CYS-MAB Conjugation Method

Abstract

This invention provides an efficient and robust method for producing cysteine-manipulated antibody conjugates that offer high yield and uniform product quality. [Solution] The present disclosure provides a method for capping, reducing, and oxidizing a cys-mAb to provide a homogeneous material for subsequent conjugation reactions. In one embodiment, the present disclosure provides a method for preparing an antibody conjugate or antibody fragment conjugate, comprising the steps of: a) obtaining a composition comprising an antibody or antibody fragment; b) exposing the antibody or antibody fragment to a cysteine ​​blocking agent (wherein the cysteine ​​blocking agent forms a stable mixed disulfide with at least one cysteine ​​residue of the antibody or antibody fragment); c) adding a reducing agent to the composition to form a reduced mixture, and causing a reduction reaction to occur such that the reduced mixture comprises a reductive antibody or reductive antibody fragment.

Description

[Technical Field]

[0001] This disclosure relates to a method for capping, reducing, and oxidizing cys-mAbs to provide homogeneous material for subsequent conjugation reactions. [Background technology]

[0002] Conjugated biomolecules are a diverse range of substances containing multiple precursor molecules, at least one of which originates from a biological system. These biologically derived components are, in most cases, produced using recombinant DNA technology. In the pharmaceutical industry, conjugated biomolecules are being studied as treatments for various medical conditions. In these cases, conjugates can offer numerous therapeutic benefits by combining the useful properties of two or more precursor molecules into a single entity.

[0003] One particularly successful class of pharmaceutical bioconjugates is antibody conjugates, also known as antibody-drug conjugates. These molecules typically consist of antibodies derived from mammalian cell cultures and synthetic molecules with biological or pharmacological activity. Several antibody-drug conjugates have been approved for cancer treatment, and many more are in clinical and preclinical development. To date, all approved antibody-drug conjugates are manufactured using non-specific chemistry to produce a mixture of conjugate molecules.

[0004] Site-directed antibody conjugate-based pharmaceuticals have synthetic molecules bound to specific sites within the antibody molecule, offering therapeutic benefits as well as good quality control and / or shelf life. Therefore, considerable effort has been made to develop methods for generating site-directed antibody conjugates. A common approach to site-directed conjugation is the use of cysteine-mutated antibodies (Cys-mAb or Thiomab) that introduce a novel cysteine ​​amino acid into the primary structure of the antibody. This modified cysteine ​​can then be used as a site for conjugating synthetic molecules.

[0005] Site-directed conjugation with Cys-mAb proteins requires a modified cysteine ​​side chain in the form of a reduced thiol. However, when antibodies are isolated from mammalian cell cultures, the modified cysteine ​​is generally "capped" as a mixed disulfide with cytoplasmic thiols such as glutathione. Therefore, even if reactive synthetic molecules are added directly, the modified cysteine ​​side chain is not available for the reaction, and no conjugate is formed.

[0006] A single-step selective reduction is highly desirable for cap removal, but this is currently not feasible due to the chemical similarity between the mixed disulfides and structural disulfides in the antibody. Adding a reducing agent to Cys-mAb to remove the cap may also reduce some of the other disulfides in the antibody, potentially resulting in thiols that react undesirably and form conjugates.

[0007] Therefore, there is still a need for an efficient and robust method for producing cysteine-manipulated antibody conjugates that offer high yield and uniform product quality. [Overview of the project] [Means for solving the problem]

[0008] In one embodiment, the present disclosure provides a method for preparing an antibody conjugate or antibody fragment conjugate, comprising the steps of: a) obtaining a composition comprising an antibody or antibody fragment; b) exposing the antibody or antibody fragment to a cysteine ​​blocking agent (wherein the cysteine ​​blocking agent forms a stable mixed disulfide with at least one cysteine ​​residue of the antibody or antibody fragment); c) adding a reducing agent to the composition to form a reduced mixture and causing a reduction reaction so that the reduced mixture comprises a reductive antibody or reductive antibody fragment; d) adding an oxidizing agent to the reduced mixture to form an oxidized mixture and causing an oxidation reaction so that the oxidized mixture comprises an oxidized antibody or oxidized antibody fragment; and e) adding an activating moiety to the oxidized mixture to form a conjugation mixture and causing a conjugation reaction so that an antibody conjugate or antibody fragment conjugate is formed.

[0009] In one embodiment, cation exchange chromatography is performed after step b) and before step c) to remove excess cysteine ​​blocking agent. In another embodiment, a buffer exchange step is performed after step c) and before step d) to remove the reducing agent. In one embodiment, the buffer exchange step is ultrafiltration / dialysis filtration. In another embodiment, a purification step is performed following step e) to remove the activativist portion. In one embodiment, the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or hydrophobic interaction chromatography ("HIC") followed by ultrafiltration / dialysis filtration.

[0010] In another aspect, the present disclosure provides a method of preparing an antibody conjugate or antibody fragment conjugate, comprising: a) obtaining a composition comprising a mixed disulfide comprising an antibody or an antibody fragment; b) adding a reducing agent to the composition to form a reduction mixture, and allowing a reduction reaction to proceed such that the reduction mixture comprises a reduced antibody or a reduced antibody fragment; c) adding an oxidizing agent to the reduction mixture to form an oxidation mixture, and allowing an oxidation reaction to proceed such that the oxidation mixture comprises an oxidized antibody or an oxidized antibody fragment; and d) adding an active chemical moiety to the oxidation mixture to form a conjugation mixture, and allowing a conjugation reaction to proceed such that the antibody conjugate or antibody fragment conjugate is formed.

[0011] In one aspect, after step a) and before step b), cation exchange chromatography is performed to remove excess cysteine blocking agent. In one aspect, after step b) and before step c), a buffer exchange step is performed to remove the reducing agent. In one embodiment, the buffer exchange step is ultrafiltration / diafiltration. In one aspect, subsequent to step d), a purification step is performed to remove the active chemical moiety. In one embodiment, the purification step comprises hydrophobic interaction chromatography ("HIC"), ultrafiltration / diafiltration, or ultrafiltration / diafiltration after hydrophobic interaction chromatography ("HIC").

[0012] In another aspect, the present disclosure provides a method of preparing an antibody conjugate or antibody fragment conjugate, comprising: a) obtaining a composition comprising a mixed disulfide comprising an antibody or an antibody fragment; b) adding a reducing agent to the composition to form a reduction mixture, and allowing a reduction reaction to proceed such that the reduction mixture comprises a reduced antibody or a reduced antibody fragment; c) adding an oxidizing agent to the reduction mixture to form an oxidation mixture, and allowing an oxidation reaction to proceed such that the oxidation mixture comprises an oxidized antibody or an oxidized antibody fragment; and d) adding an active chemical moiety to the oxidation mixture to form a conjugation mixture, and allowing a conjugation reaction to proceed such that the antibody conjugate or antibody fragment conjugate is formed.

[0013] In one aspect, after step a) and before step b), a buffer exchange step is performed to remove the reducing agent. In one aspect, subsequent to step c), a purification step is performed to remove the active chemical moiety. In one embodiment, the buffer exchange step is ultrafiltration / diafiltration. In one embodiment, the purification step comprises hydrophobic interaction chromatography ("HIC"), ultrafiltration / diafiltration, or ultrafiltration / diafiltration after hydrophobic interaction chromatography ("HIC").

[0014] In one embodiment, the mixed disulfide is an antibody or antibody fragment having a capped free cysteine. In one embodiment, the antibody or antibody fragment having a capped free cysteine comprises a cap selected from the group consisting of cysteine, cysteamine, cystamine, and glutathione. In one embodiment, the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3''-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS"). In one embodiment, the ratio of the reducing agent to the antibody or antibody fragment is 2 to 4:1 (mol / mol). In one embodiment, the oxidizing agent is dehydroascorbic acid ("DHAA"). In one embodiment, the ratio of the oxidizing agent to the antibody or antibody fragment is 3 to 6:1 (mol / mol). In one embodiment, the active chemical moiety is a halogen-containing peptide, and the halogen is selected from the group consisting of Br, I, and Cl. In one embodiment, the ratio of the active chemical moiety to the antibody or antibody fragment is 2 to 3:1 (mol / mol).

[0015] In one embodiment, the antibody or antibody fragment comprises a cysteine residue at a position selected from the group consisting of D70 of an antibody light chain relative to a reference sequence (SEQ ID NO: 7), E276 of an antibody heavy chain relative to a reference sequence (SEQ ID NO: 8), and T363 of an antibody heavy chain relative to a reference sequence (SEQ ID NO: 8). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [Figure 1]A typical Cys mAb (IgG1) consists of four polypeptide chains (two light chains and two heavy chains) linked by 16 natural disulfide bonds (shown in green). Further cysteine ​​introduced into the antibody by manipulation has two additional disulfides (shown in orange). While it is highly desirable that the manipulated disulfides be selectively reduced in the presence of the other 16 natural disulfides, this is not possible (in a single step). [Figure 2] The final selective reduction can be carried out in two steps: (1) The reduction step ensures that the operational disulfide is completely "uncapped," and some native disulfide bonds are also cleaved. (2) The oxidation step returns IgG1 to its native structure. To prevent "re-capping," clearance of the thiols released in the reduction step (R-SH) by UF / DF is required prior to oxidation. [Figure 3] "Capless" Cys mAbs can be readily and selectively conjugated with alkylating agents (e.g., peptide bromoacetamide derivatives). [Figure 4] Direct comparison of the reduction performance of matched pairs (CA+TCEP) and mismatched pairs (MES+TCEP). [Figure 5] Time-dependent formation of "capless" Cys mAbs. Reaction conditions: 10 g / L of cysteamine-capped Cys mAb, 3.5 equivalents of phosphine (TPPTS, TPPDS, or TCEP) in a specific buffer at pH 5.0, at room temperature. The reaction mixture was monitored by cation exchange chromatography and quantified at 280 nm. In this figure, only "capless" Cys mAbs are plotted. [Figure 6] In cysteamine-based conjugation processes, mAb-peptide conjugates with a uniform PAR2 content (e.g., ≥95%) can be obtained using nearly stoichiometric amounts of reduction, oxidation, and alkylation reagents. [Modes for carrying out the invention]

[0017] This disclosure provides a method for capping, reducing, and oxidizing cys-mAbs to provide homogeneous material for subsequent conjugation reactions. The method demonstrates a robust method for producing cysteine-manipulated antibody conjugates that provide high yield and uniform product quality.

[0018] "Free cysteine" has been found to be a suitable binding site for the conjugation of various characterizing groups. As used herein, free cysteine ​​refers to a cysteine ​​residue that is not involved in the normal disulfide bond between two cysteines in one or two polypeptides. Typically, free cysteine ​​is cysteine ​​introduced into the target polypeptide sequence by site-selective mutagenesis, although some proteins may instead contain cysteine ​​at an appropriate position. As described in the background art, added cysteine ​​can be a suitable binding site for characterizing groups to proteins. By introducing a cysteine ​​residue, free cysteine ​​is usually obtained because there is no partner in the protein to form a disulfide bond.

[0019] In one embodiment, the present disclosure provides a method for preparing an antibody conjugate or antibody fragment conjugate, comprising the steps of: a) obtaining a composition comprising an antibody or antibody fragment; b) exposing the antibody or antibody fragment to a cysteine ​​blocking agent (wherein the cysteine ​​blocking agent forms a stable mixed disulfide with at least one cysteine ​​residue of the antibody or antibody fragment); c) adding a reducing agent to the composition to form a reduced mixture and causing a reduction reaction so that the reduced mixture comprises a reductive antibody or reductive antibody fragment; d) adding an oxidizing agent to the reduced mixture to form an oxidized mixture and causing an oxidation reaction so that the oxidized mixture comprises an oxidized antibody or oxidized antibody fragment; and e) adding an activating moiety to the oxidized mixture to form a conjugation mixture and causing a conjugation reaction so that an antibody conjugate or antibody fragment conjugate is formed.

[0020] In one embodiment, cation exchange chromatography is performed after step b) and before step c) to improve the homogeneity of the antibody and remove excess cysteine ​​blocking agent. In another embodiment, a buffer exchange step is performed after step c) and before step d) to remove released caps (thiols) and reducing agents. In one embodiment, the buffer exchange step is ultrafiltration / dialysis filtration. In another embodiment, a purification step is performed following step e) to remove excess activatable moieties and improve the purity of the antibody or antibody fragment conjugate. In one embodiment, the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or ultrafiltration / dialysis filtration after hydrophobic interaction chromatography ("HIC").

[0021] In another embodiment, the present disclosure provides a method for preparing an antibody conjugate or antibody fragment conjugate, comprising the steps of: a) obtaining a composition comprising a mixed disulfide containing an antibody or antibody fragment; b) adding a reducing agent to the composition to form a reducing mixture and causing a reduction reaction so that the reducing mixture contains a reducing antibody or a reducing antibody fragment; c) adding an oxidizing agent to the reducing mixture to form an oxidizing mixture and causing an oxidation reaction so that the oxidizing mixture contains an oxidized antibody or an oxidized antibody fragment; and d) adding an activating moiety to the oxidizing mixture to form a conjugation mixture and causing a conjugation reaction so that an antibody conjugate or antibody fragment conjugate is formed.

[0022] In one embodiment, cation exchange chromatography is performed after step a) and before step b) to remove excess cysteine ​​blocking agent. In another embodiment, a buffer exchange step is performed after step b) and before step c) to remove cap and excess reducing agent. In one embodiment, the buffer exchange step is ultrafiltration / dialysis filtration. In another embodiment, a purification step is performed following step d) to remove the activativist portion. In one embodiment, the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or hydrophobic interaction chromatography ("HIC") followed by ultrafiltration / dialysis filtration.

[0023] In another embodiment, the present disclosure provides a method for preparing an antibody conjugate or antibody fragment conjugate, comprising the steps of: a) obtaining a composition comprising a mixed disulfide containing an antibody or antibody fragment; b) adding a reducing agent to the composition to form a reducing mixture and causing a reduction reaction so that the reducing mixture contains a reducing antibody or a reducing antibody fragment; c) adding an oxidizing agent to the reducing mixture to form an oxidizing mixture and causing an oxidation reaction so that the oxidizing mixture contains an oxidized antibody or an oxidized antibody fragment; and d) adding an activating moiety to the oxidizing mixture to form a conjugation mixture and causing a conjugation reaction so that an antibody conjugate or antibody fragment conjugate is formed.

[0024] In one embodiment, a buffer exchange step is performed after step a) and before step b) to remove the reducing agent. In another embodiment, a purification step is performed following step c) to remove the activativist portion. In one embodiment, the buffer exchange step is ultrafiltration / dialysis filtration. In one embodiment, the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or ultrafiltration / dialysis filtration after hydrophobic interaction chromatography ("HIC").

