SEQ ID NO: 90, respectively. The IgG4 domain of SEQ ID NO: 81 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO: 83 (SHH signal peptide), SEQ ID NO: 85 (IFN signal peptide) and SEQ ID NO: 87 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO: 84, SEQ ID NO: 86 and SEQ ID NO: 88, respectively. The IgG4 domain of SEQ ID NO: 89 is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQ ID NO: 91 (SHH signal peptide), SEQ ID NO: 93 (IFN signal peptide) and SEQ ID NO: 95 (CETP signal peptide) using the DNA sequences shown in SEQ ID NO: 92, SEQ ID NO: 94 and SEQ ID NO: 96, respectively.

[0848] Suitable antibody mutants having a cysteine ​​residue at the N-terminus of the heavy chain are produced by recombinant DNA expression of a pre-heavy chain chimeric polypeptide comprising 1) an antibody heavy chain polypeptide adjacent to a signal peptide obtained from a secreted or transmembrane protein, which is cleaved before the mature peptide having the N-terminal cysteine ​​residue, and 2) an antibody heavy chain polypeptide having an N-terminal cysteine ​​residue.

[0849] Suitable antibody variants having a cysteine ​​residue at the N-terminus of the light chain are produced by recombinant DNA expression of a pre-light chain chimeric polypeptide comprising 1) an antibody light chain polypeptide adjacent to a signal peptide obtained from a secreted or transmembrane protein, which is cleaved before the mature peptide having the N-terminal cysteine ​​residue, and 2) an antibody light chain polypeptide having an N-terminal cysteine ​​residue.

[0850] The trastuzumab heavy and light chains are obtained by expressing the pre-heavy and pre-light chimeric polypeptides in cells under conditions that cause secretion and cleavage of the signal peptide. The pre-heavy and pre-light chain polypeptides may be expressed in either prokaryotic or eukaryotic host cells. Preferably, mammalian host cells are transfected with an expression vector encoding the pre-heavy and pre-light chain polypeptides.

[0851] The protein sequences added to the N-terminus of the aforementioned antibody heavy chain, pre-heavy chain, light chain, and pre-light chain variants are shown here for the recombinant antibody trastuzumab, but are generally applicable to any recombinant antibody. The DNA sequences encoding trastuzumab and its variants may be constructed and expressed in mammalian cells by co-transfecting DNA vectors for the heavy and light chains and variants derived therefrom, as described in U.S. Patent No. 5,821,337 ("Immunoglobulin Variants"), which is incorporated herein by reference. The amino acid sequences of the light and heavy chains of wild-type trastuzumab are shown in SEQ ID NO: 128 and SEQ ID NO: 129, respectively.

[0852] Suitable examples of trastuzumab light chains and their pre-Fc chimeric polypeptides having an N-terminal cysteine ​​residue are shown in SEQ ID NO: 130 to SEQ ID NO: 165. Suitable examples of trastuzumab heavy chains and their pre-Fc chimeric polypeptides having an N-terminal cysteine ​​residue are shown in SEQ ID NO: 166 to SEQ ID NO: 201.

[0853] The trastuzumab light chains with N-terminal sequences C, CP, CPP, CPR, CPS, CDKT, CDKTHT, CVE and CDTPPP are shown in SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158 and SEQ ID NO: 162, respectively. The light chain of SEQ ID NO: 130 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 131 (SHH signal peptide), SEQ ID NO: 132 (IFN signal peptide) and SEQ ID NO: 133 (CETP signal peptide). The light chain of SEQ ID NO: 134 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 135 (SHH signal peptide), SEQ ID NO: 136 (IFN signal peptide) and SEQ ID NO: 137 (CETP signal peptide). The light chain of SEQ ID NO: 138 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 139 (SHH signal peptide), SEQ ID NO: 140 (IFN signal peptide) and SEQ ID NO: 141 (CETP signal peptide). The light chain of SEQ ID NO: 142 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 143 (SHH signal peptide), SEQ ID NO: 144 (IFN signal peptide) and SEQ ID NO: 145 (CETP signal peptide). The light chain of SEQ ID NO: 146 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 147 (SHH signal peptide), SEQ ID NO: 148 (IFN signal peptide) and SEQ ID NO: 149 (CETP signal peptide). The light chain of SEQ ID NO: 150 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 151 (SHH signal peptide), SEQ ID NO: 152 (IFN signal peptide) and SEQ ID NO: 153 (CETP signal peptide). The light chain of SEQ ID NO: 154 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 155 (SHH signal peptide), SEQ ID NO: 156 (IFN signal peptide) and SEQ ID NO: 157 (CETP signal peptide).The light chain of SEQ ID NO: 158 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 159 (SHH signal peptide), SEQ ID NO: 160 (IFN signal peptide) and SEQ ID NO: 161 (CETP signal peptide). The light chain of SEQ ID NO: 162 is obtained by expressing the pre-light chain chimeric polypeptides shown in SEQ ID NO: 163 (SHH signal peptide), SEQ ID NO: 164 (IFN signal peptide) and SEQ ID NO: 165 (CETP signal peptide).

[0854] Trastuzumab heavy chains with N-terminal sequences C, CP, CPP, CPR, CPS, CDKT, CDKTHT, CVE and CDTPPP are shown in SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194 and SEQ ID NO: 198, respectively. The heavy chain of SEQ ID NO: 166 is obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 167 (SHH signal peptide), SEQ ID NO: 168 (IFN signal peptide) and SEQ ID NO: 169 (CETP signal peptide). The heavy chain of SEQ ID NO: 170 is obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 171 (SHH signal peptide), SEQ ID NO: 172 (IFN signal peptide) and SEQ ID NO: 173 (CETP signal peptide). The heavy chain of SEQ ID NO: 174 is obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 175 (SHH signal peptide), SEQ ID NO: 176 (IFN signal peptide) and SEQ ID NO: 177 (CETP signal peptide). The heavy chain of SEQ ID NO: 178 is obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 179 (SHH signal peptide), SEQ ID NO: 180 (IFN signal peptide) and SEQ ID NO: 181 (CETP signal peptide). The heavy chain of SEQ ID NO: 182 is obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 183 (SHH signal peptide), SEQ ID NO: 184 (IFN signal peptide) and SEQ ID NO: 185 (CETP signal peptide). The heavy chain of SEQ ID NO: 186 is obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 187 (SHH signal peptide), SEQ ID NO: 188 (IFN signal peptide) and SEQ ID NO: 189 (CETP signal peptide). The heavy chain of SEQ ID NO: 190 can be obtained by expressing the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 191 (SHH signal peptide), SEQ ID NO: 192 (IFN signal peptide) and SEQ ID NO: 193 (CETP signal peptide).The heavy chain of SEQ ID NO: 194 is provided by the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 195 (SHH signal peptide), SEQ ID NO: 196 (IFN signal peptide) and SEQ ID NO: 197 (CETP signal peptide). The heavy chain of SEQ ID NO: 198 is provided by the pre-heavy chain chimeric polypeptides shown in SEQ ID NO: 199 (SHH signal peptide), SEQ ID NO: 200 (IFN signal peptide) and SEQ ID NO: 201 (CETP signal peptide).

[0855] Suitable host cells include 293 human lung cells (ATCC CRL-1573) and CHO-K1 hamster ovary cells (ATCC CCL-61), obtained from the American Type Culture Collection (Rockville, Md.). Cells are grown at 37° C. in an atmosphere of 95% air; 5% carbon dioxide. 293 cells are maintained in Minimum Essential Medium (Eagle) with 2 mM L-glutamine and Earle's solution, adjusted to contain 1.5 g / L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate 90%; fetal bovine serum 10%. CHO-K1 cells are maintained in Ham's F12K medium with 2 mM L-glutamine, adjusted to contain 1.5 g / L sodium bicarbonate 90%; fetal bovine serum 10%. Other suitable host cells include CV1 monkey kidney cells (ATCC CCL-70), COS-7 monkey kidney cells (ATCC CRL-1651), VERO-76 monkey kidney cells (ATCC CRL-1587), HELA human cervical cells (ATCC CCL-2), W138 human lung cells (ATCC CCL-75), MDCK canine kidney cells (ATCC CCL-34), BRL3A rat liver cells (ATCC CRL-1442), BHK hamster kidney cells (ATCC CCL-10), MMT060562 mouse mammary gland cells (ATCC CCL-51), and CD8 + T cells (described in US Ser. No. 08 / 258,152, which is incorporated herein by reference in its entirety).

[0856] Examples of suitable expression vectors are pCDNA3.1(+), shown in SEQ ID NO: 97, and pSA, shown in SEQ ID NO: 98. Plasmid pSA contains the following DNA sequence elements: 1) pBluescriptIIKS(+) (nucleotides 912-2941 / 1-619, GenBank Accession No. X52327), 2) the human cytomegalovirus promoter, enhancer, and splice donor of the first exon (nucleotides 63-912, GenBank Accession No. K03104), 3) the splice acceptor of the second exon of human α1-globin (nucleotides 6808-6919, GenBank Accession No. J00153), 4) the polyadenylation site of the SV40 T antigen (nucleotides 2770-2533, Reddy et al. (1978) Science 200, 494-502), and 5) the SV40 origin of replication (nucleotides 5725-5578, Reddy et al., ibid.). Other suitable expression vectors include plasmids pSVeCD4DHFR and pRKCD4 (U.S. Pat. No. 5,336,603), plasmid pIK.1.1 (U.S. Pat. No. 5,359,046), plasmid pVL-2 (U.S. Pat. No. 5,838,464), and plasmid pRT43.2F3 (described in U.S. Ser. No. 08 / 258,152, which is incorporated by reference in its entirety).

[0857] Suitable expression vectors for human IgG pre-Fc polypeptides may be constructed by ligation of a HindIII-PspOM1 vector fragment made from SEQ ID NO:98 with a HindIII-EagI insert fragment made from SEQ ID NOs:4, 6, 8, 12, 14, 16, 20, 22, 24, 28, 30, 32, 36, 38, 40, 44, 46, 48, 52, 54, 56, 60, 62, 64, 68, 70, 72, 76, 78, 80, 84, 86, 88, 92, 94, and 96.

[0858] Suitable selectable markers include the neomycin phosphotransferase (NEO) gene of the Tn5 transposon (Southern and Berg (1982) J. Mol. Appl. Gen. 1, 327-341), and dihydrofolate reductase (DNFR) cDNA (Lucas et al. (1996) Nucl. Acids Res. 24, 1774-1779). One example of a suitable expression vector incorporating the NEO gene is the plasmid pSA-NEO, which is constructed by ligating a first DNA fragment generated by digesting SEQ ID NO:99 with EcoRI and BglII with a second DNA fragment generated by digesting SEQ ID NO:98 with EcoRI and BglII. SEQ ID NO:99 incorporates the NEO gene (nucleotides 1551-2345, Genbank Accession No. U00004), preceded by a translation initiation sequence (Kozak (1991) J. Biol. Chem, 266, 19867-19870). Another example of a suitable expression vector incorporating the NEO gene and DHFR cDNA is the plasmid pSVe-NEO-DHFR, which is constructed by ligating a first DNA fragment generated by digesting SEQ ID NO:99 with EcoRI and BglII with a second DNA fragment generated by digesting pSVeCD4DHFR with EcoRI and BglII. Plasmid pSVe-NEO-DHFR uses the SV40 early promoter / enhancer to drive expression of the NEO gene and DHFR cDNA. Other suitable selectable markers include the XPGT gene (Mulligan and Berg (1980) Science 209, 1422-1427), and the hygromycin resistance gene (Sugden et al. (1985) Mol. Cell. Biol. 5, 410-413).

[0859] In one embodiment, cells are transfected by the calcium phosphate method of Graham et al. (1977) J. Gen. Virol. 36, 59-74. The DNA mixture (10 ug) was diluted with 0.5 ml of 1 mM Tris-HCl, 0.1 ml EDTA and 227 mM CaCl 2 The DNA mixture contains (in a ratio of 10:1:1) expression vector DNA, selectable marker DNA, and DNA encoding the VA RNA gene (Thimmappaya et al. (1982) Cell 31, 543-551). This mixture is dissolved in 0.5 mL of 50 mM Hepes (pH 7.35), 280 mM NaCl, and 1.5 mM NaPO 4 is added dropwise. The DNA precipitate is allowed to form, suspended, and added to cells grown to confluency in 100 mm plastic tissue culture dishes for 10 minutes at 25°C. After 4 hours at 37°C, the medium is aspirated and 2 ml of 20% glycerol in PBS is added for 0.5 minutes. The cells are then washed with serum-free medium, fresh medium is added, and the cells are incubated for 5 days.

[0860] In another embodiment, cells are transiently transfected by the dextran sulfate method of Somparyrac et al. (1981) Proc. Nat. Acad. Sci. 12, 7575-7579. Cells are grown to maximum density in spinner flasks, concentrated by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet. After 4 hours at 37°C, the DEAE-dextran is aspirated and 20% glycerol in PBS is added for 1.5 minutes. The cells are then washed with serum-free medium and reintroduced into the spinner flask with fresh medium containing 5 micrograms / ml bovine insulin and 0.1 micrograms / ml bovine transferrin and incubated for 4 days.

[0861] After transfection by either method, the conditioned medium is centrifuged and filtered to remove host cells and debris. The sample containing the Fc domain is then concentrated and purified by any selected method, e.g., dialysis, and / or column chromatography (see below). To identify the Fc domain in the cell culture supernatant, the cell medium is removed 24-96 hours after transfection, concentrated, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence or absence of a reducing agent such as dithiothreitol.

[0862] For non-amplified expression, the plasmids are transfected into human 293 cells (Graham et al., J. Gen. Virol. 36:59 74 (1977)) using a highly efficient procedure (Gorman et al., DNA Prot. Eng. Tech. 2:3 10 (1990)). The medium is replaced with serum-free medium and harvested daily for up to 5 days. For non-amplified expression, the plasmids are transfected into human 293 cells (Graham et al., J. Gen. Virol. 36:59 74 (1977)) using a highly efficient procedure (Gorman et al., DNA Prot. Eng. Tech. 2:3 10 (1990)). The medium is replaced with serum-free medium and harvested daily for up to 5 days. The Fc domain is purified from cell culture supernatant using HiTrap Protein A HP (Pharmacia). The eluted Fc domain is buffer exchanged into PBS using a Centricon-30 (Amicon), concentrated to 0.5 ml, and sterile filtered at 4° C. using a Millex-GV (Millipore). A series of consecutive amino acids Examples of strings of contiguous amino acids referred to herein include, but are not limited to, strings of contiguous amino acids comprising binding domains, such as secreted or transmembrane proteins, intracellular binding domains, and antibodies (whole or part thereof), and modifications thereof. The following are some non-limiting examples: 1) Immunoglobulins The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), univalent antibodies, multivalent antibodies, and antibody fragments (e.g., Fab and / or single-arm antibodies), so long as they exhibit the desired biological activity.

[0863] The "class" of an antibody refers to the type of constant domain or constant region possessed by the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0864] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 linear antibodies; single-chain antibody molecules (eg, scFv); and multispecific antibodies formed from antibody fragments.

[0865] As used herein, the terms "full length antibody," "intact antibody," and "complete antibody" are used interchangeably to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain that includes an Fc region as defined herein.