[0025] In one embodiment, the mixed disulfide is an antibody or antibody fragment having capped free cysteine. In one embodiment, the antibody or antibody fragment having capped free cysteine ​​includes a cap selected from the group consisting of cysteine, cysteamine, cystamine, and glutathione. In one embodiment, the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3”-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS"). In one embodiment, the reducing agent to antibody or antibody fragment ratio is 2 to 4:1 (mol / mol). In one embodiment, the oxidizing agent is dehydroascorbic acid ("DHAA"). In one embodiment, the oxidizing agent to antibody or antibody fragment ratio is 3 to 6:1 (mol / mol). In one embodiment, the activating moiety is a halogen-containing peptide, the halogen selected from the group consisting of Br, I, and Cl. In one embodiment, the ratio of activator partial antibody to antibody or antibody fragment is 2-3:1 (mol / mol).

[0026] In one embodiment, the antibody or antibody fragment contains a cysteine ​​residue at a position selected from the group consisting of D70 of the antibody light chain relative to the reference sequence (SEQ ID NO: 7), E276 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8), and T363 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8).

[0027] A conjugation site that is "applicable to conjugation" means that, under specified chemical conditions, the side chain of an amino acid residue at the selected conjugation site reacts with the desired additional functional moiety (or a linker covalently bonded to the additional functional moiety), resulting in a covalent bond (directly or via a linker) with the side chain of the additional functional moiety as the main reaction product.

[0028] A disulfide is a covalent bond between two sulfur atoms that can exist in different (or the same) molecules. In proteins, cysteine ​​residues can be linked by disulfide bonds, also known as cystines.

[0029] For free cysteine ​​to be an effective target for conjugated reactions, it must be in its reduced form. Proteins containing free cysteine ​​can be difficult to produce for the same reason and are therefore often obtained as mixed disulfides containing a small organic moiety. A mixed disulfide is a molecule containing a disulfide similar to the disulfide bond between two cysteine ​​amino acid residues contained in polypeptide sequences (which may or may not be the same). The small organic moiety is referred to herein as Cap, and therefore the mixed disulfide is a protein-SS-Cap molecule. In this application, the term “mixed disulfide” is used for a molecule containing a disulfide bond linking two distinct entities that are not both polypeptides, although the molecule may further contain “ordinary” disulfide bonds in addition to the mixed disulfide.

[0030] In one embodiment, the method of the present invention includes a step of reducing protein-SS-Cap molecules, since the protein to be conjugated is obtained in the form of a composition of protein-SS-Cap molecules.

[0031] As described above, Cap typically originates from a small organic moiety containing at least one sulfur atom that is part of the disulfide bond of the mixed disulfide. Such an organic moiety can exist as a monomer in a reduced form or as a dimer in an oxidized form. Thus, in a mixed disulfide, -S-Cap is the oxidized form of half of the monomer or dimer. In one embodiment, -S-Cap originates from cysteine / cystine, cysteamine / cystamine (which is decarboxylated cystine), or glutathione (G-SH) / glutathione disulfide (GS-SG), and thus the mixed disulfide is selected in one embodiment from protein-SS-cys, protein-SS-cyst, or protein-SSG (where cys refers to half of cystine, cyst refers to half of cystamine, and G refers to half of glutathione disulfide). In other words, in one embodiment, Cap of protein-SS-Cap originates from cysteine, cysteamine, or glutathione.

[0032] In certain embodiments, the cap is [ka] It is selected from the group consisting of the following.

[0033] As described above, the purpose of reduction is to obtain a molecule containing free reduced cysteine ​​(-SH) that is reactive in conjugated reactions.

[0034] In one embodiment, the mixed disulfide is a protein-SS-Cap molecule (where protein-S is derived from a protein containing free cysteine).

[0035] To obtain a protein containing reactive sulfur atoms, a reducing agent is added to a mixed disulfide composition, and the mixture is incubated and reduced to obtain, for example, a reduced protein in the form of protein-SH. The steps described herein are: a) obtaining a mixed disulfide composition containing protein; b) adding a reducing agent to the protein composition; and c) reducing the composition to obtain a solution containing reduced protein (P-SH).

[0036] The reducing agent can be selected from several available reducing agents, and although only a few reducing agents are mentioned herein, those skilled in the art know that a very large repertoire of reducing agents can be selected.

[0037] In one embodiment, the reducing agent is a redox buffer selected from the group consisting of glutathione, gamma-glitamylcysteine, cysteineglycine, cysteine, N-acetylcysteine, cysteamine, and lipamide. In one embodiment, a thiol disulfide redox catalyst such as an enzyme such as glutaredoxin is included. In one embodiment, the reducing agent is selected from small molecule reducing agents such as DTT. In one embodiment, the reducing agent is a phosphine, such as an aromatic phosphine, such as a triarylphosphine, such as a substituted triarylphosphine such as tris(2-carboxyethyl)phosphine ("TCEP"), triphenylphosphine-3,3',3"-trisulfonic acid trisodium sodium (TPPTS), or triphenylphosphine-3,3'-disulfonic acid disodium sodium (TPPDS).

[0038] When the mixed disulfides are reduced, a solution containing the reducing protein (P-SH) is obtained. Before subsequent conjugation, it may be beneficial to remove the reducing agent and / or released Cap molecules. In one embodiment, an optional step may be included to remove small molecules, such as molecules with a molecular weight of less than 10 kDa, from the solution containing the reducing protein (P-SH). In one embodiment, molecules with a molecular weight of less than 10 kDa are removed from the solution containing the reducing protein by diafiltration.

[0039] In a conjugation reaction, the chemical moiety is covalently bonded to the sulfur atom of the free cysteine ​​of the reduced protein (protein-SH). The chemical moiety may be a moiety suitable for conjugation to a protein, such as a property modifier. The property modifier may be a chemical moiety capable of modifying one or more characteristics of the target protein. In one embodiment, the chemical moiety is a property modifier such as a chemical moiety that can stabilize the protein, prolong its cyclic half-life, or increase its potency. In one embodiment, the chemical moiety is an extender. To effectively induce conjugation, the chemical moiety may be used in an activated form. In the method of the present invention described herein, the activated chemical moiety is added to a solution containing a reduced protein, and a conjugated protein is prepared by conjugation of the chemical moiety to the reduced protein. Therefore, the method of the present invention includes the further step of adding the activated chemical moiety to a solution containing a reduced protein, causing a conjugation reaction to occur, and obtaining the preparation of the conjugated protein.

[0040] The chemical moiety may be a portion suitable for conjugation to a protein, such as a property modification portion. The property modification portion may be a chemical moiety capable of modifying one or more characteristics of the target protein. In one embodiment, the chemical moiety is a peptide and / or ligand having or not having a linker sequence. In one embodiment, the chemical moiety is a property modification group, such as a chemical moiety that can stabilize a protein, prolong its cyclic half-life, or increase its potency. In one embodiment, the chemical moiety is an albumin binder. To effectively induce conjugation, an activated form of the chemical moiety may be used. In the method of the present invention described herein above, the activated chemical moiety is mixed with a reduced protein, and a protein conjugated via a sulfur atom is prepared by conjugation of the chemical moiety to the reduced protein.

[0041] The chemical moiety is preferably an active chemical moiety, which means a moiety capable of reacting with protein-SH to form a protein-S chemical moiety molecule. Examples of such active chemical moieties include electrophilic alkylating reagents containing a maleimide group or a haloacetyl group, which are known in this field.

[0042] In one embodiment, the active chemical moiety is a halogenated chemical moiety such as a halogenated peptide ligand. Examples of halogenated chemical moieties include Br, I, or CI.

[0043] In one embodiment, the active chemical portion is a halogenated albumin binder (AB-halo).

[0044] To effectively reduce mixed disulfides, an excess of reducing agent in molar concentration is usually applied. By adding a reducing agent to a composition of mixed disulfides, a reduced mixture is obtained. The amount of reducing agent can be expressed in terms of equivalents of the amount of mixed disulfides, and if the amount of reducing agent is 1 equivalent of the amount of mixed disulfides, the molar concentrations of the mixed disulfides and the reducing agent in the mixture are equal.

[0045] In one embodiment, the amount of reducing agent added is approximately 2 to 4 molar equivalents of the molar amount of the mixed disulfide.

[0046] To reduce the amount of excess reduction of the antibody or antibody fragment, it is advantageous to reduce the required amount, which is possible if the process steps are optimized as described herein. To carry out an effective reduction reaction using a smaller amount of reducing agent, the remaining reaction conditions must be carefully selected, as provided by the present invention.

[0047] The reduction of mixed disulfides takes several minutes to several hours, depending on the conditions. Those skilled in the art know that different conditions produce different effects, and therefore, the time and conditions required to completely or nearly completely reduce mixed disulfides are described in more detail in the following examples.

[0048] The reducing agent may be added to the mixed disulfide composition as a concentrate, or it may be added simply by adding the agent as a solid powder. The reducing agent is mixed with the mixed disulfide composition to initiate the reduction. This mixture can be called the reducing mixture.

[0049] For a sufficiently effective process, the reduction should result in a reduction of at least 80% of the total amount of mixed disulfides, for example, at least 90%. The reduction is considered satisfactory if the amount of mixed disulfides is at most 20%, for example, at most 10%, of the amount of mixed disulfides in the reducing mixture. In a preferred embodiment, about 5% of the mixed disulfides can be left unreduced in the solution containing the reducing protein, resulting in a reduction of about 95% of the mixed disulfides. In a further embodiment, the amount of mixed disulfides left for a suitable time for an efficient process is at most 2%.

[0050] Reduction may occur within at least 15 minutes, for example, at least 30 minutes, or for example, at least 1 hour. In one embodiment, the reducing mixture is left for 2 to 10 hours, for example, 3 to 6 hours, or about 3 to 4 hours, after the addition of the reducing agent.

[0051] In one embodiment, the reduction is carried out for a maximum of 24 hours, for example, a maximum of 12 hours, for example, a maximum of 8 hours, for example, a maximum of 6 hours, for example, a maximum of 4 hours.

[0052] In one embodiment, the reduction can be carried out at a temperature of 1 to 50°C, for example, at room temperature, for example, 18 to 25°C. In an alternative embodiment, the reduction can be carried out at a lower temperature, for example, below 10°C, for example, about 4 to 6°C.

[0053] Before proceeding with the conjugation process, the reduced protein can be separated from the reducing mixture from small organic molecules of excess reducing agent and / or mixed disulfides, such as HS-Cap of proteins having capped free cysteine. This optional step may involve removing low molecular weight molecules, such as molecules with a molecular weight of less than 10 kDa.

[0054] Those skilled in the art will know of various methods for removing low molecular weight compounds, such as filtration using a suitable membrane. In one embodiment, this method includes a buffer exchange step (ultrafiltration / diafiltration).

[0055] The effectiveness of the diafiltration process, for example, the amount of small molecules and excipients removed, is related to the volume of filtrate produced and the volume of retained solution. It should also be noted that the term "remove" in this context should be interpreted as "reducing the concentration" of low molecular weight molecules, and that excipients are typically present after the diafiltration process (or alternative process step) that "removes" low molecular weight molecules.

[0056] Before proceeding with the conjugation process, the reduced protein is oxidized to reduce the amount of excess reduced antibody or antibody fragment.

[0057] To effectively oxidize an excess of antibody or antibody fragment, an excess of oxidizing agent in molar concentration is typically applied. An oxidized mixture is obtained by adding an oxidizing agent to a composition of excess of antibody or antibody fragment. The amount of oxidizing agent can be expressed in equivalent amounts to the amount of excess of antibody or antibody fragment, and if the amount of oxidizing agent is 1 equivalent of the amount of mixed disulfide, then the molar concentrations of the mixed disulfide and the reducing agent in the mixture are equal.

[0058] In one embodiment, the amount of oxidizing agent added is approximately 3 to 6 molar equivalents of the molar amount of the excess reduced antibody or antibody fragment.

[0059] The oxidation of an over-reduced antibody or antibody fragment can take several minutes or several hours, depending on the conditions. Those skilled in the art know that different conditions produce different effects, and therefore, the time and conditions required to completely or nearly completely oxidize an over-reduced antibody or antibody fragment are described in more detail in the following examples.

[0060] The oxidizing agent may be added as a concentrate to the excessively reduced antibody or antibody fragment composition, or it may be added simply by adding the agent as a solid powder. The oxidizing agent is mixed with the excessively reduced antibody or antibody fragment composition to initiate oxidation. This mixture can be called an oxidized mixture.

[0061] For a sufficiently effective process, oxidation should result in the oxidation of at least 80% of the total amount of excess-reduced antibody or antibody fragment, for example, at least 90%. Oxidation is considered satisfactory when the amount of excess-reduced antibody or antibody fragment is at most 20%, for example, at most 10% of the amount of excess-reduced antibody or antibody fragment in the oxidation mixture. In a preferred embodiment, about 5% of excess-reduced reductate antibody or antibody fragment can be left in the solution containing the oxidized protein, and about 95% of the excess-reduced antibody or antibody fragment can be obtained. In a further embodiment, the amount of excess-reduced reductate antibody or antibody fragment left at a suitable time for an efficient process is at most 2%.

[0062] Oxidation can occur within at least 15 minutes, for example, at least 30 minutes, or for example, at least 1 hour. In one embodiment, the oxidized mixture is left to stand for 2 to 10 hours, for example, 3 to 6 hours, or about 3 to 4 hours, after the addition of the oxidizing agent.

[0063] In one embodiment, oxidation is carried out for a maximum of 24 hours, for example, a maximum of 12 hours, for example, a maximum of 8 hours, for example, a maximum of 6 hours, for example, a maximum of 4 hours.

[0064] In one embodiment, oxidation can be carried out at 1 to 50°C, for example, at room temperature, for example, 18 to 25°C. In an alternative embodiment, reduction can be carried out at a lower temperature, for example, below 10°C, for example, about 2 to 8°C. In one embodiment, the oxidizing agent is dehydroascorbic acid.

[0065] As described above, according to this method, conjugation is carried out by adding the active chemical moiety to a solution containing the reduced protein.

[0066] If prior reduction is incomplete, the ratio of reducing protein to mixed disulfides can hinder high-yield conjugated reactions. Furthermore, the presence of excess reducing agent and released Cap molecules can also interfere with conjugated reactions.

[0067] Furthermore, the relative ratio of reactants, such as the reduced protein to the active chemical moiety, also affects the effectiveness of the reaction.

[0068] In one embodiment, the molar concentration of the active chemical moiety may be at least equal to, or twice, the molar concentration of the protein to be conjugated. This can also be expressed in equivalents, for example, at least 10, e.g., 8, e.g., 6, e.g., 4, e.g., 2, or e.g., 1 equivalent relative to the protein to be conjugated. Since the active chemical moiety can be an expensive resource, it is advantageous to reduce the amount required, which is possible if the preceding steps are optimized, as described herein. To carry out an effective conjugation reaction using a small amount of the active chemical moiety, the remaining reaction conditions must be carefully selected, as provided by the present invention. In one embodiment, the amount of the active chemical moiety is at most 8 equivalents of the protein, e.g., at most 6 equivalents of the protein to be conjugated, e.g., at most 4 equivalents, e.g., at most 3 equivalents, e.g., at most 2.5 equivalents, e.g., at most 2 equivalents, e.g., at most 1.5 equivalents.

[0069] The binding of the oxidized protein to the chemical moiety may take several minutes or several hours, depending on the conditions. Those skilled in the art will know that different conditions produce different effects, and therefore the time required for complete or near-complete conjugation varies based on the conditions, which are described in more detail below. According to the method of the present invention, the conjugation reaction is considered satisfactory when the amount of the starting material, such as reduced protein, reaches 10% or less, for example, 5% or less, preferably 2% or less.

[0070] The active chemical moiety may be added as a concentrate to a solution containing oxidized protein, or it may be added simply by adding the drug as a solid powder. In one embodiment, the active chemical moiety is dissolved in a suitable solution before being added to the solution containing oxidized protein. Alternatively, the chemical moiety may be activated in solution before the conjugation reaction.

[0071] The term "half-life extension portion" refers to a "vehicle" covalently bound or conjugated to a pharmaceutically acceptable portion, domain, or Fc domain and / or pharmaceutically active portion, which, compared to the unconjugated pharmaceutically active portion, prevents or reduces chemical modifications that would reduce the in vivo protein degradation or other activity of the pharmaceutically active portion, improves other pharmacokinetic properties such as increased half-life or absorption rate (but not limited to these), reduces toxicity, improves solubility, enhances the biological activity and / or target selectivity of the pharmaceutically active portion against the target of interest, increases manufacturability, and / or reduces the immunogenicity of the pharmaceutically active portion (e.g., peptide or non-peptide portion). Polyethylene glycol (PEG) is an example of a useful half-life extension portion. Other examples of half-life extension portions according to the present invention include ethylene glycol copolymers, carboxymethylcellulose, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene maleic anhydride copolymers, polyamino acids (e.g., polylysine), dextran n-vinylpyrrolidone, poly-n-vinylpyrrolidone, propylene glycol homopolymers, propyrenoxide polymers, ethylene oxide polymers, polyoxyethylated polyols, polyvinyl alcohols, linear or branched glycosylated chains, polyacetals, long-chain fatty acids, long-chain hydrophobic aliphatic groups, and immunoglobulin F. cDomains (e.g., Feige et al., Modified peptides as therapeutic agents, U.S. Patent No. 6,660,843), albumin (e.g., human serum albumin; e.g., Rosen et al., Albumin fusion proteins, U.S. Patent No. 6,926,898 and U.S. Patent Application Publication No. 2005 / 0054051; Bridon et al., Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components, U.S. Patent No. 6,887,470), transthyretin (TTR; e.g., Walker et al., Use of transthyretin peptide / protein fusions to increase the serum half-life of pharmacologically active Examples include peptides / proteins (see U.S. Patent Application Publication No. 2003 / 0195154A1; see also U.S. Patent Application Publication No. 2003 / 0191056A1), or thyroxine-binding globulin (TBG).

[0072] Other embodiments of the useful half-life extension portion of the present invention include peptide ligands or small (non-peptide organic) molecule ligands that have binding affinity to long half-life serum proteins under physiological conditions of temperature, pH, and ionic strength. Examples include albumin-binding peptides or small molecule ligands, transthyretin-binding peptides or small molecule ligands, thyroxine-binding globulin-binding peptides or small molecule ligands, antibody-binding peptides or small molecule ligands, or other peptides or small molecules that have affinity to long half-life serum proteins. (See, for example, Blaney et al., Method and compositions for increasing the serum half-life of pharmacologically active agents by binding to transthyretin-selective ligands, U.S. Patent No. 5,714,142; Sato et al., Serum albumin binding moieties, U.S. Patent Application Publication 2003 / 0069395A1; Jones et al., Pharmaceutical active conjugates, U.S. Patent No. 6,342,225). A “long half-life serum protein” is one of hundreds of different proteins dissolved in mammalian plasma, including so-called “carrier proteins” (e.g., albumin, transferrin, and haptoglobin), fibrinogen and other blood coagulation factors, complement components, immunoglobulins, enzyme inhibitors, precursors of substances such as angiotensin and bradykinin, and many other types of proteins. The present invention encompasses the use of any single species of pharmaceutically acceptable half-life extension moieties (e.g., those described herein, but not limited to them), or the use of a combination of two or more different half-life extension moieties.

[0073] The recombinant polypeptide and nucleic acid methods used herein, including in the examples, are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) or Current Protocols in Molecular Biology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons 1994), both of which are incorporated herein by reference for any purpose.

[0074] The headings used herein are for structural purposes only and should not be construed as limiting the subjects described.

[0075] In this specification, unless otherwise defined, scientific and technical terms used in connection with this application shall have the meanings generally understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0076] In general, the cell and tissue cultures, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as the nomenclature and techniques used in connection with hybridization, as described herein, are well known and commonly used in the art. Unless otherwise stated, the methods and techniques of this application are generally carried out in accordance with common methods well known in the art, and such methods and techniques are described in the various general and specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990) (these are incorporated herein by reference). Enzymatic reactions and purification methods are carried out according to the manufacturer's instructions, as is generally achievable in the art, or as described herein. The terminology used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as their laboratory procedures and techniques, are well known and commonly used in the art. Standard methods may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as for patient treatment.

[0077] The present invention is not limited to the specific methodologies, protocols, reagents, etc., described herein, and should therefore be understood to be subject to change. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0078] Unless otherwise described in the examples or elsewhere, all numbers indicating quantities of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” When used in relation to a percentage, the term “approximately” may mean ±1%.

[0079] Unless otherwise stated, “a” and “an” as used herein mean, by convention, “one or more.”

[0080] As used herein, the terms “amino acid” and “residue” are used interchangeably and, when used in relation to peptides or polypeptides, refer to both naturally occurring and synthetic amino acids, as well as amino acid analogs, amino acid mimes, and non-naturally occurring amino acids that are chemically similar to naturally occurring amino acids.

[0081] "Naturally occurring amino acids" are amino acids encoded by the genetic code, as well as amino acids encoded by the genetic code that are modified after synthesis (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs are compounds having the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methylmethionine sulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but will retain the same basic chemical structure as naturally occurring amino acids.

[0082] "Amino acid mimetic compounds" are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids. Examples include methacryloyl or acryloyl derivatives of amides, β-imino acids, γ-imino acids, and δ-imino acids (such as piperidine-4-carboxylic acid).

[0083] "Non-natural amino acids" are compounds that have the same basic chemical structure as naturally occurring amino acids but are not incorporated into the elongated polypeptide chain by translational complexes. "Non-natural amino acids" also include, but are not limited to, amino acids that result from the modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, 20 common amino acids) but are not naturally incorporated into the elongated polypeptide chain by translational complexes. A list of examples of non-natural amino acids that can be inserted into polypeptide sequences or used in place of wild-type residues in polypeptide sequences includes, but are not limited to, β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. Examples include citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), and Nα-methylarginine (NM eR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolisine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-Tetrahydroisoquinoline (Tic), 2-Indanylglycine (IgI), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-amino-Phe), 4-guanidinophenylalanine (Guf), glycyllysine (abbreviated as "K(Nε-glycyl)", "K(glycyl)", or "K(gly)"), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methylvaline (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine N (Cha), 4-methylphenylalanine (MePhe), β,β-diphenylalanine (BiPhA), aminobutyric acid (Abu), 4-phenylphenylalanine (or biphenylalanine, 4Bip), α-aminoisobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidine acid, aminocaproic acid, aminoheptanoic acid, aminopimeric acid, desmosine, diaminopimeric acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, Examples include N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino-O-phthalic acid (4APA), and other similar amino acids, as well as any of the derivatized forms of those specifically listed, which can take L-form or D-form, with abbreviations in parentheses.

[0084] The term “isolated nucleic acid molecule” refers to a polymer or analogue of single or double-stranded deoxyribonucleotide or ribonucleotide bases, read from the 5' end to the 3' end, from which at least about 50 percent of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other substances naturally found with the nucleic acid when the whole nucleic acid is isolated from a cell source have been removed. Preferably, the isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other molecules that are found in the natural environment of the nucleic acid and are assumed to interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic, or research use.

[0085] The term “isolated polypeptide” refers to a polypeptide, peptide, lipid, carbohydrate, polynucleotide, or polypeptide isolated from at least about 50 percent of other substances from which the polypeptide is naturally found when isolated from source cells. The isolated polypeptide is preferably substantially free of any other contaminating polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, or research use.

[0086] The compositions of the present invention, which include a drug or peptide directly or indirectly linked, attached to, or bound to another antibody or antibody fragment via a linker portion, are "conjugated" or "conjugated" molecules.

[0087] The term "codes" refers to a polynucleotide sequence that codes for one or more amino acids. The term does not require a start or stop codon.

[0088] The terms “identical” and “identity” percentages, as used in relation to two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical. “Identity percentage” means the percentage of residues that are identical between amino acids or nucleotides in the comparison molecule, and is calculated based on the size of the smallest of the molecules being compared. In such calculations, any alignment gaps (if any) can be addressed by specific mathematical models or computer programs (i.e., “algorithms”). Methods that may be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, AM, ed.), (1988) New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., (1987) Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., (1988) SIAM J. Applied Math. 48:1073.

[0089] When calculating the identity percentage, the sequences to be compared are aligned in a way that gives the greatest possible match between them. The computer program used to determine the identity percentage is the GCG program package, which includes GAP (Devereux et al., (1984) Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides to determine the sequence identity percentage. The sequences are aligned so that their respective amino acids or nucleotides match best (the "match span" determined by the algorithm). A gap-start penalty (calculated as 3 × mean diagonal, where "mean diagonal" is the average of the diagonals of the comparison matrix used, and "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap-extension penalty (usually 1 / 10th of the gap-start penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used with the algorithm. In certain embodiments, standard comparison matrices (see Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; see Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0090] The following are recommended parameters for determining the identity percentage of polypeptide or nucleotide sequences using the GAP program: Algorithm: Needleman et al., 1970, J.Mol.Biol.48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992 (above); Gap penalty: 12 (however, no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0

[0091] Using a specific alignment scheme to align two amino acid sequences, only short regions of the two sequences may match, and these aligned short regions may have very high sequence identity even if there is no significant correlation between the two full-length sequences. Therefore, if desired, the selected alignment method (e.g., the GAP program) can be adjusted to obtain alignment across at least 50 consecutive amino acids of the target polypeptide.

[0092] As used herein, “antigen-binding protein” means any protein that specifically binds to a particular target antigen. This term encompasses intact antibodies containing at least two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and mutants. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments. Antigen-binding proteins also include domain antibodies such as nanobodies and scFv, which are further described below.

[0093] Generally, an antigen-binding protein is said to "specifically bind" to its target antigen when it exhibits essentially background binding to non-target molecules. However, antigen-binding proteins that specifically bind to a target can cross-react with target antigens originating from different species. Typically, an antigen-binding protein has a dissociation constant (KD) of ≤10, as measured by surface plasma resonance techniques (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or binding equilibrium exclusion methods (KinExA, Sapidyne, Boise, Idaho). -7 The M protein specifically binds to the target. Antigen-binding proteins are measured using the method described, and their KD is ≤5 × 10⁻⁶. -9When M is present, it binds specifically to the target with "high affinity," and the KD measured using the described method is ≤ 5 × 10 -10 When M is present, it specifically binds to the target with "very high affinity".

[0094] The "antigen-binding region" refers to a protein or a portion of a protein that specifically binds to a particular antigen. For example, the portion of an antigen-binding protein that contains amino acid residues that interact with the antigen and give the antigen its specificity and affinity is called the "antigen-binding region." Antigen-binding regions typically contain one or more "complementary binding regions" ("CDRs") of immunoglobulins, single-chain immunoglobulins, or antibodies of camelid animals. A particular antigen-binding region also contains one or more "framework" regions. The "CDR" is an amino acid sequence that contributes to the specificity and affinity of antigen binding. The "framework" regions can facilitate binding between the antigen-binding region and the antigen by helping to maintain the proper three-dimensional structure of the CDR.

[0095] A "recombinant protein" is a protein produced using a recombinant technique, that is, by the expression of recombinant nucleic acids as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

[0096] The term “antibody” refers to any isotype of intact immunoglobulin, or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Thus, an “antibody” is a type of antigen-binding protein. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains. An antibody may originate from only a single source, or it may be a “chimera,” i.e., different parts of its antibody may originate from two different antibodies, as further described below. Antigen-binding proteins, antibodies, or binding fragments may be produced in a hybridoma by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody.

[0097] The term "light chain" as used in reference to antibodies or fragments thereof includes the full-length light chain and fragments thereof that have a variable region sequence sufficient to confer binding specificity. The full-length light chain contains a variable region domain (VL) and a constant region domain (CL). The variable region domain of the light chain is located at the amino terminus of the polypeptide. The light chain includes a kappa chain and a lambda chain.

[0098] The term "heavy chain" as used in reference to antibodies or fragments thereof includes the full-length heavy chain and the fragment thereof having a variable region sequence sufficient to confer binding specificity. The full-length heavy chain comprises a variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, the CH domain is located at the carboxyl terminus, and CH3 is located closest to the carboxyl terminus of the polypeptide. The heavy chain may be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0099] As used herein, the term “immunologically functional fragment” (or simply “fragment”) of an antibody or immunoglobulin chain (heavy or light chain) is an antigen-binding protein that contains a portion of an antibody (regardless of how the portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain but has the ability to specifically bind to an antigen. Such a fragment is biologically active in that it specifically binds to a target antigen and can compete with other antigen-binding proteins, including intact antibodies, for specific binding to a given epitope.

[0100] These biologically active fragments can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab fragments, Fab' fragments, and F(ab')2 fragments.

[0101] In another embodiment, these may be Fv, a domain antibody, and scFv, which may be derived from the antibodies of the present invention.

[0102] For example, it is further intended that the functional portion of an antigen-binding protein disclosed herein, such as one or more CDRs, can be covalently bonded to a second protein or small molecule to create a therapeutic agent that targets a specific target in the body, giving it bifunctional therapeutic properties or extending its serum half-life.

[0103] A "Fab fragment" consists of one light chain and one heavy chain with a CH1 group and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0104] The "Fc" region contains two heavy chain fragments, each containing the CH2 and CH3 domains of the antibody. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0105] A "Fab' fragment" consists of one light chain and a portion of a heavy chain that includes the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains. As a result, an F(ab')2 molecule can be formed by creating an interchain disulfide bond between the two heavy chains of two Fab' fragments.

[0106] The "F(ab')2 fragment" consists of two light chains and two heavy chains that include a portion of the constant region between the CH1 and CH2 domains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0107] The "Fv region" includes variable regions derived from both heavy and light chains, but lacks a steady region.

[0108] A "single-chain antibody" or "scFv" is an Fv molecule in which the heavy chain and light chain variable regions are linked by a flexible linker to form a single polypeptide chain in which the antigen-binding region forms. scFv is discussed in detail in International Publication No. 88 / 01649, and in U.S. Patent Nos. 4,946,778 and 5,260,203, which are incorporated by reference.

[0109] A "domain antibody" or "single-chain immunoglobulin" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. Examples of domain antibodies include Nanobodies®. In some cases, two or more VH regions are covalently linked via a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0110] A "bivalent antigen-binding protein" or "bivalent antibody" contains two antigen-binding regions. In some cases, the two binding regions have the same antigen specificity. Bivalent antigen-binding proteins and bivalent antibodies can be bispecific; see below for more information.

[0111] A "multispecific antigen-binding protein" or "multispecific antibody" targets multiple antigens or epitopes.