[0866] A "blocking" or "antagonist" antibody is one that significantly inhibits (either partially or completely) the biological activity of the antigen to which it binds.

[0867] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to an antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to an antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0868] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0869] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops (hypervariable loops). In general, naturally occurring four-chain antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter being those of highest sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within CDR regions referred to as truncated CDRs or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3 (see Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al., supra.

[0870] "Framework" or "FR" refers to the residues of a variable domain excluding the hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0871] The phrase "N-terminally truncated heavy chain" as used herein refers to a polypeptide comprising a portion but not all of a full-length immunoglobulin heavy chain, where the missing portion is typically in the N-terminal region of the heavy chain. The missing portion may include, but is not limited to, the variable domain, CH1, and part or all of the hinge sequence. Generally, when the wild-type hinge sequence is not present, the remaining constant domain of the N-terminally truncated heavy chain will comprise a component that can be linked to another Fc sequence (i.e., a "first" Fc polypeptide as described herein). For example, the component may be a modified residue or an added cysteine ​​residue that can form a disulfide bond.

[0872] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0873] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and in regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0874] In vivo binding to human FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with mutant Fc regions. WO2000 / 42072 (Presta) describes antibody variants that improve or attenuate binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0875] "Hinge region," "hinge sequence," and variations thereof, as used herein, include their meanings known in the art, as described, for example, in Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202.

[0876] Unless otherwise indicated, the term "multivalent antibody" is used throughout this specification to refer to an antibody that comprises three or more antigen-binding sites. A multivalent antibody is preferably one that has been engineered to have three or more antigen-binding sites, and is generally not a native sequence IgM or IgA antibody.

[0877] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight association that can essentially be covalently linked, for example in an scFv. The three HVRs of each variable domain are represented by V H -V L It is in this configuration that they interact to define an antigen-binding site on the surface of the dimer. Collectively, the six HVRs, or a subset of them, confer antigen-binding specificity to the antibody. However, even a single variable domain (or even a half-Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although usually with lower affinity than the entire binding site.

[0878] The "Fab" fragment contains the variable and constant domains of the light chain and the variable domain and first constant region (CH1) of the heavy chain. F(ab') 2Antibody fragments comprise a pair of Fab fragments, which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are known in the art.

[0879] The phrase "antigen-binding arm" as used herein refers to the portion of an antibody fragment that has the ability to specifically bind to a target molecule of interest. Typically and preferably, an antigen-binding arm is a complex of immunoglobulin polypeptide sequences, e.g., HVRs and / or immunoglobulin light and heavy chain variable domain sequences.

[0880] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L Fv polypeptides generally comprise a V domain, where these domains are present in a single polypeptide chain. H and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0881] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H and V L ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H(Diabodies include diabodies, which are generally not specifically disclosed in the literature.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0882] The term "linear antibody" refers to the antibodies described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which together with complementary light chain polypeptides form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0883] 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 and / or bind the same epitope, except for possible variant antibodies, e.g., including naturally occurring mutations or variants that arise during the manufacture of a monoclonal antibody preparation, which are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0884] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0885] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0886] A "human antibody" is one having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or obtained from a non-human source that utilizes a repertoire of human antibodies or other sequences encoding human antibodies. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0887] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0888] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0889] "Affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0890] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more HVRs thereof, compared to a parent antibody that does not possess such alterations, which alterations increase the affinity of the antibody for the antigen.

[0891] An antibody having a "biological characteristic" of a designated antibody is an antibody that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies that bind to the same antigen.

[0892] A "functional antigen-binding site" of an antibody is a site capable of binding to a target antigen. The antigen-binding affinity of an antigen-binding site is not necessarily as strong as the parent antibody from which it is derived, but the ability to bind to an antigen must be measurable using any of the various known methods for assessing the binding of an antibody to an antigen. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent antibody herein does not have to be quantitatively the same. For multimeric antibodies herein, the number of functional antigen-binding sites can be assessed using ultracentrifugation analysis, as described in Example 2 of US Patent Application Publication No. 2005 / 0186208. According to this analysis method, different ratios of target antigen to multimeric antibody are combined and the average molecular weight of the complex is calculated assuming different numbers of functional binding sites. These theoretical values ​​are compared to actual experimental values ​​obtained to assess the number of functional binding sites.

[0893] A "species-dependent antibody" is an antibody that has a binding affinity for an antigen from a first mammalian species that is stronger than the binding affinity it has for a homologue of that antigen from a second mammalian species. Typically, a species-dependent antibody will "specifically bind" to a human antigen (i.e., approximately 1×10 -7 M or less, preferably about 1×10 -8 Less than about 1×10 -9 The species-dependent antibody has a binding affinity to a homologue of the antigen from a second non-human mammalian species that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than its binding affinity to the human antigen (having a binding affinity (Kd) value of M or less). The species-dependent antibody can be any of the various types of antibodies defined above. In some embodiments, the species-dependent antibody is a humanized or human antibody.

[0894] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-polyacrylamide gel electrophoresis, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see Flatman et al., J. Chromatogr. B 848:79-87 (2007). 2) Extracellular proteins Extracellular proteins play important roles, inter alia, in the formation, differentiation, and maintenance of multicellular organisms. A review of various extracellular proteins of interest is provided in U.S. Patent No. 6,723,535 (Ashkenazi et al.), issued April 20, 2004, which is incorporated by reference.

[0895] The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically regulated by information received from other cells and / or the immediate environment. This information is often conveyed by secreted peptides (e.g., mitogens, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones), which are received and interpreted by a variety of cellular receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules usually pass through the secretory pathway of the cell to reach their site of action in the extracellular environment.

[0896] Secreted proteins have various industrial applications, including pharmaceuticals, diagnostics, biosensors, and bioreactors. Most currently available protein drugs, such as thrombolytic agents, interferons, interleukins, erythropoietin, colony-stimulating factors, and various other cytokines, are secreted proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts to identify novel naturally secreted proteins have been undertaken by both industry and academia. Many efforts have been focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in the literature (see, for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Patent No. 5,536,637).

[0897] Membrane-bound proteins and receptors can play important roles, inter alia, in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g. proliferation, migration, differentiation or interaction with other cells, is typically governed by information received from other cells and / or the immediate environment. This information is often transmitted by secreted polypeptides (e.g., mitogens, survival factors, cytotoxic factors, differentiation factors, neuropeptides and hormones), which are received and interpreted by a variety of cellular receptors or membrane-bound proteins. Such membrane-bound proteins and cellular receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cell adhesion molecules such as selectins and integrins. For example, the transmission of signals that regulate cell growth and differentiation is regulated in part by the phosphorylation of various cellular proteins. Protein tyrosine kinases, the enzymes that catalyze the process, can also act as growth factor receptors. Examples include fibroblast growth factor and nerve growth factor receptors.

[0898] Membrane-bound proteins and receptor molecules have a variety of industrial applications, including pharmaceutical and diagnostic applications. For example, receptor immunoadhesins can be used as therapeutic agents to block receptor-ligand interactions. Membrane-bound proteins can also be used to screen for potential peptide or small molecule inhibitors of the relevant receptor / ligand interactions. 3) Intein-based C-terminal synthesis For example, as described in U.S. Patent No. 6,849,428, issued February 1, 2005, inteins are the protein equivalents of self-splicing RNA introns (Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)) that catalyze their own excision from precursor proteins while fusing flanking protein sequences known as exteins (reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, FB Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153 (1996)).

[0899] Studies of the intein splicing mechanism have led to the development of a protein purification system that uses thiol-induced cleavage of a peptide bond at the N-terminus of the Sce VMA intein (Chong et al., Gene 192(2):271-281 (1997)). Purification with this intein-mediated system results in a bacterially expressed protein with a C-terminal thioester (Chong et al., (1997)). In one application described for isolating a cytotoxic protein, a bacterially expressed protein with a C-terminal thioester is fused to a chemically synthesized peptide with an N-terminal cysteine ​​using "native chemical ligation" chemistry (Evans et al., Protein Sci. 7:2256-2264 (1998);Muir et al., Proc. Natl. Acad. Sci. USA 95:6705-6710 (1998)).

[0900] This technique, called "intein-mediated protein ligation (IPL)", has significantly advanced protein semisynthesis. However, the general applicability of IPL has been limited by the requirement of a chemically synthesized peptide as the ligation partner, since it is difficult to obtain chemically synthesized peptides with more than about 100 residues.

[0901] For example, as described in U.S. Patent No. 6,849,428, IPL technology was significantly expanded when expressed proteins were generated with a defined N-terminus, e.g., a cysteine, allowing the fusion of one or more proteins expressed from a host cell, e.g., bacteria, yeast, mammalian cells. In one non-limiting example, a modified RIR1 Methanobacterium thermoautotrophicum intein was used that cleaves either at the C-terminus or the N-terminus, allowing the release of bacterially expressed proteins during one-column purification, thus completely eliminating the need for proteases.

[0902] Intein technology is an example of one route to obtain the components. In one embodiment, the subunits of the compounds of the invention are obtained by transfecting suitable cells capable of expressing and secreting mature chimeric peptides, where such polypeptides include, for example, an adhesin domain adjacent to an isolatable C-terminal intein domain (see, for example, U.S. Patent No. 6,849,428, issued February 1, 2005, by Evans et al., which is incorporated by reference). Cells, such as mammalian or bacterial cells, are transfected using known recombinant DNA techniques. The secreted chimeric polypeptides can then be isolated, for example, in the case of an intein-chitin binding domain, using a chitin-derivatized resin (see, for example, U.S. Patent No. 6,897,285, issued May 24, 2005, by Xu et al., which is incorporated by reference), and then treated under conditions that allow thiol-mediated cleavage and release of the subunits with C-terminal thioester ends at this point. Thioester-terminated adhesin subunits are readily converted to C-terminal cysteine-terminated subunits.

[0903] For example, after the intein autocleavage reaction, a thioester intermediate is generated that allows for the simple addition of cysteine, selenocysteine, homocysteine ​​or homoselenocysteine, or derivatives of cysteine, selenocysteine, homocysteine ​​or homoselenocysteine ​​to the C-terminus by native chemical ligation. Methods for adding cysteine, selenocysteine, homocysteine ​​or homoselenocysteine, or derivatives of cysteine, selenocysteine, homocysteine ​​or homoselenocysteine ​​to the C-terminus by native chemical ligation are useful in aspects of the present invention and are described in U.S. Patent Application No. 2008 / 0254512 (Capon), published October 16, 2008, the entire contents of which are incorporated herein by reference. kit Another aspect of the present invention provides kits comprising the compounds disclosed herein and pharmaceutical compositions comprising these compounds. The kits may comprise diagnostic or therapeutic agents in addition to the compounds or pharmaceutical compositions. The kits may also comprise instructions for use in diagnostic or therapeutic methods. In diagnostic embodiments, the kits comprise the compounds or pharmaceutical compositions thereof, and diagnostic agents. In therapeutic embodiments, the kits comprise the antibodies or pharmaceutical compositions thereof, and one or more therapeutic agents, such as additional antineoplastic, antitumor or chemotherapeutic agents. general technique The following description is primarily directed to the production of a series of consecutive amino acids or polypeptides of interest by culturing cells transformed or transfected with a vector containing the encoding nucleic acid. Of course, it is contemplated that alternative methods well known in the art may be used. For example, the amino acid sequence or a portion thereof may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)). In vitro protein synthesis may be performed using manual methods or automatically. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using the manufacturer's instructions. Various portions of the series of consecutive amino acids or polypeptides of interest may be chemically synthesized, either individually or in combination, to produce the full-length series of consecutive amino acids or polypeptides of interest using chemical or enzymatic methods. 1. Selection and transformation of host cells Host cells are transfected or transformed with an expression or cloning vector described herein for production and cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences. Culture conditions, e.g., medium, temperature, pH, etc., can be selected by one of ordinary skill in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0904] Methods of eukaryotic cell transfection and prokaryotic cell transformation, e.g., CaCl 2 , CaPO 4, liposome-mediated and electroporation are known to those skilled in the art. Depending on the host cell used, transformation is performed using standard methods appropriate for such cells. Calcium treatment using calcium chloride as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells as described in Shaw et al., Gene, 23:315 (1983), and WO 89 / 05859 published June 29, 1989. For mammalian cells that do not have such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) is used. General aspects of transformation of mammalian cell host systems are described in U.S. Pat. No. 4,399,216. Transformation into yeast is typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods of introducing DNA into cells may also be used, such as nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, such as polybrene, polyornithine. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0905] Here, suitable host cells for cloning or expressing DNA in vectors are prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as gram-negative or gram-positive microorganisms, such as Enterobacteriaceae, for example, E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella tryphimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, described in DD 266,710, published April 12, 1989), Pseudomonas, such as P. aeruginosa, and Streptomyces. These examples are illustrative and not limiting. Strain W3110 is one particularly preferred host or parent host, as it is a common host strain for recombinant DNA product fermentation. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to provide genetic mutations in genes encoding proteins endogenous to the host, examples of such hosts include E. coli W3110 strain 1A2 with the complete genotype tonA; E. coli W3110 strain 9E4 with the complete genotype tonA ptr3; E. coli W3110 strain 9E5 with the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan r E. coli W3110 strain 27C7 (ATCC 55,244) with the complete genotype tonA ptr3 phoA E15(argF-lac) 169 degP ompT rbs7 ilvG kanr E. coli W3110 strain 37D6, which has a non-kanamycin resistant degP deletion mutation; E. coli W3110 strain 40B4, which is a 37D6 strain with a non-kanamycin resistant degP deletion mutation; and E. coli strains with mutant periplasmic proteases as disclosed in U.S. Patent No. 4,946,783, issued August 7, 1990. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0906] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for coding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9:968-975 (1991)), such as K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), K. thermotolerans and K. marxianus; yarrowia (EP402,226); Pichia pastoris (EP183,070); Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida genus; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces genus, e.g. occidentalis (EP 394,538 published October 31, 1990); and filamentous fungi such as Neurospora, Penicillium spp., Tolypocladium (WO 91 / 00357 published January 10, 1991); and Aspergillus hosts such as A.Suitable yeasts include A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 (1984)), and A. niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Suitable methylotrophic yeasts include, but are not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species illustrative of this yeast class is given in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0907] Suitable host cells for expressing the glycosylated string of consecutive amino acids or polypeptides of interest are derived from multicellular organisms. Examples of invertebrate cells include insect cells, such as Drosophila S2 and Spodoptera Sf9, and plant cells. Examples of useful mammalian host cell lines are Chinese Hamster Ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 cell lines transformed with SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell lines (293 cells, or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL51). The selection of an appropriate host cell is deemed to be within the skill of the art. 2. Selection and Use of Replication-competent Vectors A nucleic acid (e.g., cDNA or genomic DNA) encoding a series of contiguous amino acids or a polypeptide of interest may be inserted into a replicable vector for cloning (amplification of the DNA) or expression. A variety of vectors are publicly available. The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, the DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0908] A string of consecutive amino acids or a polypeptide of interest may be produced recombinantly not only directly but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or another polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector or may be part of the coding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence, for example, selected from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be the yeast invertase leader, the alpha factor leader (including the alpha factor leaders of Saccharomyces and Kluyveromyces, the latter of which is described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published April 4, 1990), or a signal described in WO 90 / 13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences, such as signal sequences from secretory peptides of the same or related species, as well as viral secretory leaders, may be used to direct secretion of the protein.

[0909] Both expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2mu plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells.

[0910] Expression and cloning vectors typically contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics, such as ampicillin, neomycin, methotrexate or tetracycline, or other toxins, (b) complement an auxotrophic deficiency, or (c) supply a vital nutrient that is not available from complex media, such as the gene encoding D-alanine racemase of Bacilli.

[0911] Examples of suitable selectable markers for mammalian cells are those that allow the identification of cells competent to take up the encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is used is a CHO cell line deficient in DHFR activity, constructed and grown as described in Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow on tryptophan, eg, ATCC No. 44076, or PEP4-1 (Jones, Genetics, 85:12 (1977)).

[0912] Expression and cloning vectors usually contain a promoter operably linked to the coding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase, tryptophan (trp) promoter systems (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), as well as hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)). Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence operably linked to the coding DNA.

[0913] Examples of promoter sequences suitable for use with yeast hosts include those encoding 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)), or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Re.g., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trioserate isomerase, phosphoglucose isomerase, and glucokinase.

[0914] Other yeast promoters are inducible promoters with the added advantage of transcription being controlled by growth conditions, such as the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[0915] Transcription from the vector in mammalian host cells is controlled by promoters derived, for example, from the genomes of viruses such as polyoma virus, infectious epithelioma virus (UK 2,211,504 published July 5, 1989), adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), from heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoters, and from heat shock promoters, to the extent that such promoters are compatible with the host cell system.

[0916] Transcription of DNA encoding a continuous string of amino acids or a polypeptide of interest by higher eukaryotes may be enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to enhance its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, enhancers from eukaryotic cell viruses will be used. Examples include the SV40 enhancer (100-270 bp) on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced ​​into the vector at the 5' or 3' position of the coding sequence, but is preferably located at the 5' position from the promoter.

[0917] Expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are usually obtained from the 5', and occasionally the 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA that encode a contiguous series of amino acids or a polypeptide of interest.

[0918] Other methods, vectors, and host cells suitable for adapting the synthesis of a series of consecutive amino acids or polypeptides in recombinant vertebrate cell culture are described in Gething et al., Nature 293:620-625 (1981); Mantei et al., Nature, 281:4046 (1979); EP 117,060; and EP 117,058. 3. Detection of gene amplification / expression Gene amplification and / or expression may be measured directly in a sample using appropriately labeled probes based on the sequences provided herein, for example, by conventional Southern blotting, Northern blotting to quantify mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization. Alternatively, antibodies may be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies may then be labeled and an assay performed, where the duplex is bound to a surface, such that upon formation of the duplex on the surface, the presence of the antibody bound to the duplex can be detected.

[0919] Alternatively, gene expression can be measured by immunological methods that directly quantify the expression of the gene product, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be either monoclonal or polyclonal and may be raised in any mammal. Conveniently, antibodies may be raised against a series of consecutive amino acids or polypeptides of interest in native sequences, or against synthetic peptides based on the DNA sequences provided herein, or against exogenous sequences fused to DNA encoding a series of consecutive amino acids or polypeptides of interest and a specific antibody epitope. 4. Polypeptide Purification The form of the string of consecutive amino acids or polypeptide of interest can be recovered from the culture medium or from a lysate of the host cell. If membrane-bound, it can be detached from the membrane using an appropriate washing solution (e.g., Triton-X100) or by enzymatic cleavage. Cells used to express the string of consecutive amino acids or polypeptide of interest can be disrupted by a variety of physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0920] It may be desired to purify the string of consecutive amino acids or polypeptides of interest from recombinant cell proteins or polypeptides. Examples of suitable purification procedures include the following: fractionation on an ion exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on silica or cation exchange resins, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration, e.g., using Sephadex G-75; protein A sepharose columns to remove contaminants such as IgG; and metal chelation columns to bind epitope tag forms. Various methods of protein purification may be used, such methods being known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification steps selected will depend, for example, on the production method used and the properties of the particular string of consecutive amino acids or polypeptides of interest to be produced. Example of expression of a series of consecutive amino acids or polypeptide components of interest in E. coli A DNA sequence encoding the desired amino acid sequence or polypeptide of interest is first amplified using selected PCR primers. The primers should contain restriction enzyme sites corresponding to those in the selected expression vector. A variety of expression vectors may be used. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)), which contains genes for resistance to ampicillin and tetracycline. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector preferably contains sequences encoding antibody resistance genes, a trp promoter, a polyhis leader (containing the first six STII codons, a polyhis sequence, and an enterokinase cleavage site), a specific amino acid sequence / polypeptide coding region of interest, a lambda transcription termination region, and an argU gene.

[0921] The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibody-resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

[0922] The selected clones can be grown overnight in liquid medium, such as LB broth supplemented with antibody. The overnight culture can then be used to inoculate a larger scale culture. The cells are then grown to a desired optical density while the expression promoter is turned on.

[0923] After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by centrifugation can be lysed using various agents known in the art, and the lysed amino acid sequence or polypeptide of interest can then be purified using a metal chelate column under conditions that tightly bind the protein.

[0924] The primers contain restriction enzyme sites corresponding to those in the selected expression vector, as well as other useful sequences that provide efficient and reliable translation initiation, fast purification on metal chelate columns, and proteolytic removal by enterokinase. The PCR amplified, poly-His tagged sequences can be ligated into an expression vector that is used to transform an E. coli host, for example, based on strain 52 (W3110 fuhA (tonA) Ion galE rpoHts (htpRts) clpP (lacIq). Transformants can be grown at 30°C with shaking in LB containing 50 mg / ml carbenicillin until the OD600 reaches 3-5. The cultures can then be transferred to C RAP medium (3.57 g (NH 4 ) 2 SO 4, 0.71 g sodium citrate-2H 2 0, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycaseSF in 500 mL water, and 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO 4 The cells were diluted 50-100 fold into PBS (prepared by mixing PBS and PBS-PAGE) and grown at 30°C for approximately 20-30 hours with shaking. A sample was removed, expression was confirmed by SDS-PAGE analysis, and the bulk cells were centrifuged to pellet the cells. The cell pellet was frozen until purification and refolding.

[0925] E. coli paste (6-10 g pellet) from 0.5-1 L fermentations was suspended in 10 volumes (w / v) of 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfate and sodium tetrathionate were added to final concentrations of 0.1 M and 0.2 M, respectively, and the solution was stirred overnight at 4°C. This process results in a denatured protein in which all cysteine ​​residues have been blocked by sulfitrization. The solution was centrifuged at 40,000 rpm for 30 minutes in a Beckman Ultracentifuge. The supernatant was clarified by diluting with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtering through a 0.22 micron filter. The clarified extract was loaded onto a 5 mil Qiagen Ni-NTA metal chelate column equilibrated in metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole, pH 7.4 (Calbiochem, Utrol grade). Proteins were eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4° C. Protein concentrations were estimated by absorbance at 280 nm using the extinction coefficient calculated based on the amino acid sequence. Expression of a series of consecutive amino acids or polypeptides in mammalian cells This general example illustrates the preparation of a glycosylated form of a desired amino acid sequence of interest or polypeptide component by recombinant expression in mammalian cells.

[0926] The vector pRK5 (see EP 307,247, published March 15, 1989) can be used as an expression vector. Optionally, the coding DNA is ligated into pRK5 using ligation methods such as those described in Sambrook et al., supra, with selected restriction enzymes allowing the DNA to be inserted.

[0927] In one embodiment, the host cell selected may be a 293 cell. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in a medium such as DMEM supplemented with fetal bovine serum and, optionally, nutrient components and / or antibodies. Approximately 10 μg of ligated vector DNA is mixed with approximately 1 μg of DNA encoding the VA RNA gene [Thimmappaya et al., Cell 31:543 (1982)] and incubated in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl 2 To this mixture, add 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO 4 Dropwise addition of 10 ml of 20% glycerol in PBS is allowed to occur at 25° C. for 10 minutes to form a precipitate. The precipitate is suspended, and 293 cells are added and allowed to stand at 37° C. for approximately 4 hours. The medium is aspirated off, and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum-free medium, new medium is added, and the cells are incubated for approximately 5 days.

[0928] Approximately 24 hours after transfection, the medium was removed and replaced with medium (alone) or 200 μCi / ml of 35 S-cysteine ​​and 200 μCi / ml 35The medium is replaced with medium containing S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel is dried and exposed to film for a selected period of time to reveal the presence of the amino acid sequence or polypeptide component of interest. The cultures containing the transfected cells may be further incubated (in serum-free medium) and the medium is tested in a selected bioassay.

[0929] In an alternative method, the desired amino acid sequence or polypeptide component of the nucleic acid may be transiently introduced into 293 cells using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximum density in spinner flasks and 700 μg of ligated vector is added. Cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for 4 hours. Cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and reintroduced into a spinner flask containing tissue culture medium, 5 μg / ml bovine insulin, and 0.1 μg / ml bovine transferrin. After approximately 4 days, the conditioned medium is centrifuged and filtered to remove cells and debris. The sample containing the expressed amino acid sequence or polypeptide component of interest can then be concentrated and purified by any selected method, such as, for example, dialysis and / or column chromatography.

[0930] In another embodiment, the amino acid sequence or polypeptide component of interest can be expressed in CHO cells. 4 CHO cells can be transfected using known reagents such as DEAE-dextran or DEAE-Dextran. The cell cultures are incubated as described above and the medium is replaced with medium (alone) or 35The medium is replaced with a medium containing a radioactive label, such as S-methionine. After determining the presence of the amino acid sequence or polypeptide component of interest, the medium may be replaced with serum-free medium. Preferably, the medium is incubated for 6 days and then the conditioned medium is harvested. The medium containing the expressed amino acid sequence or polypeptide component of interest can be concentrated and purified by a selected method.

[0931] Epitope-tagged amino acid sequences or polypeptide components of interest may also be expressed in host CHO cells. The amino acid sequences or polypeptide components of interest may be subcloned from the pRK5 vector. The subclone insert may be subjected to PCR to fuse in frame with a selected epitope tag, e.g., a poly-his tag, into a baculovirus expression vector. The poly-his tagged amino acid sequence or polypeptide component of interest insert may then be subcloned into an SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, CHO cells may be transfected (as described above) with the SV driven vector. Labeling may be performed as described above to reveal expression. The medium containing the expressed poly-His tagged amino acid sequence or polypeptide component of interest is then concentrated and cultured by any method of choice, e.g., Ni 2+ - It can be purified by chelate affinity chromatography.

[0932] In one embodiment, the amino acid sequences or polypeptide components of interest are expressed as IgG constructs (immunoadhesins) in which the coding sequences for a soluble form of each protein (e.g., the extracellular domain) are fused to an IgG1 constant region sequence including the hinge, CH2 and CH2 domains, and / or a poly-His tagged form.

[0933] After PCR amplification, each DNA is subcloned into a CHO expression vector using standard techniques described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). The CHO expression vector is constructed to have compatible restriction sites 5' and 3' to the DNA of interest to allow convenient shuttling of the cDNA. The vector used for expression in CHO cells is that described in Lucas et al., Nucl. Acids Res. 24:9 1774-1779 (1996) and uses the SV40 early promoter / enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression allows selection of stably maintained plasmids after transfection. Expression of a series of consecutive amino acids or polypeptides in yeast The following method describes the recombinant expression in yeast of a desired amino acid sequence of interest or a polypeptide component.

[0934] First, a yeast expression vector is constructed for intracellular production or secretion of a string of consecutive amino acids from the ADH2 / GAPDH promoter. DNA encoding the desired amino acid sequence or polypeptide component of interest, a selected signal peptide, and a promoter are inserted into appropriate restriction enzyme sites in a selected plasmid to direct intracellular expression of the amino acid sequence or polypeptide component of interest. For secretion, DNA encoding the string of consecutive amino acids can be cloned into a selected plasmid along with DNA encoding the ADH2 / GAPDH promoter, yeast α-factor secretion signal / leader sequence, and linker sequences (if necessary) to express the string of consecutive amino acids.

[0935] Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmid and cultured in a selected fermentation medium. The transformed yeast supernatants can be precipitated with 10% trichloroacetic acid and separated by SDS-PAGE, after which the gel can be analyzed by staining with Coomassie blue.

[0936] The recombinant amino acid sequence or polypeptide component of interest can then be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and concentrating the medium using a selected cartridge filter. The concentrate containing the amino acid sequence or polypeptide component of interest may be further purified using a selected column chromatography resin. Expression of a series of consecutive amino acids or polypeptides in baculovirus-infected insect cells The following method describes the recombinant expression of a string of consecutive amino acids in baculovirus-infected insect cells.

[0937] The desired nucleic acid encoding a series of consecutive amino acids is fused upstream of an epitope tag contained in a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (e.g., Fc region of IgG). A variety of plasmids may be used, including those obtained from commercially available plasmids, such as pVL1393 (Novagen). Briefly, the amino acid sequence or polypeptide component of interest, or the desired portion of the amino acid sequence or polypeptide component of interest (e.g., a sequence encoding the extracellular domain of a transmembrane protein), is amplified by PCR using primers complementary to the 5' and 3' regions. The 5' primer may contain flanking (selected) restriction enzyme sites. The product is then digested with the selected restriction enzyme and subcloned into an expression vector.

[0938] Recombinant baculoviruses can be generated by co-transfecting the aforementioned plasmids and BaculoGold™ viral DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using Lipofectin (commercially available from GIBCO-BRL). After incubation at 28°C for 4-5 days, released viruses are harvested for further amplification. Viral infection and protein expression are performed as described in O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).

[0939] The expressed poly-His tagged amino acid sequence or polypeptide component of interest can then be purified by PCR using, for example, Ni 2+ -chelate affinity chromatography. Extracts are prepared from recombinant virus-infected Sf9 cells according to Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are sonicated in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 The cells are washed with 100 mM NaCl (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8), resuspended and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. 2+ - Prepare an NTA agarose column (commercially available from Qiagen) with a bed volume of 5 mL, wash with 25 mL of water, and equilibrate with 25 mL of loading buffer. Load the filtered cell extract onto the column at 0.5 mL / min. Measure the column at baseline A. 280with loading buffer until 1000 s, at which point fraction collection begins. The column is then washed with secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0) to elute non-specifically bound proteins. 280 After reaching baseline, the column is developed with a 0-500 mM imidazole gradient in the secondary wash buffer. 1 mL fractions are collected and analyzed by SDS-PAGE and silver staining or Ni-conjugated to alkaline phosphatase. 2+ The eluted His was analyzed by Western blot using -NTA (Qiagen). 10 -Fractions containing the tagged sequences are pooled and dialyzed against loading buffer.

[0940] Alternatively, purification of IgG-tagged (or Fc-tagged) amino acid sequences can be carried out by known chromatographic methods, including, for example, Protein A or Protein G column chromatography.

[0941] Fc-containing constructs of proteins can be purified from conditioned medium as follows: The conditioned medium is loaded onto a 5 ml Protein A column (Pharmacia) equilibrated in 20 mM sodium phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions in tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly purified protein is then desalted and placed into the storage buffer described above for poly-His tagged proteins. Protein homogeneity is confirmed by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.

[0942] Examples of pharmaceutical compositions Non-limiting examples of such compositions and dosages are as follows.