[0112] Bispecific, dual-specific, or bifunctional antigen-binding proteins or antibodies are hybrid antigen-binding proteins or antibodies, each possessing two distinct antigen-binding sites. Bispecific antigen-binding proteins and antibodies are a type of polyspecific antigen-binding protein or polyspecific antibody, and can be generated by various methods, including, but not limited to, hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321 and Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which may be located on the same or different protein targets.

[0113] When used in relation to antigen-binding proteins (e.g., antibodies), the term “competing” means that competition between antigen-binding proteins is determined by an assay in which the antigen-binding protein (e.g., an antibody or an immunologically functional fragment thereof) blocks or inhibits, under test, the specific binding of a reference antigen-binding protein to a common antigen. Many types of competitive binding assays can be used, including, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J.Immunol. 137:3614-3619), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIA with I-125 labeling (see, e.g., Morel et al., 1988, Molec.Immunol. 25:7-15), and solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al.) This includes methods such as those described in (see Moldenhauer et al., 1990, Virology 176:546-552) and directly labeled RIAs (see Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays use purified antigen bound to a solid surface or cells containing either of these antigens, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Further details on methods for determining competitive binding are provided in the examples herein.Typically, when there is an excess of competing antigen-binding proteins, these proteins inhibit the specific binding of the reference antigen-binding protein to the common antigen by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some cases, binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% or more.

[0114] The term "antigen" refers to a molecule or part of a molecule that has the ability to be bound by a selective binder, such as an antigen-binding protein (e.g., an antibody), and that can be used in an animal to produce an antibody capable of binding to that antigen. An antigen may have one or more epitopes that have the ability to interact with different antigen-binding proteins, such as antibodies.

[0115] The term "epitope" refers to a part of a molecule that is bound by an antigen-binding protein (e.g., an antibody). The term includes any determinant that has the ability to specifically bind to an antigen-binding protein, such as an antibody. Epitopes can be continuous or discontinuous (discontinuous) (for example, in a polypeptide, amino acid residues that are not continuous in the polypeptide sequence but are linked within the molecule are bound by the antigen-binding protein). A structural epitope is one that is present in the three-dimensional structure of an active protein but not in a denatured protein. In certain embodiments, an epitope may be mimicky in that it contains a three-dimensional structure similar to the epitope used to generate the antigen-binding protein, but does not contain, or contains only some of, the amino acid residues found in that epitope used to generate the antigen-binding protein. Epitopes are most commonly found in proteins, but may occasionally be found in other types of molecules, such as nucleic acids. Epitope determinants may include chemically active surface classification molecules such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and may possess specific three-dimensional structural properties and / or specific charge properties. Generally, antigen-binding proteins specific to a particular target antigen preferentially recognize epitopes present on the target antigen in complex mixtures of proteins and / or macromolecules.

[0116] As used herein, “substantially pure” means that the described molecular species is the dominant species present, i.e., more abundant on a molar basis than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition containing at least 50% (on a molar basis) of all macromolecular species in which the species of interest is present. In other embodiments, a substantially pure composition comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all macromolecular species present in the composition. In other embodiments, the species of interest is purified to the point of substantial homogeneity, and no contaminants can be detected in the composition by conventional detection methods, so that the composition consists of a single detectable macromolecular species.

[0117] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides constituting a polynucleotide may be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. Modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and modifications of internucleotide bonds such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladates, and phosphoramidates.

[0118] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. In some embodiments, oligonucleotides have a base length of 10 to 60. In other embodiments, oligonucleotides have a base length of 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40. Oligonucleotides may be single-stranded or double-stranded for use, for example, in the construction of mutant genes. Oligonucleotides may be sense oligonucleotides or antisense oligonucleotides. Oligonucleotides may include labels, including radiolabeling, fluorescent labeling, hapten, or antigenic labeling for detection assays. Oligonucleotides may be used, for example, as PCR primers, cloning primers, or hybridization probes.

[0119] "Isolated nucleic acid molecule" means DNA or RNA that originates from a genome, mRNA, cDNA, or synthesis, or any combination thereof, in which the isolated polynucleotide does not contain all or some of the naturally occurring polynucleotides, or is ligated to polynucleotides that are not naturally ligated. For the purposes of this disclosure, "nucleic acid molecule containing" a particular nucleotide sequence should be understood not to include an intact chromosome. An isolated nucleic acid molecule "containing" a particular nucleic acid sequence may, in addition to that particular sequence, contain up to 10 or even up to 20 other sequences encoding proteins or parts thereof, or may contain operablely ligated regulatory sequences that control the expression of the coding region of the described nucleic acid sequence, and / or may contain vector sequences.

[0120] Unless otherwise specified, the left end of any single-stranded polynucleotide sequence discussed herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction in which a nascent RNA transcript is added from 5' to 3' is referred to as the transcription direction. A sequence region on a DNA strand having the same sequence as the RNA transcript, located 5' to the 5' end of the RNA transcript, is referred to as the "upstream sequence." A sequence region on a DNA strand having the same sequence as the RNA transcript, located 3' to the 3' end of the RNA transcript, is referred to as the "downstream sequence."

[0121] The term “regulatory sequence” refers to a polynucleotide sequence that can influence the expression and processing of the coding sequence to which it is ligated. The properties of such a regulatory sequence may depend on the host organism. In certain embodiments, a regulatory sequence for prokaryotes may include a promoter, a ribosome binding site, and a transcription termination sequence. For example, a regulatory sequence for eukaryotes may include a promoter containing one or more recognition sites for a transcription factor, a transcription enhancer sequence, and a transcription termination sequence. A “regulatory sequence” may also include a leader sequence and / or a fusion partner sequence.

[0122] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to introduce protein-coding information into a host cell.

[0123] The terms “expression vector” or “expression construct” refer to a vector containing nucleic acid sequences suitable for transforming host cells and which induce and / or control the expression of one or more heterogeneous coding regions operably ligated thereto (in cooperation with the host cell). An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, sequences that affect RNA splicing of coding regions operably ligated thereto.

[0124] As used herein, “operably ligated” means that the components to which this term applies are in a relationship that enables them to perform their intrinsic function under appropriate conditions. For example, in a vector, an “operably ligated” regulatory sequence to a protein-coding sequence is ligated thereto such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the regulatory sequence.

[0125] The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence to express the target gene. This term includes offspring of a parent cell, regardless of whether their morphology or genetic makeup is identical to that of the original parent cell, as long as the target gene is present.

[0126] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetic forms of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also encompass amino acid polymers modified, for example, by the addition of carbohydrate residues to form glycoproteins, or by phosphorylation. Polypeptides and proteins include molecules that can be produced by naturally occurring and non-recombinant cells, or by genetically engineered or recombinant cells, and which have the amino acid sequence of a naturally occurring protein, or molecules that have the deletion, addition, and / or substitution of one or more amino acids from a naturally occurring sequence. Specifically, the terms “polypeptide” and “protein” include antigen-binding proteins, antibodies, or sequences that have the deletion, addition, and / or substitution of one or more amino acids from an antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to a full-length protein. Such fragments may also include modified amino acids compared to a full-length protein. In certain embodiments, the fragments have an amino acid length of approximately 5 to 500. For example, the fragments may have an amino acid length of at least 5, at least 6, at least 8, at least 10, at least 14, at least 20, at least 50, at least 70, at least 100, at least 110, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450. Useful polypeptide fragments include immunologically functional fragments of antibodies that contain a binding domain.

[0127] The term “isolated protein” means that the protein in question (1) does not contain at least some other proteins that would normally be expected to be found with it, (2) substantially does not contain other proteins from the same source, such as the same species, (3) is expressed by cells of a different species, (4) has at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other substances that would naturally accompany it removed, (5) is operably bound (by covalent or non-covalent interactions) to polypeptides that would not naturally accompany it, or (6) does not occur naturally. Generally, “isolated proteins” constitute at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. It is preferable that isolated proteins substantially do not contain proteins or polypeptides or other contaminants that would be found in their natural environment and would be expected to interfere with their therapeutic, diagnostic, preventive, research, or other use.

[0128] A "variant" of a polypeptide (e.g., an antigen-binding protein such as an antibody) contains an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another polypeptide sequence. Variants include fusion proteins.

[0129] A polypeptide "derivative" is a polypeptide that has been chemically modified in some way, different from a variant resulting from insertion, deletion, or substitution, for example, by conjugation to another chemical moiety (e.g., antigen-binding proteins such as antibodies).

[0130] As used herein in relation to biological substances such as polypeptides, nucleic acids, and host cells, the term “natural origin” refers to substances found in nature.

[0131] As used herein, the terms "subject" or "patient" may refer to any mammal. In a typical embodiment, the subject or patient is a human.

[0132] "Conservative amino acid substitutions" may include substitutions of native amino acid residues (i.e., residues present at a given position in the wild-type polypeptide sequence) with non-native residues (i.e., residues not present at a given position in the wild-type polypeptide sequence), where the substitution has little to no effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions also typically include non-native amino acid residues incorporated by chemical peptide synthesis rather than by synthesis in a biological system. These include peptide mimetic compounds and other forms in which the amino acid moiety is reversed or inverted.

[0133] Naturally occurring residues can be classified into the following classes based on their common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basicity: Asn, Gln, His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0134] Further groups of amino acids can also be clearly explained by the principles described, for example, Creighton (1984) PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES (2nd Ed. 1993), WH Freeman and Company. In some cases, it may be useful to further characterize substitutions based on two or more such properties (for example, substitutions at "less polar" residues such as thr residues can be highly conserved substitutions in appropriate circumstances).

[0135] A conservative permutation may involve exchanging one member of such a class for another member of the same class. A non-conservative permutation may involve exchanging one member of such a class for a member of another class.

[0136] Synthetic amino acid residues, rare amino acid residues, or modified amino acid residues known to have similar physiological and chemical properties to those in the above classifications can be used to "conservatively" substitute specific amino acid residues in a sequence. For example, a D-Arg residue may function as a substitute for a typical L-Arg residue. In some cases, a particular substitution can be described in relation to two or more of the above classes (for example, a substitution with a small hydrophobic residue means the substitution of one amino acid with a residue found in both of the above classes, or with another synthetic, rare, or modified residue known in the art to have similar physiological and chemical properties to such a residue that satisfies both definitions).

[0137] A “vector” refers to a delivery medium that (a) promotes the expression of a nucleic acid sequence encoding a polypeptide, (b) promotes the production of a polypeptide therefrom, (c) promotes the gene transfer / transformation of a target cell using it, (d) promotes the replication of a nucleic acid sequence, (e) promotes the stability of a nucleic acid, (f) promotes the detection of nucleic acids and / or transformed / transformed cells, and / or (g) otherwise imparts a beneficial biological and / or physiological function to the nucleic acid encoding the polypeptide. A vector can be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.

[0138] Recombinant expression vectors can be designed to express proteins in prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., insect cells, yeast cells, or mammalian cells using baculovirus expression vectors). In one embodiment, the host cell is a non-human mammalian host cell. Typical host cells include those typically used for cloning and expression, such as Escherichia coli strains TOP10F', TOP10, DH10B, DH5a, HB101, W3110, BL21(DE3) and BL21(DE3)pLysS, BLUESCRIPT (Stratagene), mammalian cell lines CHO, CHO-K1, HEK293, 293-EBNApIN vector (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)), and pET vector (Novagen, Madison). This includes Wis. Alternatively, recombinant expression vectors can be transcribed and translated in vitro using, for example, a T7 promoter regulatory sequence and T7 polymerase and an in vitro translation system. The vector preferably contains a promoter upstream of the cloning site containing the nucleic acid sequence encoding the polypeptide. Examples of switchable promoters include the lac promoter, T7 promoter, trc promoter, tac promoter, and trp promoter.

[0139] In various embodiments, the vector comprises an operably linked nucleotide sequence that modulates the expression of a target polypeptide. The vector may contain or be linked to any suitable promoter, enhancer, and other expression promoters. Examples of such elements include potent expression promoters (e.g., human CMV IE promoter / enhancer, RSV promoter, SV40 promoter, SL3-3 promoter, MMTV promoter, or HIV LTR promoter, EF1 alpha promoter, CAG promoter), effective poly(A) termination sequences, origins of replication for plasmid products in E. coli, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polylinkers). The vector may also contain an inductive promoter in contrast to a constitutive promoter such as CMV IE. In one embodiment, a nucleic acid is provided comprising a sequence encoding a target polypeptide operably linked to a tissue-specific promoter that promotes the expression of the sequence in metabolism-related tissues such as liver or pancreatic tissue.

[0140] In another aspect of this disclosure, a host cell comprising the nucleic acids and vectors disclosed herein is provided. In various embodiments, the vector or nucleic acid is incorporated into the host cell genome, and in other embodiments, the vector or nucleic acid is extrachromosomal.

[0141] Recombinant cells, such as yeast cells, bacterial cells (e.g., E. coli), and mammalian cells (e.g., immortalized mammalian cells), are provided, containing such nucleic acids, vectors, or combinations thereof. In various embodiments, cells are provided, containing non-integrated nucleic acids, such as plasmids, cosmids, phagemids, or linear expression elements, which contain sequences encoding the expression of a target polypeptide.

[0142] Vectors containing nucleic acid sequences encoding target polypeptides provided herein can be introduced into host cells by transformation or gene transfer. Methods for transforming cells with expression vectors are well known.

[0143] The nucleic acid encoding the target can be positioned and / or delivered to a host cell or host animal by a viral vector. Any suitable viral vector having this capability can be used. The viral vector may contain any number of viral polynucleotides, either alone or in combination with one or more viral proteins that facilitate the delivery, replication, and / or expression of the nucleic acid of the present invention in the desired host cell. The viral vector may be a polynucleotide containing all or part of the viral genome, a viral protein / nucleic acid complex, a virus-like particle (VLP), or an intact viral particle containing nucleic acids encoding viral nucleic acids and polypeptides. The viral vector, being a viral particle, may include wild-type viral particles or modified viral particles. The viral vector may be a vector that requires the presence of another vector or wild-type virus for replication and / or expression, such as an adenovirus vector amplicon (for example, the viral vector may be a helper-dependent virus). Typically, such viral vectors consist of wild-type viral particles or viral particles whose protein and / or nucleic acid content has been modified to facilitate the transgene capacity or expression of nucleic acids (an example of such vectors is the herpesvirus / AAV amplicon). Typically, viral vectors are similar to and / or derived from viruses that normally infect humans. Suitable viral vector particles in this regard include, for example, adenovirus vector particles (including any virus of the family Adenoviridae or any virus derived from a virus of the family Adenoviridae), adeno-associated virus vector particles (AAV vector particles), or other parvovirus and parvovirus vector particles, papillomavirus vector particles, flavivirus vectors, alphavirus vectors, herpesvirus vectors, poxvirus vectors, and retrovirus vectors (including lentivirus vectors).

[0144] The target polypeptide expressed as described herein can be isolated using standard protein purification methods. The target polypeptide can be isolated from cells that naturally express it, or from cells that have been engineered to express it, such as cells that do not naturally express the target polypeptide.

[0145] Protein purification methods, as well as related materials and reagents, that can be used to isolate target polypeptides are known in the art. Further purification methods that may be useful for isolating target polypeptides can be found in references such as Bootcov MR, 1997, Proc. Natl. Acad. Sci. USA 94:11514-9, Fairlie WD, 2000, Gene 254:67-76.

[0146] The antigen-binding proteins provided are polypeptides into which one or more complementarity-determining regions (CDRs) described herein are incorporated and / or linked. In some antigen-binding proteins, the CDRs are incorporated into a “framework” region, which aligns the orientation of the CDRs, resulting in the achievement of appropriate antigen-binding properties of the CDRs. The specific antigen-binding proteins described herein are antibodies or derived from antibodies. In other antigen-binding proteins, the CDR sequence is incorporated into a different type of protein backbone.

[0147] Generally, the antigen-binding proteins provided typically comprise one or more CDRs described herein (e.g., one, two, three, four, five, or six). In some cases, the antigen-binding protein comprises (a) a polypeptide structure and (b) one or more CDRs inserted into and / or linked to the polypeptide structure. The polypeptide structure can take on a variety of different forms. For example, the polypeptide structure may be or comprise a framework of a naturally occurring antibody or a fragment or variant thereof, or it may be entirely synthetic. Examples of various polypeptide structures are further described below.

[0148] In certain embodiments, the polypeptide structure of the antigen-binding protein is an antibody or derived from an antibody. Therefore, examples of the specific antigen-binding proteins provided include, but are not limited to, monoclonal antibodies, bispecific antibodies, domain antibodies such as minibodies and Nanobodies®, synthetic antibodies (sometimes referred to herein as “antibody mimes”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, and parts or fragments thereof of each. In some cases, the antigen-binding protein is an immunological fragment of a complete antibody (e.g., Fab, Fab', F(ab')2). In other cases, the antigen-binding protein is an scFv using a CDR derived from the antibody of the present invention.

[0149] In another embodiment, an antigen-binding protein is provided which has an in vitro or in vivo (e.g., when administered to a human subject) half-life of at least 1 day. In one embodiment, the antigen-binding protein has a half-life of at least 3 days. In various other embodiments, the antigen-binding protein has a half-life of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 days or more. In another embodiment, the antigen-binding protein is derivatized or modified to have a longer half-life compared to an underivativeated or unmodified antibody. In another embodiment, the antigen-binding protein includes point mutations to increase the serum half-life. Further details regarding such variants and derivatization forms are provided below.

[0150] Some of the antigen-binding proteins offered typically have structures associated with naturally occurring antibodies. These antibody structural units typically consist of one or more tetramers, each composed of two identical caplets of polypeptide chains, although some mammalian species also produce antibodies with only a single heavy chain. In a typical antibody, each pair or caplet contains one full-length "light" chain (approximately 25 kDa in certain embodiments) and one full-length "heavy" chain (approximately 50–70 kDa in certain embodiments). Each individual immunoglobulin chain is composed of several "immunoglobulin domains," each consisting of approximately 90–110 amino acids and exhibiting a characteristic folding pattern. These domains are the basic units that make up the antibody polypeptide. The amino-terminus of each chain typically contains a variable domain responsible for antigen recognition. The carboxy-terminus is evolutionarily more conserved than the other end of the chain and is referred to as the "constant region" or "C region." Human light chains are generally classified into copper and lambda light chains, each containing one variable domain and one constant domain. Heavy chains are typically classified into muon, delta, gamma, alpha, or epsilon chains, each defining the antibody isotypes IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, but is not limited to IgG1, IgG2, IgG3, and IgG4. The IgM subtype includes IgM and IgM2. The IgA subtype includes IgA1 and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains, the IgG and IgE isotypes contain two heavy chains and two light chains, and the IgM isotype contains five heavy chains and five light chains. The C region of the heavy chain typically contains one or more domains that can perform effector functions. The number of heavy chain constant region domains depends on the isotype. For example, the IgG heavy chain contains three C region domains, each known as CH1, CH2, and CH3. The offered antibody may have any of these isotypes and subtypes.In certain embodiments, the antibody is of the IgG1 subtype, IgG2 subtype, or IgG4 subtype.

[0151] In the full-length light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also includes a "D" region of approximately 10 or more amino acids. See, for example, Fundamental Immunology, 2nd ed., Ch.7 (Paul, W., ed.) 1989, New York: Raven Press (for all purposes, the entire text is incorporated herein by reference). The variable region of each light / heavy chain pair typically forms an antigen-binding site.

[0152] In the antibodies provided herein, the variable regions of the immunoglobulin chains generally exhibit an identical overall structure, including a relatively conserved framework region (FR) linked by three hypervariable regions (more often referred to as “complementarity-determining regions” or CDRs). The CDRs derived from the two chains of each heavy / light chain pair described above are typically aligned by the framework region to form a structure that specifically binds to a particular epitope of the antigen. From the N-terminus to the C-terminus, both the naturally occurring light and heavy chain variable regions typically follow the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.) or Chothia & Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883.

[0153] The present invention relates to a composition comprising an antigen-binding protein having at least one conjugation site. The conjugation site must be applicable to the conjugation of a functional moiety (e.g., a drug, ligand, or peptide) to be added by a defined conjugation chemistry through the side chains of amino acid residues at the conjugation site. Achieving site-specific conjugation with high selectivity for antigen-binding proteins according to the present invention requires consideration of various design criteria. First, preferred conjugation or binding chemistry must be defined or specified. Functional moieties can be conjugated or bound to a selected conjugation site of an antigen-binding protein by combining various conjugation chemistry known in the art. For example, a maleimide-activated conjugation partner targeting an accessible cysteine ​​thiol on an antigen-binding protein is one embodiment, but many conjugation or binding chemical reactions targeting standard or non-standard, e.g., unnatural amino acid side chains within the antigen-binding protein sequence can be used according to the present invention.

[0154] Chemical reactions for chemoselective conjugation include copper(I)-catalyzed azide-alkyne[3+2] bipolar cycloaddition, Staudinger ligation, other acyl transition processes (S→N;X→N), oximation, hydrazone bond formation, and other suitable organic chemical reactions, such as cross-coupling using water-soluble palladium catalysts. (For example, Bong et al., Chemoselective Pd(0)-catalyzed peptide coupling in water, Organic Letters 3(16):2509-11(2001); Dibowski et al., Bioconjugation of peptides by palladium-catalyzed CC cross-coupling in water,Angew.Chem.Int.Ed.37(4):476-78(1998);DeVasher et al.,Aqueous-phase,palladium-catalyzed cross-coupling of aryl bromides under mild conditions,using water-soluble,sterically demanding alkylphosphines,J.Org.Chem.69:7919-27(2004);Shaugnessy et al. al.,J.Org.Chem,2003,68,6767-6774;Prescher,JA and Bertozzi CR,Chemistry in living system, Nature Chemical Biology 1(1);13-21(2005)).

[0155] As described above, conjugation (or covalent bonding) to antigen-binding proteins is carried out by amino acid residues at the conjugation site, such as cysteinyl residues (but not limited to cysteinyl residues) in the side chain. The amino acid residues at the selected internal conjugation site, such as cysteinyl residues, are either amino acid residues that occupy the same amino acid residue position in the native Fc domain sequence, or they can be manipulated into the Fc domain sequence by substitution or insertion.

[0156] The selection of the arrangement of conjugation sites within the entire antigen-binding protein is another important aspect of selecting internal conjugation sites according to the present invention. Any exposed amino acid residue on an antigen-binding protein can be a potently useful conjugation site, and if not previously present at a selected conjugation site in the antigen-binding protein sequence, it can be mutated to cysteine ​​or some other reactive amino acid for site-selective binding. However, this approach does not take into account potential steric hindrance that may disrupt the activity of the conjugate partner or limit the reactivity of the manipulated mutation.

[0157] In one embodiment, the antigen-binding protein is an antibody or a functional fragment thereof. In one embodiment, the antibody or functional fragment thereof contains cysteine ​​or a non-standard amino acid substitution at one or more conjugation sites selected from the group consisting of D70 of the antibody light chain relative to the reference sequence (SEQ ID NO: 7), E276 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8), and T363 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8). To clarify, "D70 of the antibody light chain relative to the reference sequence (SEQ ID NO: 7)" is the same substitution site as AHo position D88 and Kabat position D70 of the light chain of antibody 5G12.006; "E276 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8)" is the same substitution site as AHo position E384 and Kabat position E285 of the heavy chain of antibody 5G12.006; and "T363 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8)" is the same substitution site as AHo position T487 and Kabat position T382 of the heavy chain of antibody 5G12.006.

[0158] [Table 1]

[0159] [Table 2]

[0160] [Table 3]

[0161] [Table 4]

[0162] [Table 5]

[0163] [Table 6]

[0164] [Table 7]

[0165] [Table 8]

[0166] The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. The specific antibodies disclosed herein contain one or more amino acid sequences that are identical to, or substantially identical to, one or more amino acid sequences of the CDRs shown in Tables 4A and 4B. The system described by Kabat et al. above is used for such CDRs.

[0167] The structure and properties of CDRs contained in naturally occurring antibodies are described above. Briefly, in conventional antibodies, CDRs are incorporated into a framework of variable regions of the heavy and light chains that constitute the region responsible for antigen binding and recognition. The variable region contains at least three heavy or light chain CDRs (see above (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, MD; Chothia and Lesk, 1987, J.Mol.Biol.196:901-917; also see Chothia et al., 1989, Nature 342:877-883)) within a framework region (named framework regions 1-4 (FR1, FR2, FR3, and FR4) by Kabat et al., 1991; also see Chothia and Lesk, 1987). However, the CDRs provided herein can be used not only to define the antigen-binding domain of conventional antibody structures, but also incorporated into various other polypeptide structures described herein.

[0168] The antigen-binding proteins provided include monoclonal antibodies. Monoclonal antibodies can be produced by immortalizing spleen cells isolated from transgenic animals after completion of an immunization schedule, for example, using any method known in the art. Spleen cells can be immortalized by fusing them with myeloma cells to generate hybridomas, for example, using any method known in the art. Myeloma cells for use in the hybridoma generation fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and have enzyme deficiencies that prevent them from growing in specific selective media that support the proliferation of only the desired fusion cells (hybridoms). Examples of cell lines suitable for use in mouse fusion include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XXO Bul. Examples of cell lines used in rat fusion include R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion include U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0169] In some cases, hybridoma cell lines are generated by immunizing animals (e.g., transgenic animals possessing human immunoglobulin sequences) with an immunogen, collecting spleen cells from the immunized animals, fusing the collected spleen cells to a myeloma cell line, thereby generating hybridoma cells, establishing hybridoma cell lines from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies binding to target polypeptides. Such hybridoma cell lines and the monoclonal antibodies they produce are embodiments of the present invention.

[0170] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any method known in the art. Hybridomas or mAbs can be further screened to identify mAbs with specific characteristics.

[0171] Chimeric antibodies and humanized antibodies based on the aforementioned sequences are also provided. Monoclonal antibodies for use as therapeutic agents may be modified in various ways before use. One example is a chimeric antibody, which is an antibody consisting of protein segments derived from different antibodies that are covalently linked to produce a functional immunoglobulin light chain or immunoglobulin heavy chain or an immunofunctional portion thereof. Generally, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. For methods relating to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567 and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA 81:6851-6855. These documents are incorporated herein by reference. Regarding CDR porting, see, for example, U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101.

[0172] Generally, the goal of creating chimeric antibodies is to create a chimera in which the number of amino acids derived from the intended patient species is maximized. One example is a "CDR-implanted" antibody, which contains one or more complementarity-determining regions (CDRs) derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the antibody chain is identical or homologous to the corresponding sequence in an antibody derived from a different species or belonging to a different antibody class or subclass. In human use, variable regions or selected CDRs derived from rodent antibodies are often implanted into human antibodies, thereby replacing the naturally occurring variable regions or CDRs of the human antibody.

[0173] One useful type of chimeric antibody is "humanized" antibody. Generally, humanized antibodies are generated from monoclonal antibodies initially produced in non-human animals. Typically, specific amino acid residues of this monoclonal antibody derived from the non-antigen recognition portion of the antibody are modified to be homologous to the corresponding residues in a human antibody of the corresponding isotype. Humanization can be carried out using various methods, for example, by replacing the corresponding region of a human antibody with at least a portion of a rodent variable region (see, for example, U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-1536).

[0174] In one aspect, the CDRs of the light chain variable region and heavy chain variable region of an antibody provided herein are grafted into framework regions (FRs) derived from antibodies derived from the same or different phylogenetic species. For example, V, which is the heavy chain variable region and light chain variable region, H 1, V H 2, V H 3, V H 4, V H 5, V H 6, V H 7, V H 8, V H 9, V H 10, V H 11, V H 12 and / or V L 1 and V LThe CDR of 2 can be transplanted into a consensus human FR. To create a consensus human FR, several FRs derived from human heavy or light chain amino acid sequences can be aligned to identify the consensus amino acid sequence. In other embodiments, the heavy or light chain FRs disclosed herein are exchanged with FRs derived from different heavy or light chains. In one embodiment, rare amino acids in the FRs of the antibody's heavy and light chains are not exchanged, and the remaining FR amino acids are exchanged. A “rare amino acid” is a specific amino acid that is located in a position where it is not normally found in the FR. Alternatively, a transplantable variable region derived from one heavy or light chain may be used with a constant region different from the constant region of a particular heavy or light chain disclosed herein. In other embodiments, the transplantable variable region is part of a single-chain Fv antibody.

[0175] In certain embodiments, hybrid antibodies can be generated by using a constant region derived from a non-human species together with a human variable region.

[0176] Fully human antibodies are also provided. Methods are available for producing fully human antibodies ("fully human antibodies") that are specific to a given antigen without exposing humans to the antigen. One particular means provided for carrying out the production of fully human antibodies is the "humanization" of the mouse humoral immune system. The introduction of a human immunoglobulin (Ig) locus into a mouse inactivated by the endogenous Ig gene is one means of producing fully human monoclonal antibodies (mAbs) in a mouse, an animal that can be immunized with any desired antigen. By using fully human antibodies, immunogenicity and allergic reactions that may be caused by administering mice or mouse-derived mAbs to humans as therapeutic agents can be minimized.

[0177] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that can produce a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more consecutive amino acids and are optionally conjugated to carriers such as haptens. See, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551-2555, Jakobovits et al., 1993, Nature 362:255-258, and Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of such a method, the transgenic animal is produced by inactivating the endogenous mouse immunoglobulin loci that encode the heavy and light chains of mouse immunoglobulins and inserting a large fragment of human genomic DNA containing the loci that encode human heavy and light chain proteins into the mouse genome. Next, partially modified animals having fewer complements than the complete complement of the human immunoglobulin locus are crossbred to obtain animals with the desired fully modified immune system. Upon administration of the immunogen, these transgenic animals produce antibodies that are immune-specific to the immunogen but have an amino acid sequence containing a variable region, and are human rather than mouse. For further details of such methods, see, for example, International Publication No. 96 / 33735 and International Publication No. 94 / 02602. Further methods relating to transgenic mice for the production of human antibodies are described in U.S. Patent Nos. 5,545,807, 6,713,610, 6,673,986, 6,162,963, 5,545,807, 6,300,129, 6,255,458, 5,877,397, 5,874,299 and 5,545,806, PCT Publication International Publication No. 91 / 10741 and International Publication No. 90 / 04036, and European Patent No. 546073B1 and European Patent Application Publication No. 546073A1.