[0943] A composition comprising a compound comprising a stretch of consecutive amino acids, including consecutive amino acids having the sequence of etanercept (e.g., Enbrel), may comprise mannitol, sucrose, and tromethamine. In one embodiment, the composition is in the form of a lyophilizate. In one embodiment, the composition is reconstituted, for example, with Bacteriostatic Water for Injection (BWFI) USP (containing 0.9% benzyl alcohol). In one embodiment, the compound is administered to a subject to alleviate signs and symptoms, induce major clinical response, inhibit progression of structural damage, and improve physical function in subjects with moderate to severe active rheumatoid arthritis. The compound may be initiated in combination with methotrexate (MTX) or used alone. In one embodiment, the compound is administered to a subject to reduce signs and symptoms of moderate to severe active polyarticular-course juvenile rheumatoid arthritis in a subject who has had an inadequate response to one or more DMARDs. In one embodiment, the compound is administered to a subject to reduce signs and symptoms, inhibit progression of active arthritic structural damage, and improve physical function in a subject with psoriatic arthritis. In one embodiment, the compound is administered to a subject to reduce signs and symptoms in a subject with active ankylosing spondylitis. In one embodiment, the compound is administered to a subject for the treatment of chronic moderate to severe plaque psoriasis. In one embodiment, if the subject has rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis, the compound is administered at 25-75 mg per week used as one or more subcutaneous (SC) injections. In a further embodiment, the compound is administered at 50 mg per week in the form of a single SC injection. In one embodiment, if the subject has plaque psoriasis, the compound is administered at 25-75 mg twice a week or every 4 days, 3 months apart, followed by a taper to a maintenance dose of 25-75 mg per week. In a further embodiment, the compound is administered at a dose of 50 mg twice a week or every 4 days, 3 months apart, followed by a taper to a maintenance dose of 50 mg per week. In one embodiment, the dose is 2x to 100x lower than the doses described herein.In one embodiment, if the subject has active polyarticular JRA, the compound may be administered at a dose of 0.2-1.2 mg / kg per week (up to a maximum of 75 mg per week). In a further embodiment, the compound is administered at a dose of 0.8 mg / kg per week (up to a maximum of 50 mg per week). In some embodiments, the dose is 2x to 100x lower than the doses listed above.

[0944] A composition comprising a compound comprising a stretch of consecutive amino acids, including consecutive amino acids having the sequence of infliximab (e.g., Remicade), may comprise sucrose, polysorbate 80, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate. Preservatives are absent in one embodiment. In one embodiment, the composition is in the form of a lyophilizate. In one embodiment, the composition is reconstituted, for example, with water for injection (BWFI) USP. In one embodiment, the pH of the composition is 7.2 or about 7.2. In one embodiment, the compound is administered to a subject with rheumatoid arthritis at a dose of 2-4 mg / kg used as an intravenous infusion, followed by a similar dose at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. In a further embodiment, the compound is administered at a dose of 3 mg / kg used as an intravenous infusion, followed by a similar dose at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. In one embodiment, the dose is titrated up to 10 mg / kg or every 4 weeks. In one embodiment, the compound is administered in combination with methotrexate. In one embodiment, the compound is administered to a subject with Crohn's disease or fistulizing Crohn's disease at a dose of 2-7 mg / kg used as an induction regimen for 0, 2 and 6 weeks, followed by a maintenance regimen of 4-6 mg / kg every 8 weeks to treat moderate to severe active Crohn's disease or fistulizing disease. In a further embodiment, the compound is administered at a dose of 5 mg / kg used as an induction regimen for 0, 2 and 6 weeks, followed by a maintenance regimen of 5 mg / kg every 8 weeks to treat moderate to severe active Crohn's disease or fistulizing disease. In one embodiment, the dose is titrated up to 10 mg / kg. In one embodiment, the compound is administered to a subject with ankylosing spondylitis at a dose of 2-7 mg / kg used as an intravenous infusion, followed by similar doses 2 and 6 weeks after the first infusion, then every 6 weeks thereafter. In a further embodiment, the compound is administered at a dose of 5 mg / kg used as an intravenous infusion, followed by similar doses 2 and 6 weeks after the first infusion, then every 6 weeks thereafter.In one embodiment, the compound is administered to a subject with psoriatic arthritis at a dose of 2-7mg / kg used as an intravenous infusion, followed by a similar dose at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. In a further embodiment, the compound is administered at a dose of 5mg / kg used as an intravenous infusion, followed by a similar dose at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. In one embodiment, the compound is administered with methotrexate. In one embodiment, the compound is administered to a subject with ulcerative colitis at a dose of 2-7mg / kg used as an induction regimen at 0, 2 and 6 weeks, followed by a maintenance regimen of 2-7mg / kg every 8 weeks to treat moderate to severe active ulcerative colitis. In further embodiments, the compound is administered to subjects with ulcerative colitis at a dose of 5 mg / kg used as an induction regimen for weeks 0, 2, and 6, followed by a maintenance regimen of 5 mg / kg every 8 weeks. In some embodiments, the dose is 2x to 100x lower than the doses listed above to treat individual diseases.

[0945] In each of the composition embodiments described herein, when the composition is in lyophilized form, it may be reconstituted with, for example, a sterile aqueous solution, sterile water, sterile water for injection (USP), bacteriostatic sterile water for injection (USP), and equivalents thereof known to those of skill in the art.

[0946] It will be understood that in administering any of the instant compounds of the present invention, the compound may be administered in isolated form, in the form of a carrier, as part of a pharmaceutical composition, or in any suitable vehicle. Dosage When a dosage range is recited herein, for example, 1-10 mg / kg per week, it is understood that the invention disclosed herein also contemplates doses at each integer value between the upper and lower limits, and tenths thereof, etc. Thus, in the case of the given example, the invention contemplates doses of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 mg / kg, etc. up to 10 mg / kg.

[0947] In embodiments, the compounds of the invention may be administered as a single dose or may be administered as multiple doses.

[0948] In general, the daily dosage for treating a disorder or condition by the aforementioned methods will generally range from about 0.01 to about 10.0 mg / kg of body weight of the subject to be treated.

[0949] Variations may be made based on the above dosage ranges by a physician of ordinary skill in the art, taking into account known considerations, for example, the weight, age, and condition of the person being treated, the severity of the disease, and the particular route of administration chosen.

[0950] The disclosed compounds are also expected to act synergistically with associated concomitant results at effective dosages required. Pharmaceuticals It is understood that the term "pharmaceutically acceptable carrier" includes excipients, carriers or diluents. The particular carrier, diluent or excipient used will depend on the means and purpose for which the active ingredient is being applied.

[0951] For parenteral administration, a solution containing the compound of the present invention or a pharma- ceutically acceptable salt thereof in a sterile aqueous solution may be used. Such aqueous solutions should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

[0952] The compositions of the present invention may be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as solutions (e.g., injectable and infusible solutions), dispersions or suspensions. The preferred form depends on the intended mode of administration and therapeutic application. Some compositions are in the form of injectable or infusible solutions. The mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the compound is administered by intravenous infusion or injection. In another embodiment, the compound is administered by intramuscular or subcutaneous injection.

[0953] For therapeutic use, the compositions disclosed herein can be administered in a variety of ways, including in soluble form by bolus injection, continuous infusion, sustained release from an implant, oral ingestion, local injection (e.g., intracardiac, intramuscular), systemic injection, or other suitable techniques well known in the pharmaceutical art. Other methods of pharmaceutical administration include, but are not limited to, oral, subcutaneous, transdermal, intravenous, intramuscular, and parenteral administration methods. Typically, soluble compositions include purified compounds in combination with physiologically acceptable carriers, excipients, or diluents. Such carriers are non-toxic to recipients at the dosages and concentrations employed. Preparation of such compositions can involve combining the compounds with buffers, antioxidants, carbohydrates including glucose, sucrose, or dextrins, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Neutral buffered saline or saline mixed with congenital serum albumin are exemplary appropriate diluents. The product may be formulated as a lyophilizate using appropriate excipient solutions (eg, sucrose) as diluents.

[0954] Other derivatives include the compounds / compositions of the invention covalently attached to non-proteinaceous polymers. Attachment to the polymer is generally performed so as not to interfere with the desired biological activity of the compound, for example, the binding activity of the compound to a target. The non-proteinaceous polymer is usually a hydrophilic synthetic polymer, i.e., a polymer not found in nature. However, naturally occurring polymers produced recombinantly or by in vitro methods are useful, as are polymers isolated from nature. Hydrophilic polyvinyl polymers fall within the scope of the present invention, for example, polyvinyl alcohol and polyvinylpyrrolidone. Particularly useful are polyalkylene ethers, such as polyethylene glycol, polypropylene glycol, polyoxyethylene esters or methoxypolyethylene glycol; polyoxyalkylenes, such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched and unbranched polysaccharides, including sugar monomers, D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid (galacturonate, etc.). acid), D-mannuronic acid (e.g., polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose, and neuraminic acids, including homo- and heteropolysaccharides, such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrin, glycogen, or polysaccharide subunits of acidic mucopolysaccharides, such as hyaluronic acid; polymers of sugar alcohols, such as polysorbitol and polymannitol; and heparin or heparon.

[0955] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the compound of the present invention. A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of a compound may vary depending on factors such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve the desired prophylactic result. Typically, the prophylactically effective amount is less than the therapeutically effective amount, since a prophylactic amount is used in subjects before or at an early stage of disease.

[0956] All combinations of the various elements disclosed herein are within the scope of the invention.

[0957] The present invention will be better understood in connection with the following experimental details, however, those skilled in the art will readily appreciate that the specific experiments detailed are merely illustrative of the invention, as more fully described in the claims which follow. EXAMPLES

[0958] Experimental details Example 1 TNR1B-Alkyne-Azide-Fc6 TNR1B-alkyne-azide-Fc6 was prepared by reaction of alkyne-modified TNR1B (TNF receptor 1B) with azide-modified Fc6 as follows: TNR1B-azide-alkyne-Fc6 is prepared using the same principle by reaction of azide-modified TNR1B with alkyne-modified Fc6.

[0959] Alkyne-modified TNFR1B (TNR1B-Alk) was synthesized by cystyl-propargylamide, HSCH 2 CH[NH 2 ]CONHCH 2 C≡CH 3(Figure 1) and azide-modified TNR1B (TNR1B-Az) was prepared by cleavage of the TNR1B-intein (TNR1B-Mth fusion protein) with cystyl-3-azidopropylamide, HSCH 2 CH[NH 2 ]CONH(CH 2 ) 3 NH 2 It was prepared by cleavage of the TNR1B-intein by

[0960] TNR1B-intein and Fc6 are described in U.S. Patent Application No. 11 / 982,085, published October 16, 2008, which is incorporated herein by reference in its entirety.

[0961] The TNR1B-intein fusion protein was generated using the vector pCDNA3-TNR1B-Mth, the sequence of which is shown in SEQ ID NO: 100. The initially expressed pre-TNR1B-intein chimeric polypeptide, containing the TNR1B extracellular domain joined at its C-terminus by a peptide bond to the N-terminus of the Mth RIR1 self-splicing intein at the autocleavage site, is shown in SEQ ID NO: 101. Cleavage of the homologous TNR signal sequence by a cellular signal peptidase forms the mature TNR1B-intein fusion protein, which is secreted into the cell culture medium, the sequence of which is shown in SEQ ID NO: 102.

[0962] The Fc6 protein was expressed using the vector pCDNA3-SHH-IgG1-Fc11, the sequence of which is shown in SEQ ID NO: 103. The initially expressed pre-Fc6 polypeptide is shown in SEQ ID NO: 104. Cleavage of the heterologous Sonic Hedgehog (SHH) signal sequence by a cellular signal peptidase forms the mature Fc6 protein, the sequence of which is secreted into the cell culture medium, is shown in SEQ ID NO: 105.

[0963] Protein production was carried out by transient expression in serum-free suspension culture-adapted CHO-DG44 cells. Transient transfections were performed using polyethyleneimine as a transfection agent complexed with DNA under high-density conditions as described by Rajendra et al., J. Biotechnol. 153, 22-26 (2011). Seed train cultures were maintained in TubeSpin® bioreactors 50 tubes obtained from TPP (Trasadingen, CH) and scaled up in volume to generate sufficient biomass for transfection. Transfections were performed in 0.5-1.0 L cultures. Cultures at this scale were maintained in 2 L or 5 L Schott bottles with ventilated caps. The bottles were humidified and kept under CO 2 The medium was stirred at 180 rpm in a Kuehner incubator shaker with a controlled 5% CO. Cell cultures were harvested after 10 days, centrifuged and sterile filtered prior to purification.

[0964] Cystyl-propargylamide and cystyl-3-azidopropylamide were prepared as follows: Boc-Cys(Trt)-OH, (C 6 H 5 ) 3 CSCH 2 CH[NHCO 2 C(CH 3 ) 3 ]CO 2 H; Propargylamine, HC≡CCH 2 NH; 3-Azidopropylamine, NH 2 CH 2 CH 2 CH 2 N 3EDC, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; and HOBt, 1-hydroxybenzotriazole were obtained from AnaSpec (Freemont, CA) or CPC Scientific (San Jose, CA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). For the synthesis of cystyl-propargylamide, CHCl solutions of Boc-Cys(Trt)-OH (100 mM) and propargylamine (100 mM) were made at 100 mM each in EDC, HOBt, and triethylamine. For the synthesis of cystyl-3-azidopropylamide, 3-azidopropylamine (100 mM) was substituted for propargylamine. Both reactions were worked up by the following procedure. After stirring overnight at room temperature, the reaction was quenched with excess saturated NaHCO3 in water, extracted with CH2Cl2, dried over MgSO4, filtered, evaporated and purified by column chromatography. To remove the Boc / Trt protecting groups, the intermediate product was purified by column chromatography using TFA / triisopropylsilane / H 2 The reaction mixture was dissolved in 50:50 HO (90:5:5) at a concentration of 0.05 M and stirred at room temperature for 30 min. The reaction mixture was then evaporated to dryness and stored in CHCl. 2 Cl 2 The organic layer was then extracted with water to yield the final cystyl-propargylamide product as a yellowish oil and the final cystyl-3-azidopropylamide product as a yellowish solid.

[0965] To prepare alkyne-modified TNR1B (Figure 1) or azide-modified TNR1B, TNR1B-intein protein in cell culture medium was applied to a column packed with chitin beads obtained from New England BioLabs (Beverley, MA) pre-equilibrated with buffer A (20 mM Tris-HCl, 500 mM NaCl, pH 7.5). Unbound protein was washed from the column with buffer A. Cleavage was initiated by rapid equilibration of the chitin resin in buffer B (20 mM Tris-HCl, 500 mM NaCl, pH 8.0) containing either 50 mM cystyl-propargylamide (for alkyne-modified TNR1B) or 50 mM cystyl-3-azidopropylamide (for azide-modified TNR1B), and incubation was carried out at room temperature for 24 to 96 h. The cleaved alkyne-modified TNR1B (SEQ ID NO: 106) or azide-modified TNR1B protein (SEQ ID NO: 107) was eluted from the column with Buffer A, concentrated using an Amicon Ultracel-3 Centrifugal Filter Unit from Millipore (Billerica, MA), dialyzed against Dulbecco's phosphate-buffered saline without Ca or Mg salts (PBS) obtained from UCSF Cell Culture Facility (San Francisco, CA), and stored at 4°C prior to use.