[0178] The transgenic mice described above are referred to herein as "HuMab" mice and contain a minilocus of the human immunoglobulin gene encoding unreorganized immunoglobulin sequences of the human heavy chain ([mu] and [gamma]) and [copper] light chain, along with targeted mutations that inactivate the endogenous [mu] and [copper] chain loci (Lonberg et al., 1994, Nature 368:856-859). Therefore, mice exhibit reduced expression of mouse IgM or [copper], and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations to produce high-affinity human IgG [copper] monoclonal antibodies (Lonberg et al., above; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764:536-546). HuMab mice were created by Taylor et al.,1992, Nucleic Acids Research 20:6287-6295;Chen et al.,1993,International Immunology 5:647-656;Tuaillon et al.,1994,J.Immunol.152:2912-2920;Lonberg et al.,1994,Nature 368:856-859;Lonberg,1994,Handbook of Exp.Pharmacology 113:49-101;Taylor et al.,1994,International Immunology 6:579-591;Lonberg and Huszar,1995,Intern.Rev.Immunol.13:65-93;Harding and Lonberg, 1995, Ann.NY This is described in detail in Acad.Sci.764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, and these documents are incorporated herein by reference in their entirety for all purposes.Furthermore, please refer to U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, as well as U.S. Patent No. 5,545,807, International Publication No. 93 / 1227, International Publication No. 92 / 22646, and International Publication No. 92 / 03918. The disclosures of all these documents are incorporated herein by reference in their entirety for all purposes. The techniques used to generate human antibodies in these transgenic mice are also disclosed in International Publication No. 98 / 24893 and Mendez et al., 1997, Nature Genetics 15:146-156 (these documents are incorporated herein by reference). For example, HCo7 and HCo12 transgenic mouse lines can be used to generate human monoclonal antibodies against target antigens. Further details regarding the production of human antibodies using transgenic mice are provided below.

[0179] By using hybridoma technology, antigen-specific human mAbs with desired specificity can be generated and selected from transgenic mice such as those mentioned above. Such antibodies may be cloned and expressed using appropriate vectors and host cells, or the antibodies may be collected from cultured hybridoma cells.

[0180] Fully human antibodies may also be derived from phage display libraries (as disclosed in Hoogenboom et al., 1991, J.Mol. Biol. 227:381; and Marks et al., 1991, J.Mol. Biol. 222:581). Phage display techniques mimic immunoselection through the display of an antibody repertoire on the surface of filamentous bacteriophages and the subsequent selection of the phages by their binding to a selected antigen. One such technique is described in International Publication No. 99 / 10494 (incorporated herein by reference).

[0181] Derivatives of the antigen-binding proteins described herein are also provided. Derivatized antigen-binding proteins may include any molecule or substance that confers desired properties to an antibody or fragment, such as an increased half-life for a particular application. Derivatized antigen-binding proteins may include, for example, a detectable (or labeled) moiety (e.g., a radioactive molecule, a colorimetric molecule, an antigenic molecule, or an enzyme molecule, a detectable bead (such as a magnetic or high-electron-density (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive moiety, a cytotoxic moiety, or a pharmaceutically active moiety), or a molecule that improves the stability of the antigen-binding protein for a particular application (e.g., administration to a subject such as a human subject, or other in vivo or in vitro use). Examples of molecules that can be used to derivatize antigen-binding proteins include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of antigen-binding proteins can be prepared using methods well known in the art. Certain antigen-binding proteins include PEGylated single-chain polypeptides described herein. In one embodiment, the antigen-binding protein is conjugated or linked to transthyretin (TTR) or a TTR variant. TTR or a TTR variant can be chemically modified with a chemical selected from the group consisting of, for example, dextran, poly(n-vinylpyrrolidone), polyethylene glycol, propropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol.

[0182] Other derivatives include covalent or aggregate conjugates of antigen-binding proteins with other proteins or polypeptides, such as the expression of recombinant fusion proteins containing heterologous polypeptides fused to the N-terminus or C-terminus of the antigen-binding protein. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, such as a yeast alpha factor leader, or a peptide such as an epitope tag. The fusion protein containing the antigen-binding protein may include a peptide (e.g., poly-His) added to facilitate the purification or identification of the antigen-binding protein. The antigen-binding protein can also be linked to a FLAG peptide as described in Hopp et al., 1988, Bio / Technology 6:1204 and U.S. Patent No. 5,011,912. The FLAG peptide enables rapid assays by providing an epitope to which highly antigenic and specific monoclonal antibodies (mAbs) reversibly bind, and facilitates the purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which a given polypeptide is fused with a FLAG peptide are commercially available (Sigma, St. Louis, MO).

[0183] In some embodiments, the antigen-binding protein includes one or more labels. The terms “labeling group” or “label” mean any detectable label. Preferred labeling groups include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I) a fluorescent group (e.g., FITC, rhodamine, lanthanidrine photopolymer), an enzyme group (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal-binding domain, epitope tag). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used as appropriate.

[0184] The term "effector group" refers to any group that is coupled to an antigen-binding protein and acts as a cytotoxic substance. Examples of suitable effector groups include radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I) Other suitable groups include toxin, therapeutic, or chemotherapy groups. Examples of suitable groups include calitiamycin, auristatin, geldanamycin, and mytansin. In some embodiments, the effector group is coupled to the antigen-binding protein via spacer arms of varying lengths to reduce potential steric hindrance.

[0185] Generally, labels are classified into various classes depending on the assay in which they will be detected: a) isotope labels (which may be radioactive or heavy isotopes), b) magnetic labels (e.g., magnetic particles), c) redox active moieties, d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), e) biotinylated groups, and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art.

[0186] Specific markers include optical dyes, which are not limited to chromophores, phosphors, and fluorophores, the latter of which are often specific. Fluorophores may be "small molecule" phosphors or protein phosphors.

[0187] "Fluorescent label" means any molecule that can be detected by its intrinsic fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, rhodamine, and Texas Examples include Red (Pierce, Rockford, IL), Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook by Richard P. Haugland, which is explicitly incorporated herein by reference.

[0188] Suitable protein-based fluorescent labels also include, but are not limited to, green fluorescent proteins containing GFP (Chalfie et al., 1994, Science 263:802-805) and EGFP (Clontech Laboratories, Inc., Genbank accession number U55762) from Renilla, Ptilosarcus, or Aequorea species, blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec, Canada; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), and luciferase (Ichiki et al. al., 1993, J.Immunol. 150:5408-5417), β-galactosidase (Nolan et al.) Examples include al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607) and Renira (International Publication No. 92 / 15673, International Publication No. 95 / 07463, International Publication No. 98 / 14605, International Publication No. 98 / 26277, International Publication No. 99 / 49019, U.S. Patent No. 5292658, U.S. Patent No. 5418155, U.S. Patent No. 5683888, U.S. Patent No. 5741668, U.S. Patent No. 5777079, U.S. Patent No. 5804387, U.S. Patent No. 5874304, U.S. Patent No. 5876995, U.S. Patent No. 5925558).

[0189] Nucleic acids encoding antigen-binding proteins or parts thereof as described herein are also provided, such nucleic acids include nucleic acids encoding one or both chains of an antibody, or fragments, derivatives, mutant proteins, or variants thereof; polynucleotides encoding only heavy chain variable regions or CDRs; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers for identification, analysis, mutagenesis, or amplification of polynucleotides encoding polypeptides; antisense nucleic acids that inhibit polynucleotide expression; and complementary sequences thereof. Nucleic acids may be of any length. Nucleic acids can have nucleotide lengths of, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000, or more, and / or may contain one or more additional sequences, such as regulatory sequences, and / or may be part of a longer nucleic acid, such as a vector. Nucleic acids can be single-stranded or double-stranded and may include RNA and / or DNA nucleotides, as well as their artificial variants (e.g., peptide nucleic acids). Complete heavy-chain and light-chain sequences can be formed by adding any variable regions provided herein to such constant regions. However, it should be understood that these constant-region sequences are provided only as specific examples. In some embodiments, the variable region array is concatenated with other constant region arrays known in the art.

[0190] Nucleic acids encoding specific antigen-binding proteins or parts thereof (e.g., full-length antibodies, heavy or light chains, variable domains, or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) can be isolated from B cells of mice immunized with the antigen. These nucleic acids can be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another known technique by which derivatives of antibodies and other antigen-binding proteins can be prepared. In one technique, polypeptides that are components of the desired antigen-binding protein are expressed in any suitable recombinant expression system, and the expressed polypeptides can then associate to form the antigen-binding protein.

[0191] In one embodiment, nucleic acids that hybridize to other nucleic acids under specific hybridization conditions are further provided. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, NY 1989; 6.3.1-6.3.6. Moderately stringent hybridization conditions as defined herein include a pre-washing solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer containing about 50% formamide, 6x SSC, and a hybridization temperature of about 55°C (or other similar hybridization solutions, such as those containing about 50% formamide, are used at a hybridization temperature of 42°C), as well as washing conditions performed at 60°C with 0.5x SSC and 0.1% SDS. Under stringent hybridization conditions, hybridization occurs in 6x SSC at 45°C, followed by one or more washes in 0.1x SSC, 0.2% SDS at 68°C. Furthermore, those skilled in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization so that nucleic acids containing nucleotide sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to each other typically remain hybridized.

[0192] Basic parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are described, for example, in Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (above), and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those skilled in the art, for example, based on nucleic acid length and / or base composition.

[0193] Mutations can be introduced into nucleic acids, thereby altering the amino acid sequence of the polypeptide encoded by that nucleic acid (e.g., an antibody or antibody derivative). Mutations can be introduced using any method known in the art. In one embodiment, for example, one or more specific amino acid residues are modified using a site-directed mutagenesis protocol. In another embodiment, for example, one or more randomly selected residues are modified using a random mutagenesis protocol. Regardless of the method, the mutant polypeptide can be expressed and screened for desired properties.

[0194] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the polypeptide they encode. For example, nucleotide substitutions can be performed that lead to amino acid substitutions at non-essential amino acid residue positions. Alternatively, one or more mutations can be introduced into nucleic acids that selectively alter the biological activity of the polypeptide they encode. For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative alterations include increasing, decreasing, or eliminating activity. Examples of qualitative alterations include changing the antigen specificity of an antibody. In one embodiment, a nucleic acid encoding any of the antigen-binding proteins described herein can be mutagenesized to modify its amino acid sequence using molecular biological techniques well established in the art.

[0195] In another embodiment, nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences are provided. The nucleic acid molecule may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer, or a fragment that encodes the active portion of the polypeptide.

[0196] Nucleic acid sequence-based probes can be used to detect multiple transcripts encoding, for example, a single polypeptide of nucleic acid or a nucleic acid similar to nucleic acid. Probes may include labeling groups such as radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. Such probes can be used to identify cells expressing polypeptides.

[0197] In another embodiment, a vector is provided containing a nucleic acid encoding a polypeptide or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. Recombinant expression vectors may contain nucleic acids in a form suitable for expression in host cells. Recombinant expression vectors include one or more regulatory sequences selected based on the host cell used for expression, such regulatory sequences operably ligated to the nucleic acid sequence to be expressed. Regulatory sequences may induce constitutive expression of a nucleotide sequence in many types of host cells (e.g., SV40 early gene enhancer, Rouss sarcoma virus promoter, and cytomegalovirus promoter), or induce expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences; Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science). See 236:1237 (this document is incorporated herein by reference in its entirety), as well as those that induce inducible expression of nucleotide sequences in response to specific treatments or conditions (e.g., metallothionin promoters in mammalian cells, and tet-responsive and / or streptomycin-responsive promoters in both prokaryotes and eukaryotes (see the same document)). Those skilled in the art will understand that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. Expression vectors can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides encoded by the nucleic acids described herein.

[0198] In another embodiment, host cells into which a recombinant expression vector has been introduced are provided. The host cells may be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., yeast cell, insect cell, or mammalian cell (e.g., CHO cell)). The vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or gene transfer techniques. Stable gene transfer in mammalian cells is known to be possible with only a small cell fraction, depending on the expression vector and gene transfer technique used, allowing for the integration of foreign DNA into the genome. To identify and select such integrations, a gene encoding a selection marker (e.g., for antibiotic resistance) is generally introduced into the host cell along with the target gene. Preferred selection markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells into which the introduced nucleic acid has been stably introduced can be identified, among many methods, particularly by drug selection (e.g., cells into which the selection marker gene has been incorporated will survive, while other cells will die).

[0199] Expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes, or expression cassettes, comprising at least one of the above-mentioned polynucleotides, as well as host cells comprising such expression systems or constructs, are also provided herein.

[0200] The antigen-binding proteins provided herein can be prepared by any of many conventional methods. For example, antigen-binding proteins can be produced by recombinant expression systems using any method known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).

[0201] Antigen-binding proteins can be expressed in hybridoma cell lines (for example, specifically, antibodies can be expressed in hybridomas) or non-hybridoma cell lines. Expression constructs encoding antibodies can be used for the transformation of mammalian host cells, insect host cells, or microbial host cells. Transformation can be carried out using any known method for introducing polynucleotides into host cells, including, for example, methods for packaging polynucleotides into viruses or bacteriophages by gene transfer procedures known in the art, as exemplified by U.S. Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455, and then transducing the host cells with the construct. The optimal transformation procedure used will depend on the type of host cell being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated gene transfer, calcium phosphate precipitation, polybren-mediated gene transfer, protoplast fusion, electroporation, polynucleotide encapsulation in liposomes, mixing of nucleic acids with positively charged lipids, and direct microinjection of DNA into the nucleus.

[0202] A recombinant expression construct typically comprises a nucleic acid molecule encoding a polypeptide including one or more of the following: one or more CDRs, light chain constant regions, light chain variable regions, heavy chain constant regions (e.g., C) provided herein. H 1, C H 2 and / or C H 3) and / or another scaffold portion of the antigen-binding protein. These nucleic acid sequences are inserted into a suitable expression vector using standard ligation techniques. In one embodiment, a heavy chain constant region or a light chain constant region is added to the C-terminus of a heavy chain variable region or a light chain variable region and ligated into the expression vector. The vector is typically selected to be functional in the specific host cell in which it is used (i.e., the vector is compatible with the host's cellular mechanisms and can enable gene amplification and / or expression). In some embodiments, vectors are used that utilize protein-protein interaction detection methods using protein reporters such as dihydrofolate reductase (see, for example, U.S. Patent No. 6,270,964, which is incorporated herein by reference). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies or BD Biosciences (formerly "Clontech"). Other useful vectors for antibody and fragment cloning and expression include Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84:439-44 (this document is incorporated herein by reference). Additional suitable expression vectors are discussed, for example, in Methods Enzymol., vol. 185 (DV Goeddel, ed.), 1990, New York: Academic Press.

[0203] Typically, an expression vector used in any of the host cells will contain a sequence for maintaining the plasmid and a sequence for cloning and expressing the exogenous nucleotide sequence. Such sequences are collectively referred to as “flanking sequences” and, in certain embodiments, will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including a donor splice site and an acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selection marker element. Each of these sequences will be discussed below.

[0204] Optionally, the vector may contain an oligonucleotide molecule located at the 5' or 3' end of a sequence encoding a "tag," i.e., the sequence encoding the antigen-binding protein. Such oligonucleotide sequences may encode polyHis (e.g., hexaHis) or another "tag" such as FLAG®, HA (influenza virus hemagglutinin), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide during polypeptide expression and can function as a means for affinity purification or detection of the antigen-binding protein from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as the affinity matrix. The tag can then be optionally removed from the purified antigen-binding protein by various means, such as using a specific peptidase for cleavage.

[0205] Flanking sequences can be homogeneous (i.e., from the same species and / or strain as the host cell), heterogeneous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from two or more sources), synthetic, or natural. Therefore, the source of a flanking sequence can be any prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, provided that the flanking sequence is functional in the host cellular mechanism and can be activated by the host cellular mechanism.

[0206] Flanking sequences useful in vectors may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein have been previously identified by mapping and / or restriction endonuclease digestion and can therefore be isolated from a suitable tissue source using a suitable restriction endonuclease. In some cases, the complete nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0207] Flanking sequences, whether fully known or partially known, can be obtained using polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes such as oligonucleotides and / or flanking sequence fragments from the same or different species. If the flanking sequence is unknown, a DNA fragment containing the flanking sequence can be isolated, for example, from a coding sequence or a larger DNA fragment that may contain another gene. After generating a suitable DNA fragment by digestion with a restriction endonuclease, isolation can be achieved by purification with an agarose gel, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those skilled in the art. The selection of suitable enzymes for achieving this objective will be readily apparent to those skilled in the art.

[0208] Typically, the origin of replication is part of a commercially purchased prokaryotic expression vector, and the origin assists in the amplification of the vector in host cells. If the selected vector does not contain an origin of replication site, it can be chemically synthesized based on a known sequence and bound to the vector. For example, the origin of replication of plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, varicella stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. In general, origins of replication elements are not required for mammalian expression vectors (for example, the SV40 origin is often used simply because it also contains a viral initial promoter).

[0209] Transcription termination sequences are typically located 3' to the end of the polypeptide encoding region and serve to terminate transcription. In prokaryotic cells, transcription termination sequences are usually GC-rich fragments followed by a polyT sequence. While these sequences can be readily cloned from libraries or even commercially purchased as part of vectors, they can also be readily synthesized using methods for nucleic acid synthesis, such as those described herein.

[0210] Selection marker genes encode proteins necessary for the survival and proliferation of host cells grown in a selective culture medium. Typical selection marker genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, in prokaryotic host cells; (b) proteins that compensate for deficiencies in the cellular nutritional requirements; or (c) proteins that supply essential nutrients unavailable from complex or defined media. Specific selection markers include kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Conveniently, neomycin resistance genes can also be used for selection in both prokaryotic and eukaryotic host cells.

[0211] Other selectable genes can be used to amplify the expressed genes. Amplification is the process by which genes required for the production of proteins important for proliferation or cell survival are repeated in tandem within the chromosomes of recombinant cells over generations. Examples of selectable markers suitable for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoter-less thymidine kinase gene. Mammalian cell transformants are placed under selective pressure, which is specifically adapted so that only these transformants survive due to the selectable genes present in the vector. Selective pressure is applied by culturing the transformed cells under conditions of continuously increasing the concentration of the selectable drug in the culture medium, thereby amplifying both the selectable gene and the DNA encoding another gene, such as an antigen-binding protein that binds to a target polypeptide. As a result, the amount of polypeptides, such as antigen-binding proteins, synthesized from the amplified DNA increases.

[0212] The ribosome binding site is typically required for mRNA translation initiation and is characterized by a Shine-Dalgano sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located at 3' of the promoter and 5' of the coding sequence of the polypeptide to be expressed.

[0213] In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, various pre-sequences or pro-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be modified, or a pro-sequence can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids that were not completely removed during expression, remaining at a -1 position (relative to the first amino acid of the mature protein). For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, attached to the amino terminus. Alternatively, by using several enzymatic cleavage sites, if the enzyme cleaves such regions within the mature polypeptide, the desired polypeptide may be produced in a slightly truncated form.

[0214] Expression and cloning typically involve a promoter that is recognized by the host organism and operably ligated to a molecule encoding an antigen-binding protein. A promoter is a non-transcriptional sequence located upstream (i.e., at the 5' end) of the start codon of a structural gene (generally within approximately 100–1000 bp) and controls the transcription of that structural gene. Promoters are usually classified into two classes: inductive promoters and constitutive promoters. Inductive promoters initiate an increase in transcription levels from DNA under their control in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, transcribe the gene to which they are operably ligated uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by various potential host cells are well known. A suitable promoter is operably ligated to the DNA encoding the heavy or light chain constituting the antigen-binding protein by extracting the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into a vector.

[0215] Promoterians suitable for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used in conjunction with yeast promoters. Promoterians suitable for use in mammalian host cells are well known and include, but are not limited to, those derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (such as adenovirus type 2), bovine papillomavirus, aerovirus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include, for example, heterologous mammalian promoters such as heat shock promoters and actin promoters.

[0216] Enhancer sequences can be inserted into vectors to increase transcription by higher eukaryotes of DNA encoding the light or heavy chains that make up antigen-binding proteins. Enhancers are typically cis-acting elements of DNA, usually about 10–300 bp long, that act on promoters to increase transcription. Enhancers are relatively direction and position independent and can be found at both the 5' and 3' positions relative to the transcription unit. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are commonly used. The SV40 enhancer, cytomegalovirus initial promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in the art, are exemplary enhancing elements for eukaryotic promoter activation. While enhancers can be located in the vector at either the 5' or 3' position relative to the coding sequence, they are generally located at the 5' position of the promoter. To promote the secretion of antibodies into the extracellular space, sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into the expression vector. The selection of signal peptides or leaders depends on the type of host cell producing the antibody, and native signal sequences can be replaced with heterologous signal sequences. Examples of signal peptides that are functional in mammalian host cells include: the interleukin-7 (IL-7) signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

[0217] In one embodiment, the leader sequence includes sequence number 21 (MDMRVPAQLL GLLLLWLRGA RC), which is coded by sequence number 22 (atggacatga gagtgcctgc acagctgctg ggcctgctgc tgctgtggct gagaggcgcc agatgc). In another embodiment, the leader sequence includes sequence number 23 (MAWALLLLTL LTQGTGSWA), which is coded by sequence number 24 (atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcc).

[0218] The expression vector provided may be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they may be obtained individually and conjugated to the vector. Methods used to obtain each flanking sequence are well known to those skilled in the art.

[0219] After constructing a vector and inserting nucleic acid molecules encoding the light chain, heavy chain, or both light and heavy chains that constitute the antigen-binding sequence into appropriate sites on the vector, the completed vector can be inserted into host cells suitable for amplification and / or polypeptide expression. Transformation of host cells into selectable host cells for expression vectors for antigen-binding proteins can be achieved by well-known methods, including gene transfer, infection, co-precipitation with calcium phosphate, electroporation, microinjection, lipofection, DEAE-dextran-mediated gene transfer, or other known techniques. The method of selection will, in part, be functional to the type of host cell used. Such methods and other suitable methods are well known to those skilled in the art and are shown, for example, in Sambrook et al., 2001.

[0220] When cultured under appropriate conditions, host cells synthesize antigen-binding proteins, which can then be recovered from the culture medium (if the host cells secrete them into the medium) or directly from the host cells producing them (if they do not secrete them). The selection of appropriate host cells will depend on various factors, including the desired expression level, polypeptide modifications desirable or essential for activity (such as glycosylation or phosphorylation), and the ease of folding into biologically active molecules.

[0221] Mammalian cell lines available as hosts for expression include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), which are well known in the art, including, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), and many other cell lines. In another embodiment, a B cell lineage-derived cell line can be selected that does not produce its own antibodies but has the ability to produce and secrete heterologous antibodies.

[0222] As used herein, “linker moiety” means a bioacceptable peptidyl or non-peptidyl organic group covalently bonded to an amino acid residue of a toxic peptide analog or other polypeptide chain (e.g., an immunoglobulin HC or LC or immunoglobulin Fc domain) contained in the composition of the present invention, the linker moiety covalently bonds or conjugates the toxic peptide analog or other polypeptide chain to another peptide or polypeptide chain or half-life extension moiety in the composition. In some embodiments of the composition, the half-life extension moiety described herein is conjugated, i.e., directly covalently bonded, to an amino acid residue of the toxic peptide analog itself or to a peptidyl or non-peptidyl linker moiety (including, but not limited to, aromatic or aryl linkers) covalently bonded to an amino acid residue of the toxic peptide analog. The presence of any linker moiety is optional. If present, the chemical structure of the linker moiety is not particularly important, as it primarily serves as a spacer to position, bind, connect, or display or optimize the position of one functional part in relation to one or more other functional parts of the molecule of the composition of the present invention. The presence of a linker moiety may be useful in optimizing the pharmacological activity of some embodiments of the composition of the present invention. If present, the linker can construct amino acids linked by peptide bonds. If present, the linker moiety may independently be identical or different from any other linker or linker that may be present in the composition of the present invention. In some embodiments, the linker may be a polyvalent linker that facilitates the polyvalent display of the toxic peptide analog of the present invention. Polyvalent display of such biologically active compounds can increase binding affinity and / or potency by affinity. The in vivo properties of a therapeutic agent can be altered by conjugation to a polymer or protein (i.e., specific targeting, extension of half-life, distribution profile, etc.).

[0223] Peptidyl linker. As described above, the linker portion, if present (whether within the primary amino acid sequence of the toxic peptide analog or as a linker to add a half-life extension portion to the toxic peptide analog), may actually be "peptidyl" (i.e., constructed from amino acids linked by peptide bonds), and may be constructed with a length of preferably 1 to about 40 amino acid residues, more preferably 1 to about 20 amino acid residues, and most preferably 1 to about 10 amino acid residues. Although not required, the amino acid residues in the linker are preferably from 20 standard amino acids, and more preferably from cysteine, glycine, alanine, proline, asparagine, glutamine and / or serine. It is even more preferable that the peptidyl linker be constructed from a number of sterically unhinged amino acids, such as glycine, serine and alanine linked by peptide bonds. Furthermore, if a peptidyl linker is present, it is desirable that a peptidyl linker be selected that avoids rapid proteolytic turnover in the in vivo circulation. Some of these amino acids may be glycosylated, as will be clearly understood by those skilled in the art. For example, a useful linker sequence for forming the sialylation site is X1X2NX4X5G (SEQ ID NO: 25) (where X1, X2, X4, and X5 are each any amino acid residue, independently of each other).

[0224] In other embodiments, the 1 to 40 amino acids of the peptidyl linker moiety are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Preferably, the linker is constructed from a number of sterically unhindered amino acids, such as glycine and alanine. Therefore, preferred linkers include polyglycine, polyserine, and polyalanine, or any combination thereof. Some exemplary peptidyl linkers are poly(Gly) 1~8 In particular, (Gly)3, (Gly)4 (SEQ ID NO: 26), (Gly)5 (SEQ ID NO: 27), and (Gly)7 (SEQ ID NO: 28), as well as GlySer and poly(Gly)4Ser, for example, "L15" (GGGGSGGGGSGGGGS; SEQ ID NO: 29), poly(Gly-Ala) 2~4 and poly(Ala)1~8 Other specific examples of peptidyl linkers include (Gly)5Lys (SEQ ID NO: 30) and (Gly)5LysArg (SEQ ID NO: 31). Other useful examples of peptidyl linkers are as follows: (Gly)3Lys(Gly)4(Sequence ID 32); (Gly)3AsnGlySer(Gly)2(Sequence ID 33); (Gly)3Cys(Gly)4(Sequence ID 34); and GlyProAsnGlyGly (SEQ ID NO: 35).

[0225] To illustrate the above nomenclature, for example, (Gly)3Lys(Gly)4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (sequence number 36). Other combinations of Gly and Ala are also useful.

[0226] Other preferred linkers include those identified herein as "L5" (GGGGS; or "G4S"; SEQ ID NO: 37), "L10" (GGGGSGGGGS; SEQ ID NO: 38), "L20" (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 39), "L25" (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 40), and any linkers used in the following examples.

[0227] In some embodiments of the compositions of the present invention that include a peptide linker moiety, acidic residues, such as glutamic acid or aspartic acid residues, are positioned within the amino acid sequence of the linker moiety. Examples of peptide linker sequences include the following: GGEGGG (sequence number 41); GGEEEGGG (Sequence code 42); GEEEG (sequence number 43); GEEE(sequence number 44); GGDGGG (Sequence ID 45); GGDDDGG (sequence number 46); GDDDG(sequence code 47); GDDD(array 48); GGGGSDDSDEGSDGEDGGGGS(Sequence code 49); WEWEW(sequence number 50); FEFEF(sequence number 51); EEEWWW(array_52); EEEFFF (Sequence ID 53); WWEEEWW(Sequence ID 54); or FFEEEFF (Sequence ID 55).

[0228] In other embodiments, the linker is a phosphorylated site, for example, X1X2YX4X5G (Sequence ID 56) (where X1, X2, X 4、 X1 and X5 are each independently any amino acid residue); forming X1X2SX4X5G (Sequence ID 57) (where X1, X2, X4 and X5 are each independently any amino acid residue), or X1X2TX4X5G (Sequence ID 58) (where X1, X2, X4 and X5 are each independently any amino acid residue).

[0229] The linkers shown herein are illustrative, and peptidyl linkers within the scope of the present invention may be much longer and may contain other residues. The peptidyl linker may contain, for example, cysteine, another thiol, or a nucleophile for conjugation with a half-life extension moiety. In another embodiment, the linker contains a cysteine ​​or homocysteine ​​residue, or another 2-amino-ethanethiol moiety or 3-amino-propanethol moiety for conjugation with maleimide, iodoacetamide, or a thioester, or a functionalized half-life extension moiety.

[0230] Other useful peptidyl linkers include, for example, large, flexible linkers containing random Gly / Ser / Thr sequences, such as GSGSATGGSGSTASSGSGSATH (SEQ ID NO: 59) or HGSGSATGGSGSTASSGSGSAT (SEQ ID NO: 60), which are estimated to be about the size of a PEG molecule of approximately 1 kDa. Alternatively, useful peptidyl linkers may contain amino acid sequences known in the art to form a rigid helical structure (e.g., rigid linker: -AEAAAKEAAAKEAAAKAGG- / / SEQ ID NO: 61). Furthermore, peptidyl linkers may also contain non-peptidyl segments, such as a six-carbon aliphatic molecule of the formula -CH2-CH2-CH2-CH2-CH2-CH2-. Peptidyl linkers can be modified to form the derivatives described herein.