[0966] Figure 2 shows SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of alkyne-modified TNR1B compared to cysteine-modified TNR1B (SEQ ID NO: 108) prepared with 50 mM cysteine ​​instead of cystyl-propargylamide. SDS-PAGE was performed using NuPAGE® Novex Bis-Tris Midi Gels (10%) from Invitrogen (Carlsbad, CA). Proteins were visualized using Silver Stain Plus or Bio-Safe Coomassie Stain from Bio-Rad (Hercules, CA). Alkyne-modified TNR1B (lane 3) and cysteine-modified TNR1B (lane 1) had the same Mr approx. 43,000. Furthermore, the alkyne-modified TNR1B had comparable biological activity to the cysteine-modified TNR1B as measured using a human sTNFRII / TNFRSF1B Quantikine ELISA obtained from R&D Systems (Minneapolis, MN). Preparations of cysteine-modified TNR1B (lane 2), alkyne-modified TNR1B (lane 4), or thioester-modified TNR1B (SEQ ID NO: 109) (lane 5) made in the presence of 50 mM MESNA had similar M, but less than 5% of the biological activity was observed for the preparations made in the absence of MESNA. Therefore, the alkyne-modified TNR1B prepared in the absence of MESNA was used in further studies.

[0967] Azide-modified Fc6 (Az-Fc6) was prepared by reaction of Fc6 protein with various azide-containing peptide thioesters (Figure 3) and azide-containing PEG thioesters (Figure 4). Alkyne-modified Fc6 (Alk-Fc6) was prepared by reaction of alkyne-containing thioesters with Fc6 protein.

[0968] Recombinant Fc6 protein was expressed in Chinese hamster ovary (CHO) cells as described for TNR1B-intein (see above) and purified by protein A affinity chromatography. Culture supernatant was applied to a column packed with rProtein A Fast Flow from Pharmacia (Uppsala, Sweden) pre-equilibrated with PBS. The column was washed extensively with PBS, and then Fc6 protein was eluted with 0.1 M glycine buffer at pH 2.7. Fractions were collected, pooled, dialyzed against PBS at pH 9.0 (to a final pH of 7.5) in tubes, and stored at 4° C. before use.

[0969] Table 1 shows representative azide- and alkyne-containing peptide / PEG thioesters. The thioesters were synthesized by the Fmoc / t-butyl solid-phase strategy on 2-chlorotrityl chloride resin preloaded with Fmoc-Thr(tBu)-OH. Amino acid derivatives were obtained from CPC Scientific (Sunnyvale, CA) and Fmoc-PEG n -OH derivatives were obtained from Quanta BioDesign (Powell, OH); 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), dichloromethane (DCM), trichloroacetic acid (TFA), N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N'-diisopropylethylamine (DIEA), and triisopropylsilane (TIS) were obtained from Sigma (St. Louis, MO). Standard HBTU activation was used for peptide elongation. PEG-containing peptides were prepared using Fmoc-PEG. nThe insertion of a -OH was required. As a final step in peptide elongation, the terminal α-Fmoc (9-fluorenylmethoxycarbonyl) protecting group was converted to Boc (tert-butoxycarbonyl). The peptide resin was washed with DCM and cleaved with 1% TFA / DCM to generate the fully protected peptide with a free carboxylic acid on the C-terminus. Peptide thioesters were formed by treating the crude protected peptide with DIC / HOBt / DIEA and benzyl mercaptan or thiophenol in DCM overnight. After concentration, the crude protected peptide thioesters were precipitated by complex trituration with cold ether followed by centrifugation. The peptides were then incubated at room temperature for 2 h with TFA / TIS / H2O for 2 h. 2 Deprotection was carried out by treatment of the crude protected product with O 95:2.5:2.5. After precipitation with ice-cold ether, the deprotected peptide thioester was purified by H 2 Purification by preparative RP-HPLC in O-acetonitrile (0.1% TFA) system afforded the final products with 91-95% purity and desired MS.

[0970] Azide-modified Fc6 and alkyne-modified Fc6 were prepared by native chemical coupling as follows: 2-(N-morpholino)ethanesulfonic acid (MES) was obtained from Acros (Morris Plains, NJ), tris(2-carboxyethyl)phosphine (TCEP) was obtained from Pierce (Rockford, IL), and 4-mercaptophenylacetic acid (MPAA) was obtained from Sigma-Aldrich (St. Louis, MO). Reactions were carried out by coupling various thioesters shown in Table 1 with Fc6 protein as follows: Reactions (100 uL) contained 50 mM MES buffer pH 6.5, 0.8 mM TCEP, 10 mM MPAA, peptide thioester 4 mg / ml, and Fc6 protein 0.5 mg / ml. After overnight incubation at room temperature, the reaction was adjusted to pH 7.0 with 0.05 vol / vol 1.0 M Tris-HCl, pH 9.0, purified using Protein A magnetic beads from New England BioLabs, dialyzed against 0.1 M phosphate, pH 8.0, and concentrated.

[0971] FIG. 5 shows the SDS-PAGE analysis in which Fc6 protein (lane 1) was quantitatively reacted with azido-DKTHT-thioester to give Az-DKTHT-Fc6 protein (lane 2) and azido-PEG 4 -DKTHT-thioester quantitatively reacted with Az-PEG 4 The sequence of the Az-DKTHT-Fc6 protein is shown in SEQ ID NO: 110, and the Az-PEG-DKTHT-Fc6 protein (lane 3) is obtained. 4 The sequence of -DKTHT-Fc6 is shown in SEQ ID NO: 111. PEG 4 Oligomers show a progressive increase in size to the same extent as the five amino acid DKTHT sequence, indicating that a single oxyethylene monomer unit contributes to contouring a length similar to that of a single amino acid residue, consistent with a similar fully extended conformation of approximately 3.5-4 Å (Flory (1969) Statistical Mechanics of Chain Molecules (Interscience Publishers, New York)).

[0972] TNR1B-alkyne-azide-Fc6 was synthesized by HPLC using Az-DKTHT-Fc6 protein (Figure 6) and Az-PEG 4 The α-DKTHT-Fc6 protein (FIG. 7) was prepared by reaction of alkyne-modified TNR1B with the α-DKTHT-Fc6 protein (FIG. 7). Sodium hydrogen phosphate (anhydrous) and sodium dihydrogen phosphate (monohydrate) were obtained from Acros, TCEP was from Pierce, and CuSO 4 (pentahydrate) was from Sigma-Aldrich and tris[1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) was from AnaSpec (Freemont, CA). Reactions (60 uL) were prepared in 0.1 M sodium phosphate, pH 8.0, 1.0 mM CuSO 4 , 2.0 mM TBTA, alkyne-modified TNR1B (30 ug), and unmodified Fc6 protein, Az-DKTHT-Fc6 protein, or Az-PEG 4-DKTHT-Fc6 protein (10ug). The reaction was initiated by adding 2.0mM TCEP and incubated overnight at room temperature. The reaction product was purified using Protein A magnetic beads to remove any unreacted alkyne-modified TNR1B.

[0973] Figure 8 shows SDS-PAGE analysis of the TNR1B-alkyne-azide-Fc6 product under reducing conditions. 4 In the absence of TBTA and TCEP, Az-DKTHT-Fc6 (lane 2) and Az-PEG 4 Both -DKTHT-Fc6 (lane 5) yielded a single band of M approximately 28-30,000 daltons (arrow d) corresponding to the input azide-modified Fc6 protein, with no signs of any product formation. Furthermore, there was no evidence of carryover of the input alkyne-modified TNR1B (shown in lane 1) after Protein A purification. However, CuSO 4 In the presence of TBTA and TCEP, the reaction between alkyne-modified TNR1B and Az-DKTHT-Fc6 (lane 3) and the reaction between alkyne-modified TNR1B and Az-PEG 4 Reactions between Az-DKTHT-Fc6 (lane 6) and Az-PEG-DKTHT-Fc6 (lane 7) produced two novel products of Mr ∼75,000 daltons (arrow a) and ∼65,000 daltons (arrow b). After buffer-exchange in 0.1 M phosphate at pH 8.0 to remove salt, reactions performed with the alkyne-modified TNR1B preparation produced a significant increase in yield of the Mr ∼75,000 dalton product versus the Mr ∼65,000 dalton product, whereas reactions between Az-DKTHT-Fc6 (lane 4) and Az-PEG-DKTHT-Fc6 (lane 5) produced two novel products of Mr ∼75,000 daltons (arrow a) and ∼65,000 daltons (arrow b). 4 -DKTHT-Fc6 (lane 6) gave essentially similar reaction products.

[0974] FIG. 9 shows the TNR1B-alkyne-azide-Fc6 reaction product (left panel) and TNR1B-alkyne-azide-PEG 41 shows an SDS-PAGE analysis comparing the TNR1B-alkyne-azide-Fc6 reaction product (right panel) with a TNR1B-Fc fusion protein (etanercept) of Mr ∼75,000 Daltons (lane 2), the predicted sequence of which is shown in SEQ ID NO:112, and TNR1B-alkyne-azide-PEG of Mr ∼75,000 Daltons (lane 4), the predicted sequence of which is shown in SEQ ID NO:113. 4 The -Fc6 product is essentially indistinguishable in size from etanercept (lanes 1, 3) and its sequence is shown in SEQ ID NO:114.

[0975] Figure 10 shows SDS-PAGE analysis providing further evidence confirming the requirement of alkyne and azide groups for reactivity. Reaction mixtures that contained alkyne-modified TNR1B with unmodified Fc6 protein yielded no reaction product (lane 2) compared to Fc6 alone (lane 1), and alkyne-modified TNR1B with Az-DKTHT-Fc6 yielded the expected product (lane 4) compared to Az-DKTHT-Fc6 alone (lane 3). Again, no carryover of the input alkyne-modified TNR1B (shown in lane 5) was evident after Protein A purification.

[0976] The TNR1B-alkyne-azide-Fc6 product in Figure 10 was further characterized by sequencing of its tryptic peptides by LC-MS. After SDS-PAGE, the gel was Coomassie stained and four gel regions were excised corresponding to the product of Mr ≈75,000 (arrow a), the product of Mr ≈65,000 (arrow b), the unstained region where the alkyne-modified TNR1B had migrated (arrow c), and the unreacted Az-DKTHT-Fc6 protein of Mr ≈28,000 (arrow d). The four gel slices were cut into small pieces (approximately 0.5-1.0 mm 3) and treated as follows: Ammonium bicarbonate, acetonitrile, dithiothreitol, and iodoacetamide were obtained from Sigma-Aldrich, formic acid was obtained from Pierce, and porcine trypsin (sequencing grade) was obtained from Promega (Madison, WI). To remove Coomassie stain, each gel slice was diluted with 25 mM NH 4 HCO 3 The gel was extracted with 200 μL, centrifuged to remove the supernatant, and dehydrated by adding acetonitrile for several minutes until the gel pieces became small and white. The acetonitrile was discarded, and the gel slices were dried in a Speed ​​Vac (Savant Instruments, Farmingdale, NY). The gel was then washed with 25 mM NH 4 HCO 3 Reduction and alkylation were carried out by rehydrating the gel slices in 40 uL of 10 mM dithiothreitol in 50 mM NaCl, vortexing, and incubating for 45 min at 56° C. The supernatant was then discarded and resuspended in 25 mM NH 4 HCO 3 40 uL of 55 mM iodoacetamide in 100 mM NaCl was added, the gel slices were vortexed and incubated at room temperature for 30 min in the dark. The supernatant was discarded and the gel slices were again dehydrated in acetonitrile and dried in a Speed ​​Vac. They were then incubated on ice for 60 min in 25 mM NH 4 HCO 3 Trypsin digestion was performed by rehydrating the gel slices with 25 uL of trypsin (12.5 ug / mL) in 25 mM NH 4 HCO 3 Cover with 37 °C water overnight. o The gels were incubated at 37°C for 10 min at 4°C. The supernatant was removed and the gels were then extracted twice with 30 uL of 50% acetonitrile / 0.1% formic acid in water. The organic extracts were combined with the aqueous supernatant and reduced to a volume of 10 uL in a Speed ​​Vac and then analyzed by LC-MS using a Q-Star Elite mass spectrometer (AB SCIEX, Foster City, CA).

[0977] Figure 11 summarizes the structural characterization of the TNR1B-alkyne-azide-Fc6 reaction product by mass spectrometry. As expected for the full-length TNR1B-alkyne-azide-Fc6 product, the product of M ≈75,000 contained peptides from both the alkyne-modified TNR1B and the azide-modified Fc6 parent protein. Furthermore, the peptide coverage of the alkyne-modified TNR1B sequence (upper panel) extended from the N-terminal region (EYYDQTAQMCCSK) to the C-terminal region (SMAPGAVHLPQPVST). Similarly, the peptide coverage of the azide-modified Fc6 protein sequence (lower panel) extended from the N-terminal region (DTLMISR) to the C-terminal region (TTPPPVLDSDGSFFLYSK). In contrast, the Mr 65,000 lacking the EYYDQTAQMCCSK peptide was suggested to be an N-terminally deleted version of the predicted full-length TNR1B-alkyne-azide-Fc6 product. Where the alkyne-modified TNR1B would normally migrate (arrow c), no sequence derived from the TNR1B protein was detected in the unstained region of Mr 43,000, and only sequence derived from the Fc6 protein was detected in the unreacted Az-DKTHT-Fc6 protein of Mr 28,000 (arrow d).

[0978] TNR1B-alkyne-azide-Fc6 and TNR1B-alkyne-azide-PEG in FIG. 4 The -Fc6 products were further characterized for their biological activity by measuring their ability to bind TNF-α using surface plasmon resonance (SPR). Recombinant human TNF-α protein (carrier-free) was obtained from R&D Systems and reconstituted in PBS. SPR studies were performed using a Biacore T100 instrument from Biacore AB (Uppsala, Sweden). The surface-bound ligands, TNR1B-alkyne-azide-Fc6 and TNR1B-alkyne-azide-PEG. 4-Fc6 was immobilized on a CM5 sensor chip, Series S, using the Amine Coupling Kit (BR-1000-50) from GE Healthcare (Piscataway, NJ) according to the manufacturer's instructions. TNF-α binding was performed at 25° C. in 10 mM Hepes buffer, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween-20. Binding was evaluated in duplicate at TNF-α concentrations of 0.156 nM, 0.312 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5.0 nM, 10.0 nM, 20.0 nM, and 40 nM. Data was evaluated using Biacore T100 Evaluation Software, version 2.0.3.

[0979] FIG. 12 shows TNR1B-alkyne-azide-Fc6 (left panel) and TNR1B-alkyne-azide-PEG 4 The kinetic binding curves of α-Fc6 (right panel) and α-Fc6 (left panel) are shown. Both products exhibit saturable TNF-α binding and are fit to a biexponential model (χ 2 Excellent fits were obtained using a kinetic binding data set (K 0.05). Table 2 summarizes the kinetic binding data. Approximately 40% of the binding sites for each product were found to be highly affinity, with TNR1B-alkyne-azide-Fc6 (K D =2.99×10 -10 M) TNR1B-alkyne-azide-PEG 4 -Fc6(K D =1.86×10 -10 The remaining 60% of the binding sites have a lower affinity and a dissociation constant of TNR1B-alkyne-azide-PEG 4 -Fc6(K D =5.12×10 -9 M) and TNR1B-alkyne-azide-Fc6 (K D =5.17×10 -9 M) increases high affinity binding but is equivalent to low affinity binding. 4 When the linker is assembled, TNR1B-alkyne-azide-PEG is more efficient than TNR1B-alkyne-azide-Fc6. 4These results provide convincing evidence for a high degree of cooperative (bimanual) binding of TNF-α by -Fc6.

[0980] [Table 2]

[0981] [Table 3]

[0982] Example 2 Fab'-alkyne-azide-Fc6 Fab'-alkyne-azide-Fc6 was prepared by reaction of cycloalkyne-modified Fab' with azide-modified Fc6 as follows.

[0983] Adalimumab (Humira) was obtained as a liquid formulation (50 mg / ml) from Abbott (Abbott Park, IL). Fab' fragments were prepared using IdesS protease to first generate Fab'2 fragments, followed by selective reduction of the interchain disulfides with 2-mercaptoethylamine (Figure 13). Antibody (10 mg) was exchanged into cleavage buffer (50 mM sodium phosphate, 150 mM NaCl, pH 6.6) using a Slide-A-Lyzer Mini Dialysis Unit, 10K MWCO from Pierce (Rockford, IL) and then incubated with his-tagged recombinant IdeS immobilized on agarose beads (FragIT MidiSpin columns) from Genovis (Lund, Sweden) for 1 hour at room temperature with constant mixing. The beads were removed from the digestion solution by centrifugation and washed twice with cleavage buffer, then the digestion and washing solutions were combined and applied to a HiTrap Protein A HP column from GE Life Sciences (Piscataway, NJ) to remove Fc' fragments and undigested antibodies. The flow-through fraction containing the Fab'2 fragments was reduced to Fab' fragments by adding a 1 mL aliquot to a vial containing 6 mg of 2-mercaptoethylamine (MEA) from Pierce. Reduction was performed with 10 mM EDTA to minimize reoxidation of interchain disulfides. After incubation at 37°C for 110 minutes, excess MEA was removed by buffer-exchange into PBS containing 10 mM EDTA using a PD-10 desalting column from GE Life Sciences (Piscataway, NJ). The eluate containing the Fab' fragments was concentrated using an Amicon Ultracel-3 Centrifugal Filter Unit from Millipore (Billerica, MA).

[0984] FIG. 14 shows SDS-PAGE analysis of adalimumab after cleavage with IdeS (panel A) followed by protein A chromatography and mild reduction with MEA (panel B). In the presence of a strong reducing agent (dithiothreitol) in a polyacrylamide gel, the whole antibody (lane 1) migrated as a heavy chain (arrow a) of Mr ≈55,000 and a light chain (arrow d) of Mr ≈25,000. IdeS cleaved the heavy chain (lane 2) into a C-terminal fragment (arrow b) of Mr ≈29,000 and an N-terminal fragment (arrow c) of Mr ≈26,000. The light chain and the N-terminal heavy chain fragment contain the Fab'2 domain and the C-terminal heavy chain fragment contains the Fc' domain. The protein A column efficiently removed the Fc' domain from the Fab' domain (compare lane 2 with lanes 5 and 6). Under non-reducing conditions, the Fab'2 domain migrated as a single species with Mr ≈110,000 (lane 3), whereas the Fab' domain generated by mild reduction with MEA migrated as a single species with Mr ≈55,000 (lane 4). Under reducing conditions, both the Fab'2 domain (lane 5) and the Fab' domain (lane 6) generated the same light chain (arrow d) and N-terminal heavy chain fragment (arrow c) as expected. Thus, the Fab' domain obtained by this procedure was essentially devoid of Fab'2 and Fc' domains.

[0985] Cycloalkyne-modified Fab' was linked to a bifunctional linker, DIBAC-PEG 12 -Lys(Mal), a bifunctional linker containing a maleimide group that can react with free thiol groups on the Fab' fragment (Figure 15). 12 -Lys(Mal) was prepared using an Fmoc solid-phase synthesis strategy. Lys(Mtt)-Wang resin and succinimide 3-maleimidopropanoate (Mpa-OSu) were obtained from CPC Scientific (Sunnyvale, CA), and Fmoc-N-amide-dPEG was synthesized using the Fmoc solid-phase synthesis strategy. 12The acid was obtained from Quanta BioDesign (Powell, OH) and 5-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-5-oxopentanoic acid, an acid-functionalized aza-dibenzocyclooctyne (DIBAC acid), was synthesized as described by Debets, MF et al., Chem. Commun. 46, 97-99 (2010). Fmoc-N-amide-dPEG 12 Acid and DIBAC acid were reacted sequentially with Lys(Mtt)-Wang resin to give DIBAC-PEG 12 The deprotected resin was reacted with Mpa-OSu at the free amino group on the lysine side chain to give DIBAC-PEG12-Lys(Mpa)-Wang resin. After cleavage with TFA / water (95:5), the crude product was purified by preparative RP-HPLC to give DIBAC-PEG12-Lys(Mpa)-Wang resin with a purity of 93% and the desired MS spectrum. 12 -Lys(Mal) product (DPKM) was produced.

[0986] FIG. 16 shows the DIBAC-PEG Fab′ fragment of adalimumab. 12 For purification, reactions (0.535 mL) were carried out in 0.1 M sodium phosphate at pH 7.0 and pH 7.4, each containing 5 mg of Fab' fragment and DIBAC-PEG. 12-Lys(Mal) 10 mg. After 30 h incubation at room temperature, the two reactions were combined and buffer-exchanged into 20 mM sodium acetate, 20 mM NaCl (pH 5.5) using a PD-10 column. The eluate (3.5 mL) was applied to a GE Life Sciences HiTrap SP HP cation-exchange column, which retained all unmodified Fab' and residual Fab'2. The flow-through fractions (5.5 mL) containing the purified cycloalkyne-modified Fab' (Figure 16b) were pooled, adjusted to pH 7.0 with 10x PBS (0.55 mL) and concentrated by affinity chromatography on a GE Life Sciences Protein L column (Capto L). The cycloalkyne modified Fab' was eluted from the Protein L column with 0.1 M glycine HCl, pH 2.7 (2.4 mL), neutralized with 1 / 20 volume of 1.0 M Tris HCl, pH 9.0, buffer-exchanged into PBS using a PD-10 column (3.5 mL) and concentrated to a final volume of 70 uL at a concentration of 9.5 mg / mL using an Amicon Ultracel-3 Centrifugal Filter Unit.

[0987] Various azide-modified Fc6 proteins with different lengths of PEG linkers were used in the preparation of adalimumab Fab'-cycloalkyne-azide-Fc6. Az-DKTHT-Fc6 (Figure 3) and Az-DKTHT-PEG x -Fc6 derivatives (x=12, 24, and 36) (Figure 4) were incubated in 50 mM MES, pH 6.5, 0.8 mM TCEP, 10 mM MPAA, four Az-DKTHT-PEG xReactions (2 mL) were prepared containing 5 mg / mL of each azide-thioester and 2.36 mg / mL of Fc6 protein. After 20 hours at room temperature, the reactions were neutralized with 100 uL of Tris-HCl, pH 9.0, clarified by centrifuging at 12,000xg, and applied to a 1 mL HiTrap Protein A HP column. The column was washed with 12 volumes of PBS, and then the azide-modified Fc6 protein was eluted with 2.0 mL of 0.1 M glycine-HCl, pH 2.7, neutralized with 1 / 20 volume of 1.0 M Tris-HCl, pH 9.0, dialyzed against three changes of PBS for 12 hours each using a Slide-A-Lyzer Mini Dialysis Unit, 10K MWCO, and concentrated using Amicon Ultracel-3 Centrifugal Filter Units.

[0988] FIG. 17 shows the results of SDS-PAGE of Fc6 (lane 1), Az-DKTHT-Fc6 (lane 2), and Az-DKTHT-PEG 12 -Fc6 (lane 3), Az-DKTHT-PEG 24 -Fc6 (lane 4), and Az-DKTHT-PEG 36 -Fc6 (lane 5) protein analyzed by SDS-PAGE under reducing conditions. Fc6 protein reacted quantitatively (>90%) with all four thioesters, increasing the size of the product ladder.

[0989] FIG. 18 shows the analysis by size exclusion chromatography (SEC) to confirm that the four azide-modified Fc6 protein products were of the same dimeric structure as the parent Fc6 molecule. SEC was performed using a Prominence HPLC system (Shimadzu Corp, Kyoto, Japan). A TSKgel Super SW3000 column (4.6 mm×30 cm column, 4.6 mm×5 cm guard column) was obtained from TOSOH Bioscience (Tokyo, Japan). The mobile phase, flow rate, column temperature, and detection wavelength used were 50 mM sodium phosphate, 300 mM NaCl (pH 7.4), 0.35 mL / min, 30° C., and 280 nm, respectively. The four azide-modified Fc6 protein products showed decreasing retention times with increasing size of the PEG linker, confirming their dimeric structure. All four products also showed essentially a single peak, indicating a two-handed structure in which both N-termini of the parent Fc6 dimer were modified with PEG linkers, confirmed by SDS-PAGE analysis under non-reducing conditions (see below).

[0990] The cyclooctyne-modified Fab' was reacted with all four azide-modified Fc6 molecules (Figure 19) to produce Fab'-PEGs of increasing arm length. y -Cycloalkyne-azide-PEG x A family of Fab'-PEG-Fc6 structures was generated (Figure 20). The resulting arm lengths were 12 -Fc6 (where x=0, y=12), Fab'-PEG 24 -Fc6 (where x=12, y=12), Fab'-PEG 36 -Fc6 (where x=24, y=12), and Fab'-PEG 48 -Fc6 (where x=36, y=12). Reactions (8 uL) were carried out with each of the four azide-modified Fc6 proteins (2.5 mg / ml) in the presence or absence of cycloalkyne-modified Fab' (5 mg / ml) overnight at room temperature in 0.1 M sodium phosphate, pH 7.0.

[0991] FIG. 21 shows SDS-PAGE analysis of Fab'-cycloalkyne-azide-Fc6 reactions under reducing and non-reducing conditions. In the absence of cycloalkyne-modified Fab' (lanes 5, 7, 9, and 11), all four of the azide-modified Fc6 proteins gave rise to a single band in both reducing and non-reducing gels, recognizing the two-handed structure of these dimers. In the presence of cycloalkyne-modified Fab' (lanes 4, 6, 8, and 10), all four of the azide-modified Fc6 proteins were highly consumed in the resulting reactions. Under reducing conditions, all four reactions yielded products of approximately M r 57,000-62,000 (arrow a). Fab'-PEG 12 The size of the -Fc6 product (lane 4) is approximately 1-2 kD, larger than the wild-type adalimumab heavy chain (lane 1), and the Fab'-PEG 24 -Fc6 (lane 6), Fab'-PEG 36 -Fc6 (lane 8), and Fab'-PEG 48 The size of the -Fc6 (lane 10) product further increased with the total length of the PEG linker. Under non-reducing conditions, two products were observed, the first with an M ≈155,000-160,000 (arrow a) and the second with an M ≈110,000-115,000 (arrow b). 12 The -Fc6 product (lane 4) is approximately 5 kD larger than the adalimumab whole antibody (lane 1), consistent with the expected two-handed product, and the larger Fab'-PEG 24 -Fc6 (lane 6), Fab'-PEG 36 -Fc6 (lane 8), and Fab'-PEG 48 The -Fc6 (lane 10) product further increased in size proportionately to the increase in the total length of the PEG linker.

[0992] FIG. 22 shows the Fab'-PEG 12 -Fc6, Fab'-PEG 24 -Fc6, Fab'-PEG 36 -Fc6, and Fab'-PEG 48Figure 1 shows the analysis by SEC to confirm the two-handed structure of the larger reaction products (i.e., two Fab' hands attached to one Fc6 domain) with M ≈155,000-160,000. All four reaction products showed shorter retention times than the adalimumab whole antibody, which further decreased as the size of the PEG linker increased, confirming the two-handed structure observed by SDS-PAGE analysis.

[0993] The biological activity of the Fab'-cycloalkyne-azide-Fc6 product was assessed by its ability to neutralize TNF-α-mediated cytotoxicity against mouse WEHI cells treated with actinomycin D. Mouse WEHI-13VAR cell line (ATCC CRL-2148) was obtained from American Type Culture Collection (Rockville, MD) and grown in Gibco RPMI medium 1640 (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin (10 U / ml) from Life Technologies (Grand Island, NY). TNF-α cytotoxicity assays were performed as follows: WEHI-13VAR cells were cultured at 2 × 10 per well. 4 Cells were seeded overnight in 96-well Nunc white cell culture plates obtained from Thermo Fisher (Waltham, MA) and then treated with actinomycin D (0.5 μg / ml) and TNF-α (0.2 ng / ml) obtained from Sigma (St. Louis, MO) in the absence or presence of TNFR-IgG or other inhibitors. Incubated at 37°C / CO 2After 24 hours of incubation at 5%, cell viability was determined with CellTiter-Glo Luminescent Cell Viability Assay Systems (Promega, Madison, WI), which measures the amount of ATP present in metabolically active cells and luminescence measured using a POLARstar luminometer (BMG LABTECH Inc., Cary, NC). Each inhibitor was diluted in ten three-fold serial dilutions starting at 10 μg / ml and assayed in duplicate or triplicate. Cytotoxicity data were calculated using the following equation: (luciferase reading of one sample / luciferase reading from cells treated with actinomycin D alone)×100% and presented as mean±standard deviation. Enbrel was used as a cytotoxicity positive control and Fc6 as a negative control.

[0994] FIG. 23 shows the relative abundance of Fab'-PEG compared to cycloalkyne-modified Fab' (based on an equivalent amount of input cycloalkyne-modified Fab'). 12 -Fc6, Fab'-PEG 24 -Fc6, Fab'-PEG 36 -Fc6, and Fab'-PEG 48 1 shows neutralization of TNF-α mediated cytotoxicity by Fab'-PEG-Fc6 reaction mixture. 12 -Fc6 and Fab'-PEG 24 Both -Fc6 reaction mixtures showed similar TNF-α neutralizing activity compared to that of the input cycloalkyne-modified Fab' (upper panel), and Fab'-PEG 36 -Fc6 and Fab'-PEG 48 The Fab'-PEG-Fc6 reaction mixtures showed 1.5- and 2.0-fold increases in TNF-α neutralizing activity, respectively, compared to the input cycloalkyne-modified Fab' (lower panel). Because the amounts of both products only represented 10-20% of the total cycloalkyne-modified Fab' in each reaction, as estimated by SDS-PAGE (Figure 22), the Fab'-PEG-Fc6 reaction mixtures showed 1.5- and 2.0-fold increases in TNF-α neutralizing activity, respectively, compared to the input cycloalkyne-modified Fab' (lower panel). 36 -Fc6 and Fab'-PEG 48 Both products of the -Fc6 reaction are estimated to be at least 7.5- and 10-fold larger, respectively, than the input cycloalkyne-modified Fab'.

[0995] Example 3 Fab-alkyne-azide-Fc6 Fab-alkyne-azide-Fc6 is prepared by reacting an alkyne- or cycloalkyne-modified Fab protein, which is generated by cleaving a Fab-intein fusion protein as follows, with an azide-modified Fc6. Similarly, Fab-azide-alkyne-Fc6 is prepared by reacting an alkyne- or cycloalkyne-modified Fab protein, which is generated by cleaving a Fab-intein fusion protein, with an azide-modified Fab protein.

[0996] Adalimumab Fab-intein fusion proteins are generated by co-transfecting the expression vector pFUSE2ss-DE27-Vκ-CLIg-hk (SEQ ID NO: 115) with pPUSEss-DE27-Vγ1-CHIg-hG1-Mth-1 (SEQ ID NO: 116) or pFUSEss-DE27-Vγ1-CHIg-hG1-Mth-2 (SEQ ID NO: 117).

[0997] The vector pFUSE2ss-DE27-Vκ-CLIg-hk directs expression of the pre-κ light chain of adalimumab, set forth in SEQ ID NO: 118. Cleavage of the heterologous IL-2 signal sequence by a cellular signal peptidase forms the mature κ light chain of adalimumab, set forth in SEQ ID NO: 119.

[0998] The vector pFUSEss-DE27-Vγ1-CHIg-hG1-Mth-1 directs the expression of a first type of pre-heavy chain-intein chimeric polypeptide as shown in SEQ ID NO: 120, in which the adalimumab heavy chain VH and CH1 domains are joined C- to N-terminally to the RIR1 self-splicing intein at the autocleavage site. The heterologous IL-2 signal sequence is cleaved by a cellular signal peptidase to form the mature heavy chain-intein fusion protein as shown in SEQ ID NO: 121. Together, the proteins of SEQ ID NO: 119 and SEQ ID NO: 121 comprise the adalimumab Fab-1-intein fusion protein, which is secreted into the cell culture medium.

[0999] The vector pFUSEss-DE27-Vγ1-CHIg-hG1-Mth-2 directs the expression of a second type of pre-heavy chain-intein chimeric polypeptide as shown in SEQ ID NO: 122, in which the adalimumab heavy chain VH and CH1 domains are joined C- to N-terminally to the RIR1 self-splicing intein at the autocleavage site. The heterologous IL-2 signal sequence is cleaved by a cellular signal peptidase to form the mature heavy chain-intein fusion protein as shown in SEQ ID NO: 123. Together, the proteins of SEQ ID NO: 119 and SEQ ID NO: 123 comprise the adalimumab Fab-2-intein fusion protein, which is secreted into the cell culture medium.

[1000] Protein production is carried out by transient expression in CHO-DG44 cells essentially as described in Example 1 by co-transfecting SEQ ID NO:115 with SEQ ID NO:116 to generate the adalimumab Fab-1-intein fusion protein, and SEQ ID NO:115 with SEQ ID NO:117 to generate the adalimumab Fab-2-intein fusion protein.

[1001] The alkyne-modified adalimumab Fab protein is produced by cleaving the adalimumab Fab-intein fusion protein with 50 mM cystyl-propargylamide essentially as described in Example 1. The adalimumab Fab-1-intein fusion protein is cleaved with cystyl-propargylamide to produce the alkyne-modified adalimumab Fab-1 protein, a heterodimeric protein of SEQ ID NO: 119 and SEQ ID NO: 124. The adalimumab Fab-2-intein fusion protein is cleaved with cystyl-propargylamide to produce the alkyne-modified adalimumab Fab-2 protein, a heterodimeric protein of SEQ ID NO: 119 and SEQ ID NO: 125.

[1002] The adalimumab Fab-intein fusion protein is cleaved with 50 mM cystyl-3-azidopropylamide to produce an azide-modified adalimumab Fab protein, essentially as described in Example 1. The adalimumab Fab-1-intein fusion protein is cleaved with cystyl-3-azidopropylamide to produce an azide-modified adalimumab Fab-1 protein, a heterodimeric protein of SEQ ID NO: 119 and SEQ ID NO: 126. The adalimumab Fab-2-intein fusion protein is cleaved with cystyl-3-azidopropylamide to produce an azide-modified adalimumab Fab-2 protein, a heterodimeric protein of SEQ ID NO: 119 and SEQ ID NO: 127.

[1003] The alkyne-modified adalimumab Fab-1 protein or the alkyne-modified adalimumab Fab-2 protein was co-transferred to the Az-DKTHT-Fc6 protein (Figure 6) or Az-PEG x Adalimumab Fab-1-alkyne-azide-Fc6 and adalimumab Fab-2-alkyne-azide-Fc6 are prepared by reacting with -DKTHT-Fc6 protein (Figure 7).

[1004] Tris(3-hydroxypropyltriazolylmethyl)amine (THTPA) is prepared as described in Hong et al., Angew. Chem. Int. Ed. 48, 1-7 (2009). Reactions with linker-Fc were carried out in 0.1 M sodium phosphate, pH 7.0, at a concentration of 5 mgs / mL or greater and a molar ratio of Fab-A:linker-Fc >2:1. CuSO was added to a final concentration of 0.0001 M. 4, 0.0005M THTPA is added to the reaction. The reaction is initiated by adding to final concentrations of 0.005M aminoguanidine and 0.005M sodium ascorbate. After 12-18 hours of incubation at room temperature in a closed tube, the reaction mixture is applied to a chromatographic column packed with Protein A (GE Lifesciences, NJ) to remove excess reagents and unreacted Fab-A, washed with PBS, and eluted with 0.1M glycine-HCl, pH 2.7, immediately neutralized by adding 1.0M Tris-HCl, pH 9.0. The eluted adalimumab Fab-1-alkyne-azide-Fc6 and adalimumab Fab-2-alkyne-azide-Fc6 products are dialyzed against PBS.

[1005] Adalimumab Fab-1-azide-alkyne-Fc6 and adalimumab Fab-2-azide-alkyne-Fc6 are prepared by reacting azide-modified adalimumab Fab-1 protein or azide-modified adalimumab Fab-2 protein with cycloalkyne-modified Fc6 protein.

[1006] DIBAC-PEG prepared similarly 12 -thioesters (Table 1) and other DIBAC-PEG x -thioester and DIBAC-PEG x Cycloalkyne-modified Fc6 protein is prepared essentially as described in Example 1 using the -DKTHT-thioester.

[1007] Example 4 N-terminal azide-modified Fc protein A series of azide-modified Fc proteins (N-Azide ... 3Azidoacetyl-Px-Fc) was prepared by reacting Fc6 protein with five thioesters with the sequence azidoacetyl-Px-DKTHT-thiophenol (x=0, 12, 24, 36, 48). The reaction was carried out in the absence of TCEP to minimize any reduction of the azido group to a primary amine. Azidoacetyl-Px-DKTHT-thiophenol with x=12, 24, 36 is shown in Table 1. Azidoacetyl-DKTHT-thiophenol thioesters were prepared as described in Example 1 (C 32 H 45 O 10 N 11 S[M+H] + Calculated for 776.8, found 776.3). Azidoacetyl-PEG48-DKTHT-thiophenol was prepared by solid phase by sequential condensation of Fmoc-PEG12-OH and Fmoc-PEG36-OH obtained from Quanta BioDesign (C 134 H 247 N 13 O 60 S[M+H] + The calculated value for is 3032.5, and the measured value is 3032.8). The structural formula is as follows:

[1008] Each reaction (2 mL) contained 50 mM MES pH 6.5, 10 mM mercaptophenylacetic acid, 2.2 mg Fc6, and one of the following five thioesters: azidoacetyl-DKTHT-thiophenol (5 mg), azidoacetyl-PEG12-DKTHT-thiophenol (5 mg), azidoacetyl-PEG24-DKTHT-thiophenol (10 mg), azidoacetyl-PEG36-DKTHT-thiophenol (10 mg), or azidoacetyl-PEG48-DKTHT-thiophenol (20 mg). Reactions were carried out at room temperature for 20 hours, neutralized with 0.1 mL Tris-HCl pH 9.0, centrifuged at 12,000xg, and applied to a HiTrap Protein A HP column. The column was washed with 12 volumes of PBS and then diluted with N 3The -Px-Fc protein was eluted with 0.1 M glycine HCl, pH 2.7 and neutralized with 1 / 20 volume of 1.0 M Tris HCl, pH 9.0. Peak fractions by A280 were pooled, desalted on a PD-10 column, and concentrated using Amicon Ultracel-3 Filter Centrifugal Units.

[1009] FIG. 24 shows the results of SDS-PAGE of purified N 3 -Px-Fc proteins are shown: Fc6 control (lane a), N 3 -P0-Fc (lane b), N 3 -P12-Fc (lane c), N 3 -P24-Fc (lane d), N 3 -P36-Fc (lane e), and N 3 -P48-Fc (lane f). 3 The size of the -Px-Fc protein increased with the length of the PEG linker. 3 The size of the -Px-Fc protein (Figure 24) was 3 The size of the -Px-Fc protein (Figure 17) and SDS-PAGE were indistinguishable.

[1010] Example 5 GLP1-triazole-Fc hybrid immunoglobulin A series of GLP1-triazole-Fc hybrid immunoglobulins (GLP1-P4-DN-Px-Fc) were prepared by the addition of GLP-1 (glucagon-like peptide 1) analogs further modified to have a cyclooctyne functional group to the five N-terminal groups of Example 4. 3 The GLP-1 analogs were prepared by reacting each of the GLP-1 analogs with the GLP-1 analogs HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG-PEG-Px-Fc protein. 3 -C-NH 2(HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG, SEQ ID NO: 202) corresponds to residues 7-37 of the original GLP-1 peptide, where glycine is replaced by alanine at position 8 and glutamic acid is replaced by glycine at position 22. In addition, the GLP-1 analog has a PEG3 linker and a cysteine ​​residue at the C-terminus that is used to attach to the cyclooctyne functionality. This GLP-1 analog, gly8-glu22-GLP-1(7-37)-PEG3-cys-NH 2 was prepared by SPPS (C 165 H 253 N 43 O 53 S[M+H] + Calculated value: 3720.3, measured value: 3721.3).

[1011] The cyclooctyne functionality was linked to gly8-glu22-GLP-1(7-37)-PEG3-cys-NH using DBCO-PEG4-maleimide, a heterobifunctional linker containing a maleimide group that can react with the free thiol group on the C-terminal cysteine ​​residue. 2 (Figure 25). DBCO-PEG4-maleimide (C 50 H 54 N 4 O 9 The linker was obtained from Click Chemistry Tools (Scottsdale, AZ) at a concentration of 25 mg / mL, obtained from Sigma-Aldrich (St. Louis, MO). The reaction (0.4 mL) contained 50 mM MES, pH 6.5, 5 mM EDTA, 0.45 mg gly8-glu22-GLP-1(7-37)-PEG3-cys-NH 2 peptide, and 0.9 mg / mL of DBCO-PEG4-maleimide linker. The reaction was carried out at room temperature for 30 min. Excess unreacted linker was removed using a 5 mL HiTrap desalting column obtained from GE Life Sciences. Figure 25 shows the structure of the resulting cyclooctyne-modified GLP-1 analog reaction product (GLP1-P7-DBCO).

[1012] GLP1-P7-DBCO was 3 The GLP1-P7-triazole-Px-Fc peptide was reacted individually with each of the three N-Px-Fc proteins (Figure 26) to generate a series of GLP1-P7-triazole-Px-Fc hybrid immunoglobulins (Figure 27). Each reaction (1.5 mL) contained 0.1 M sodium phosphate at pH 0.7, 0.375 mg of the GLP1-P7-DBCO peptide, and five N-Px-Fc peptides. 3 The reaction was carried out at room temperature for 3.5 hours and the reaction was purified by HiTrap Protein A HP chromatography, desalted and concentrated as described in Example 4.

[1013] Figure 28 shows purified GLP1-triazole-Fc hybrid immunoglobulins by SDS-PAGE under reducing (left) and non-reducing (right) conditions: Fc6 control (lane a), GLP1-P4-DN-P0-Fc (lane b), GLP1-P4-DN-P12-Fc (lane c), GLP1-P4-DN-P24-Fc (lane d), GLP1-P4-DN-P36-Fc (lane e), and GLP1-P4-DN-P48-Fc (lane f). The size of the GLP1-triazole-Fc hybrid immunoglobulins is N 3 -Px-Fc protein, increased with PEG linker length.

[1014] FIG. 29 shows the results of SDS-PAGE under reducing conditions of GLP1-triazole-Fc hybrid immunoglobulin and N 3 -Px-Fc protein is compared directly: Fc6 control (lane a), N 3 -P0-Fc (lane b), GLP1-P4-DN-P0-Fc (lane c), N 3 -P12-Fc (lane d), GLP1-P4-DN-P12-Fc (lane e), N 3 -P24-Fc (lane f), GLP1-P4-DN-P24-Fc (lane g), N 3 -P36-Fc (lane h), GLP1-P4-DN-P36-Fc (lane i), N 3-P48-Fc (lane j), and GLP1-P4-DN-P48-Fc (lane k). 3 The conversion rate of the -Px-Fc protein to the corresponding GLP1-P4-DN-Px-Fc hybrid immunoglobulin was approximately 95%.

[1015] Example 6 Biological activity of GLP1-triazole-Fc hybrid immunoglobulin The biological activity of GLP1-P7-triazole-Px-Fc hybrid immunoglobulin was evaluated in a cell-based assay measuring the induction of cAMP synthesis in cells expressing the human GLP-1 receptor (GLP-1R). To isolate GLP-1R-expressing cells, Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen (Grand Island, NY), fetal bovine serum (FBS), penicillin, streptomycin, and geneticin sulfate (G418) were obtained from Corning (Manassas, VA), CalPhos transfection kit was obtained from Clontech (Mountain View, CA), human GLP-1 receptor expression plasmid was obtained from GeneCopoeia (Rockville, MD), and anti-human GLP-1R-phycoerythrin monoclonal antibody was obtained from R&D Systems (Minneapolis, MN). For cAMP assays, 3-isobutyl-1-methylxanthine (IBMX) was obtained from Sigma-Aldrich (No. I5879), the cAMP Dynamic 2 kit was obtained from Cisbio Bioassays (Bedford, MA), and the GLP-1 (7-37) peptide was obtained from AnaSpec (No. 20761).

[1016] GLP-1R expressing cells were generated by transfecting human 293T embryonic kidney cells with the GLP-1R expression vector (EX-A0510-M02) using the CalPho mammalian transfection kit. Transfected cells were grown in DMEM supplemented with 10% FBS and penicillin and streptomycin (10 IU / ml), and selection of stable transfectants was performed in the same medium containing 2 mg / ml G418. GLP-1R expression was evaluated by flow cytometric analysis using anti-human GLP-1R-phycoerythrin monoclonal antibody.

[1017] For cAMP assays, 293T-GLP-1R cells were plated overnight in 384-well tissue culture treated white microtiter plates (Corning No. 3704) at a density of 5,000, 8,000 or 10,000 cells / well in 20 μL medium / well. The next day, serial dilutions of agonists (GLP-1 peptide or GLP1-triazole-Fc hybrid immunoglobulin) in 20 μL PBS containing 0.5 mM IBMX were added to the cells, and the cells were then incubated at 37° C. for 1, 4 or 24 hours. After stimulation, cAMP levels were determined by homogeneous time-resolved fluorescence (HTRF) in a ClarioStar microplate reader (BMG Labtech) using the Cisbio cAMP Dynamic 2 kit according to the manufacturer's instructions. After addition of HTRF detection reagents, anti-cAMP Mab was labeled with cryptate (20 μL) and cAMP was labeled with dye d2 (20 μL), the plate was incubated for 1 h at room temperature, and the fluorescence ratio (665 nm / 620 nm) was calculated and used to determine the cAMP concentration in the cell lysates by a four-parameter fit to a cAMP standard curve.

[1018] Figure 30 shows the results for GLP-1(7-37) peptide and GLP1-P7-DN-Px-Fc protein (x=0, 12, 24, 36, 48). All five GLP1-triazole-Fc hybrid immunoglobulins induced cAMP levels similar to GLP-1(7-37) peptide. Stimulation by GLP-1(7-37) was similar whether the cells were exposed to the agonist for 1, 4 or 24 hours, with an EC50 of about 2 nM at 24 hours, whereas stimulation by GLP1-triazole-Fc hybrid immunoglobulin increased dramatically when the cells were exposed to the agonist for a longer period, with an EC50 of about 0.4 nM at 24 hours.

[1019] Example 7 N-Terminal Cyclooctyne-Fc Protein A series of cyclooctyne-modified Fc proteins (DIBAC-P11-DN-Px-Fc), each of which has a cyclooctyne functionality at the N-terminus, were prepared by ligating the homobifunctional cyclooctyne linker to the azide-modified N 3 The linker, DIBAC-PEG11-DIBAC, shown in FIG. 31, was obtained from CPC Scientific (C 74 H 102 N 6 O 17 [M+H] + The calculated value for 1346.6 and the measured value for 1346.4) of the PEG11 portion of this lin...

Claims

1. A compound having the structure 【Chemistry 1】 (In the formula, A is a biologically active structure of the compound; Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of the one or more polypeptides comprising consecutive amino acids, the consecutive amino acids being (i) an F of an antibody; ｃ (ii) is identical to a certain series of consecutive amino acids present in a domain chain; ｃ and (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is (a) an organic acid residue, or (b) EPKSCDKTHTCPPCP, ERKCCVECPPCP, ELKTPLGDTTHTCPRPRP (EPKSCDTPPPCRPRP). ３ or ESKYGPPCPSC, or a series of consecutive amino acid residues present in these sequences, The dotted line between B and Z represents an amide bond between the N-terminal cysteine ​​or selenocysteine ​​of Z and an amino acid residue or organic acid residue of B; The solid line between A and B represents the following structure: 【Chemistry 2】 [Here, X ａ is a chemical structure containing cyclooctane fused to dihydropyridazine, R ａ connects to either A or B, and R ｂ connects to the other of A or B, R ａ and R ｂ is independently a bond or [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, branching residue, C １ ~C １０ Alkyl, C ３ ~C １０ Cycloalkane, C ２ ~C １０ Alkenes, C ５ ~C １０ Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C ２ ~C ５ Acyl, C ２ ~C ５ Acylamino, C ２ ~C ５ Acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acid, aryl, heteroaryl, carbamate, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene, 【Chemistry 3】 {where X １ is CH or N, X ２ is CH ２ or carbonyl, R ５ is aryl or alkyl; R Ｃ is H, alkyl or aryl, and [PEG(y)]z is y=1-100 and z=1-10. 【Chemistry 4】 is and wherein the chain is a chemical structure comprising one or more moieties independently selected from the group consisting of: represents a bond containing

2. X ａ But the following structure 【Chemistry 5】 (Here, R ｃ is H, alkyl or aryl. or a tautomer thereof.

3. R ａ is connected to the cyclooctane, and R ｂ 3. The compound of claim 1 or 2, wherein:

4. The solid line between A and B represents the following structure: 【Chemistry 6】 (Here, R ｃ is H, alkyl or aryl. or a tautomer thereof.

5. R ａ Or R ｂ but, i) is attached to A by a carbon-nitrogen bond or a carbon-sulfur bond; ii) is attached to A by a carbon-nitrogen bond; iii) is attached to A by a carbon-nitrogen bond that is an amide bond; iv) is attached to A by a biologically labile bond; v) is attached to A by a carbon-sulfur bond; vi) is attached to A by a succinimide-sulfur bond; vii) contains branched residues, or viii) is linked to two or more A's by branching residues; The compound according to any one of claims 1 to 4.

6. A, i) contains the structure of a compound that is an approved drug for the treatment of a subject suffering from a disease; ii) includes an organic compound structure having a molecular weight of less than 1000 Daltons, a DNA aptamer, an RNA aptamer, an oligonucleotide, or a biologically active protein; iii) includes a primary or secondary amine; iv) linked to B by a primary or secondary amine; v) aripiprazole or oseltamivir; vi) contains a secondary amine; vii) is a respiratory drug, an antiasthmatic, an analgesic, an antidepressant, an antianginal, an antiarrhythmic, an antihypertensive, an antidiabetic, an antihistamine, an anti-infective, an antibiotic, an anti-inflammatory, an anti-Parkinson's drug, an antipsychotic, an antipyretic, an antiulcer drug, an attention deficit hyperactivity disorder (ADHD) drug, a central nervous system stimulant, a decongestant, or a psychostimulant; viii) alprenolol, acebutolol, amidephrine, amineptine, amosulalol, amoxapine, amphetaminil, atenolol, atomoxetine, balofloxacin, bametan, befunolol, benazepril, benfluorex, benzoctamine, betahistine, betaxolol, bevantolol, bifemelane, bisoprolol, brinzolamide, bufeniod, butethamine, camilofine, carazolol, carticaine, carvedilol, cephaeline, ciprofloxacin, clozapine, clobenzorex, chlorprenaline, ciprofloxacin clopentamine, delapril, demexiptyline, denopamine, desipramine, desloratadine, diclofenac, dimetofrine, dioxadrol, dobutamine, dopexamine, doripenem, dorzolamide, droprenylamine, duloxetine, eltoprazine, enalapril, enoxacin, epinephrine, ertapenem, esaprazole, esmolol, etoxadrol, fasudil, fendiline, fenetylline, fenfluramine, fenoldopam, fenoterol, fenproporex, flecainide, fluoxetine, formoterol , frovatriptan, gaboxadol, garenoxacin, gatifloxacin, grepafloxacin, hexoprenaline, imidapril, indalpine, indecainide, indeloxazine hydrochloride, isoxsuprine, ispronicline, labetalol, landiolol, lapatinib, levophacetoperane, lisinopril, lomefloxacin, lotrafiban, maprotiline, mecamylamine, mefloquine, mepindolol, meropenem, metapramine, metaproterenol, methoxyphenamine, dextrorotatory methylphenidate, methylphenidate, methapranolol , metoprolol, mitoxantrone, mivazerol, moexipril, moprolol, moxifloxacin, nebivolol, nifenalol, nipradilol, norfloxacin, nortriptyline, nylidrine, olanzapine, oxamniquine, oxprenolol, oxyfedrine, paroxetine, perhexiline, phenmetrazine, phenylephrine, phenylpropylmethylamine, phoredrine, picirolex, pimethylline, pindolol, pipemidic acid, pyridocaine, practolol, pradofloxacin,Pramipexole, pramiverine, prenalterol, prenylamine, prilocaine, procaterol, pronethalol, propafenone, propranolol, propylhexedrine, protokylol, protriptyline, pseudoephedrine, reboxetine, rasagiline, (r)-rasagiline, repinotan, reproterol, rimiterol, ritodrine, safinamide, salbutamol / albutero ol, salmeterol, sarizotan, sertraline, silodosin, sotalol, soterenol, sparfloxacin, spirapril, sulfinalol, synephrine, tamsulosin, tebanicline, tianeptine, tirofiban, tretoquinol, trimetazidine, troxipide, varenicline, vildagliptin, viloxazine, biquidil or xamoterol; ix) comprises a biologically active protein; x) containing secreted proteins; xi) comprises the extracellular domain of a protein; xii) is biologically active to the extent that it has target binding activity; xiii) is an independently folding protein or part thereof; xiv) is a glycosylated protein; xv) contains intrachain disulfide bonds; xvi) binding a cytokine; xvii) binding to a cytokine that is TNFα; xviii) atrial natriuretic peptide (ANP), calcitonin, corticotropin releasing hormone (CRH), endothelin, exenatide, gastric inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), analogs of GLP-1 or GLP-2, glucagon vasoactive intestinal peptide (GVIP), ghrelin, peptide YY or secretin, or portions thereof; ixx) a series of consecutive amino acids in the sequence HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG; xx) contains at least one stretch of consecutive amino acids that is identical to a stretch of consecutive amino acids present in the heavy chain of the Fab or Fab' of the antibody; xxi) contains at least one stretch of consecutive amino acids that is identical to a stretch of consecutive amino acids present in the light chain of the Fab or Fab' of the antibody; xxii) comprising at least one Fab or Fab', or a portion of at least one Fab or Fab', of an antibody; xxiii) comprising Fab-1 or Fab'1 of the antibody, or a portion thereof; xxiv) comprising Fab-2 or Fab'2 of an antibody, or a portion thereof; xxv) comprising two Fab or Fab' arms of an antibody; xxvi) comprises at least one stretch of consecutive amino acids that is identical to a stretch of consecutive amino acids present in a single chain antibody; or xxvii) comprises at least one stretch of consecutive amino acids that is identical to a stretch of consecutive amino acids present in a TNFα receptor; The compound according to any one of claims 1 to 5.

7. A compound having the structure 【Chemistry 7】 (In the formula, Z is a protein component of the compound, the protein component comprising one or more polypeptides, at least one of the one or more polypeptides comprising consecutive amino acids, the consecutive amino acids being (i) an F of an antibody; ｃ (ii) is identical to a certain series of consecutive amino acids present in a domain chain; ｃ and (iii) having a cysteine ​​or selenocysteine ​​at the N-terminus; B is (a) an organic acid residue, or (b) the following sequence: EPKSCDKTHTCPPCP, ERKCCVECPPCP, ELKTPLGDTTHTCPRP (EPKSCDTPPPCRP) ３ or ESKYGPPCPSC, or a series of consecutive amino acids in a portion thereof, The dotted line between B and Z represents an amide bond between the N-terminal cysteine ​​or selenocysteine ​​of Z and an amino acid residue or organic acid residue of B; L is selected from the group consisting of tetrazine and trans-cyclooctene; R ａ is a chemical structure comprising a chain of one or more moieties, each of which may be selected from the group consisting of [PEG(y)]z, polyalkylene glycols, polyoxyalkylated polyols, polyvinyl alcohols, polyvinyl alkyl ethers, poly(lactic acid), poly(lactic-glycolic acid), polysaccharides, branching residues, C １ ~C １０ Alkyl, C ３ ~C １０ Cycloalkane, C ２ ~C １０ Alkenes, C ５ ~C １０ Cycloalkenes, amines, sulfur, oxygen, succinimides, maleimides, glycerol, triazoles, isoxazolidines, C ２ ~C ５ Acyl, C ２ ~C ５ Acylamino, C ２ ~C ５ Acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, amino acid, aryl, heteroaryl, carbamate, chemical structures containing cyclooctane fused to dihydropyridazine, chemical structures containing cyclooctene fused to triazole, chemical structures containing cyclooctene fused to isoxazolidine, dibenzocyclooctene, dibenzoazacyclooctene, 【Chemistry 8】 [Here, X １ is CH or N, X ２ is CH ２ or carbonyl, R ５ is aryl or alkyl; R Ｃ is H, alkyl or aryl, and [PEG(y)]z is y=1-100 and z=1-10. 【Chemistry 9】 is] (independently selected from the group consisting of:

8. L is a tetrazine, said tetrazine having the following structure: 【Chemistry 10】 where Rc is H, alkyl or aryl.

8. The compound of claim 7 having the formula:

9. L is trans-cyclooctene, the trans-cyclooctene having the following structure: 【Chemistry 11】 8. The compound of claim 7 having the formula:

10. Z comprises one C, C is a first polypeptide of the compound, the first polypeptide comprises consecutive amino acids, the consecutive amino acids being (i) F of an antibody; ｃ (ii) is identical to a certain series of consecutive amino acids present in a domain chain; ｃ 10. The compound of claim 1, which binds to a receptor and (iii) has at its N-terminus a sequence selected from the group consisting of cysteine, selenocysteine, CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP and CDTPPPCPRCP.

11. C, i) consecutive amino acids, the consecutive amino acids being the F ｃ (ii) is identical to a certain series of consecutive amino acids present in a domain chain; ｃ and (iii) having a naturally occurring cysteine-containing sequence at the N-terminus selected from the group consisting of CP, CPXCP (where X=P, R or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP and CDTPPPCPRCP. ii) a polypeptide component of the compound, the polypeptide component comprising consecutive amino acids, the consecutive amino acids being the F of (i) an antibody. ｃ (ii) is identical to a certain series of consecutive amino acids present in a domain chain; ｃ (iii) having a sequence at the N-terminus that contains a non-naturally occurring cysteine ​​or selenocysteine; iii) comprises consecutive amino acids which are identical to a certain stretch of consecutive amino acids present in an Fc domain chain of an antibody selected from the group consisting of IgG, IgM, IgA, IgD and IgE; iv) comprises consecutive amino acids, which are identical to a stretch of consecutive amino acids present in the Fc6 domain chain of an antibody; v) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in a chain other than the Fc domain chain of an antibody; vi) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in the Fab or Fab' heavy chain of an antibody; or vii) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in the light chain of a Fab or Fab' of an antibody; The compound of claim 10.

12. 12. The compound of claim 10 or 11, wherein Z further comprises a second polypeptide, the second polypeptide comprising consecutive amino acids that are identical to a stretch of consecutive amino acids present in a chain of an antibody other than an Fc domain chain of the antibody.

13. The second polypeptide comprises: i) a sequence of amino acids that is identical to a sequence of consecutive amino acids present in the heavy chain of the Fab or Fab' of the antibody; or ii) comprises consecutive amino acids that are identical to a stretch of consecutive amino acids present in the light chain of the Fab or Fab' of the antibody; 13. The compound of claim 12.

14. Z, i) comprises an antibody or a portion thereof; ii) comprising at least one Fab or Fab' of an antibody, or a portion of said at least one Fab or Fab'; iii) comprising Fab-1 or Fab'1 of the antibody, or a portion thereof; iv) comprising Fab-2 or Fab'2 of the antibody, or a portion thereof; v) containing two Fab or Fab' arms of an antibody; vi) comprises at least one stretch of consecutive amino acids that is identical to a stretch of consecutive amino acids present in a single chain antibody; or vii) a second polypeptide, wherein B is linked to Z by an amide bond between the N-terminal cysteine ​​or selenocysteine ​​of said second polypeptide of Z and an amino acid or organic acid residue of B; A compound according to any one of claims 1 to 13.

15. The N-terminus of C is i) F of the modified antibody ｃ comprises a certain series of consecutive amino acids present in a domain chain, or ii) cysteine, selenocysteine, homocysteine ​​or homoselenocysteine, or a derivative of cysteine, selenocysteine, homocysteine ​​or homoselenocysteine; The compound according to any one of claims 10 to 14.

16. A homodimer or heterodimer comprising the compound of any one of claims 1 to 15.

17. Each homodimeric or heterodimeric compound is i) capable of being bound to one another by at least one disulfide bond; ii) each compound can be linked to the other by at least one disulfide bond between said C or said second polypeptide; iii) linked to the other by at least one disulfide bond; iv) each compound is linked to the other by at least one disulfide bond between said C or said second polypeptide; 17. The homodimer or heterodimer of claim 16.

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