[0231] Non-peptidyl linker. Optionally, the non-peptidyl linker moiety is also useful for conjugating the half-life extension portion to the peptide portion of the half-life extension portion conjugate toxin peptide analog. For example, -NH-(CH2) s Alkyl linkers such as -C(O)- (wherein s=2~20) can be used. These alkyl linkers may be further substituted with any non-sterically hindered group such as lower alkyl (e.g., C1~C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An example of a non-peptidyl linker is the PEG linker (e.g., shown below): [ka] (wherein n is such that the linker has a molecular weight of about 100 to about 5000 Datons (Da), preferably about 100 to about 500 Da).

[0232] In one embodiment, the non-peptidyl linker is an aryl. The linker can be modified to form derivatives in the same manner as described herein. "Aryl" is phenyl, or phenyl in close proximity to a saturated, partially saturated, or unsaturated 3-membered, 4-membered, or 5-membered carbon bridge, wherein the phenyl or bridge is C 1~8 Alkyl, C 1~4 It is substituted with 0, 1, 2, or 3 substituents selected from haloalkyl or halo. "Heteroaryl" is an unsaturated 5-membered, 6-membered, or 7-membered monocycle, or a partially saturated or unsaturated 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, or 11-membered bicyclic ring, where at least one ring is unsaturated, and these monocycles and bicyclic rings contain 1, 2, 3, or 4 atoms selected from N, O, and S, and the ring is C 1~8 Alkyl, C 1~4 It is substituted with 0, 1, 2, or 3 substituents selected from haloalkyl and halo.

[0233] The non-peptide portion of the composition of the present invention, such as a non-peptidyl linker or a non-peptide half-life extension portion, can be synthesized by conventional organic chemical reactions.

[0234] Other embodiments of the polyvalent linker include a rigid polyheterocyclic core of controlled length. The linker is chemically identified at one end to adapt to orthogonal bonding chemistry (i.e., azido "clicks", amide bonds, thioether formation by alkylation with maleimide or haloacetamide, oxime formation, reductive amination, etc.).

[0235] The above is merely an example and does not represent a thorough treatment of various types of linkers that can be optionally used by the present invention. [Examples]

[0236] Example 1 Generation of anti-GIPR / GLP-1 peptide conjugates The anti-GIPR antibody 2G10_LC1.003 was modified to have the E70C mutation in SEQ ID NO: 151 (light chain) or the E275C mutation in SEQ ID NO: 152 (heavy chain). Anti-GIPR Cys mAb (3-12 mg / mL of IgG1 in 20 mM sodium acetate (pH 5.0)) capped with biscysteamine was partially reduced at room temperature with 2-4 equivalents of triphenylphosphine-3,3',3”-trisulfonate. The reaction progress was monitored using cation exchange chromatography (CEX) (typically completed in 1-2 hours). The liberated cysteamine was purged from the partially overreduced IgG1 by buffer exchange to 20 mM sodium acetate (pH 5.0). The resulting partially overreduced cysteamine-free Cys mAbs (3-12 mg / mL) were added to pH 7.0-7.5 by adding 4-7 equivalents of 4 mM dehydroascorbic acid and 0.5 M Na2HPO4, and then incubated at 2-8°C for reoxidation. The progress of reoxidation was monitored by reverse-phase HPLC. As soon as IgG1 was completely reformed (typically 1-3 hours), 2-3 equivalents of bromoacetyl-GLP-1 peptide (SEQ ID NO: 129) with a C-terminal linker (SEQ ID NO: 29) were added, and the reaction mixture was further incubated at 2-8°C. The progression of the conjugate reaction was monitored by LC / MS and / or CEX until a target peptide-to-antibody ratio (PAR) profile was obtained (e.g., ≥95% PAR2, <5% PAR0 + PAR1). The reaction of the reaction mixture was stopped by adjusting the pH to 5.0 with acetic acid. The desired PAR2 anti-GIPR / GLP-1 conjugate was purified by hydrophobic interaction chromatography (HIC) and subsequently formulated with UF / DF in 10% sodium acetate, 9% sucrose, pH 5.2.

[0237] [Table 9]

[0238] [Table 10]

[0239]

Table 11

[0240]

Table 12

[0241]

Table 13

[0242]

Table 14

[0243]

Table 15

[0244]

Table 16

[0245] The term "GLP-1 receptor agonist" or "GLP-1 peptide" refers to a compound having GLP-1 receptor activity. Examples of such compounds include exendin, exendin analogs, exendin agonists, GLP-1(7-37), GLP-1(7-37) analogs, and GLP-1(7-37) agonists. The GLP-1 receptor agonist compound may optionally be amidated. The terms "GLP-1 receptor agonist" and "GLP-1 receptor agonist compound" have the same meaning.

[0246]

Table 17

[0247]

Table 18

[0248] [Table 19]

[0249] [Table 20]

[0250] AEEA refers to [2-(2-amino)ethoxy)acetic acid.

[0251] EDA refers to ethylenediamine.

[0252] MPA refers to maleimidopropionic acid.

[0253] Results and Discussion Successful site-directed conjugation with Cys mAb protein (IgG1) heavily relies on the ability to selectively reduce ("uncapped") cysteine ​​residues manipulated on a disulfide-bridged IgG1 scaffold. This practically challenging process aims to reduce only the disulfide bonds of two manipulated cysteines in the presence of at least 16 native disulfide bonds that hold the IgG1 tetramer. A single-step selective reduction is highly desirable but currently unattainable (Figure 1). Instead, a two-step purely selective reduction has been developed (Figure 2).

[0254] Reduction step: Cys mAb was reacted with a reducing agent (typically phosphine) to result in complete reduction ("uncapped") of the manipulated cysteine ​​residue. Some native disulfide bonds were also reduced. The degree of this undesirable side reaction depends heavily on post-translational modifications (e.g., "capping" characteristics) and reaction parameters, including reaction conditions (e.g., temperature, type and amount of reducing agent, reaction buffer). The result is an "uncapped" and excessively reduced Cys mAb.

[0255] Acidification step: The thiol "cap" liberated in the reduction step must be removed before proceeding to prevent undesirable "recapping" of Cys mAb. The native disulfide bonds are then reconstituted in the presence of an oxidizing agent, typically dehydroascorbic acid.

[0256] This two-step protocol yields "uncapped" Cys mAb protein ready for site-specific conjugation via S-alkylation reaction (Figure 3).

[0257] To achieve a substantially homogeneous Cys mAb conjugate (PAR2; peptide to antibody ratio of 2), it is essential to keep over-reduction minimal, since restoration of native disulfide bonds via oxidation is not complete. On the other hand, under-reduction results in under-alkylated impurities (PAR1, PAR0) and is therefore equally undesirable. For an optimal reduction profile with minimal over-reduction and complete decapping, appropriate combination of several parameters is required, as described below.

[0258] A positively charged cysteine "cap" (e.g., cysteamine, CA) must be paired with a negatively charged reducing agent. Triphenylphosphine-3,3',3''-trisulfonate (TPPTS) was found to be particularly effective. Other anionic phosphines, such as TCEP and TPPDS, were functional but their performance was inferior to that of TPPTS (Figure 5). Mismatched pairs, for example mercaptoethanesulfonate (MES, a negatively charged "cap") and tris(2-carboxyethyl)phosphine (TCEP, a negatively charged reducing agent), resulted in slow, non-selective and / or incomplete reactions (Figure 4).

[0259] To maximize the attractive Coulomb interaction between oppositely charged reaction partners (e.g., positively charged cysteamine-capped IgG1 and negatively charged TPPTS), the reaction buffer must have a low ionic strength (e.g., 20 mM sodium acetate). Increasing the ionic strength of the reaction medium results in a slow and non-selective reduction (Figure 5).

[0260] The reaction pH must be sufficiently low (e.g., pH 5) to prevent the free thiol ("cap") from undergoing a secondary side reaction, such as disulfide exchange with the native disulfide of IgG1.

[0261] The appropriate combination of the above variables allows for complete capping with the use of a slightly excess reducing agent (e.g., 1.5 equivalents excess, 3.5 equivalents per total Cys mAb), resulting in minimal over-reduction. Such minimal over-reduction is easily corrected in the subsequent oxidation step using dehydroascorbic acid (DHAA, e.g., 4-6 equivalents) as a mild oxidizing agent. The resulting substantially homogeneous "capless" Cys mAb can be cleanly alkylated with a slightly excess (e.g., 0.2 equivalents excess, 2.2 equivalents per total Cys mAb) of bromoacetamide (e.g., a bromoacetamide derivative of the synthetic peptide) to obtain a PAR2 conjugate with ≥95% (Figure 6).

Claims

1. A method for preparing an antibody conjugate or antibody fragment conjugate, a) A step of obtaining a composition containing an antibody or antibody fragment; b) Exposing the antibody or antibody fragment to a cysteine ​​blocking agent (wherein the cysteine ​​blocking agent forms a stable mixed disulfide with at least one cysteine ​​residue of the antibody or antibody fragment); c) A step of adding a reducing agent to the composition to form a reducing mixture, and causing a reduction reaction to occur such that the reducing mixture contains a reducing antibody or a fragment of a reducing antibody; d) A step of adding an oxidizing agent to the reducing mixture to form an oxidizing mixture, and causing an oxidation reaction to occur such that the oxidizing mixture contains an oxidizing antibody or an oxidizing antibody fragment; and e) Adding the activating portion to the oxidation mixture to form a conjugation mixture and causing a conjugation reaction to form an antibody conjugate or antibody fragment conjugate. A method that includes this.

2. The method according to claim 1, wherein the mixed disulfide is an antibody or antibody fragment having capped free cysteine.

3. The method according to claim 2, wherein the antibody or antibody fragment having capped free cysteine ​​comprises a cap selected from the group consisting of cysteine, cysteamine, cystamine, and glutathione.

4. The method according to claim 1, wherein cation exchange chromatography is performed after step b) and before step c) to remove excess cysteine ​​blocking agent.

5. The method according to any one of claims 1 to 4, wherein the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3"-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS").

6. The method according to claim 5, wherein the ratio of reducing agent to antibody or antibody fragment is 2 to 4:1 (mol / mol).

7. The method according to any one of claims 1 to 6, wherein a buffer exchange step is performed after step c) and before step d) to remove the reducing agent.

8. The method according to claim 7, wherein the buffer exchange step is ultrafiltration / dialysis filtration.

9. The method according to any one of claims 1 to 8, wherein the oxidizing agent is dehydroascorbic acid ("DHAA").

10. The method according to claim 9, wherein the ratio of oxidizing agent to antibody or antibody fragment is 3 to 6:1 (mol / mol).

11. The method according to any one of claims 1 to 10, wherein the activation portion is a peptide containing a halogen, and the halogen is selected from the group consisting of Br, I, and Cl.

12. The method according to claim 11, wherein the ratio of activator part versus antibody or antibody fragment is 2 to 3:1 (mol / mol).

13. The method according to any one of claims 1 to 12, wherein a purification step is performed following step e) to remove the activation portion.

14. The method according to claim 13, wherein the purification step comprises hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or ultrafiltration / dialysis filtration after hydrophobic interaction chromatography ("HIC").

15. A method for preparing an antibody conjugate or antibody fragment conjugate, a) A step of obtaining a composition comprising a mixed disulfide containing an antibody or antibody fragment; b) A step of adding a reducing agent to the composition to form a reducing mixture, and causing a reduction reaction to occur such that the reducing mixture contains a reducing antibody or a fragment of a reducing antibody; c) A step of adding an oxidizing agent to the reducing mixture to form an oxidizing mixture, and causing an oxidation reaction to occur such that the oxidizing mixture contains an oxidizing antibody or an oxidizing antibody fragment; and d) Adding the activating portion to the oxidation mixture to form a conjugation mixture and causing a conjugation reaction to form an antibody conjugate or antibody fragment conjugate. A method that includes this.

16. The method according to claim 15, wherein the mixed disulfide is an antibody or antibody fragment having capped free cysteine.

17. The method according to claim 16, wherein the antibody or antibody fragment having capped free cysteine ​​comprises a cap selected from the group consisting of cysteine, cysteamine, cystamine, and glutathione.

18. The method according to claim 15, wherein cation exchange chromatography is performed after step a) and before step b) to remove excess cysteine ​​blocking agent.

19. The method according to any one of claims 15 to 18, wherein the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3"-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS").

20. The method according to claim 19, wherein the ratio of reducing agent to antibody or antibody fragment is 2 to 4:1 (mol / mol).

21. The method according to any one of claims 15 to 20, wherein a buffer exchange step is performed after step b) and before step c) to remove the reducing agent.

22. The method according to claim 21, wherein the buffer exchange step is ultrafiltration / dialysis filtration.

23. The method according to any one of claims 15 to 22, wherein the oxidizing agent is dehydroascorbic acid ("DHAA").

24. The method according to claim 23, wherein the ratio of oxidizing agent to antibody or antibody fragment is 3 to 6:1 (mol / mol).

25. The method according to any one of claims 15 to 24, wherein the activation portion is a peptide containing a halogen, and the halogen is selected from the group consisting of Br, I, and Cl.

26. The method according to claim 25, wherein the ratio of activator part versus antibody or antibody fragment is 2 to 3:1 (mol / mol).

27. The method according to any one of claims 15 to 26, wherein a purification step is performed following step d) to remove the activation portion.

28. The method according to claim 27, wherein the purification step comprises hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or ultrafiltration / dialysis filtration after hydrophobic interaction chromatography ("HIC").

29. A method for preparing an antibody conjugate or antibody fragment conjugate, a) A step of obtaining a composition comprising a reducing mixture containing a reducing antibody or a fragment of a reducing antibody; b) A step of adding an oxidizing agent to the reducing mixture to form an oxidizing mixture, and causing an oxidation reaction to occur such that the oxidizing mixture contains an oxidizing antibody or an oxidizing antibody fragment; and c) A step of adding the activating portion to the oxidation mixture to form a conjugation mixture and causing a conjugation reaction so that an antibody conjugate or antibody fragment conjugate is formed. A method that includes this.

30. The method according to claim 29, wherein the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3"-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS").

31. The method according to claim 30, wherein the ratio of reducing agent to antibody or antibody fragment is 2 to 4:1 (mol / mol).

32. The method according to any one of claims 29 to 31, wherein a buffer exchange step is performed after step a) and before step b) to remove the reducing agent.

33. The method according to claim 32, wherein the buffer exchange step is ultrafiltration / dialysis filtration.

34. The method according to any one of claims 29 to 33, wherein the oxidizing agent is dehydroascorbic acid ("DHAA").

35. The method according to claim 34, wherein the ratio of oxidizing agent to antibody or antibody fragment is 3 to 6:1 (mol / mol).

36. The method according to any one of claims 29 to 35, wherein the activation portion is a peptide containing a halogen, and the halogen is selected from the group consisting of Br, I, and Cl.

37. The method according to claim 36, wherein the ratio of activator part versus antibody or antibody fragment is 2 to 3:1 (mol / mol).

38. The method according to any one of claims 29 to 37, wherein a purification step is performed following step c) to remove the activation portion.

39. The method according to claim 38, wherein the purification step comprises hydrophobic interaction chromatography ("HIC"), ultrafiltration / dialysis filtration, or ultrafiltration / dialysis filtration after hydrophobic interaction chromatography ("HIC").

40. The method according to any one of claims 1 to 39, wherein the antibody or antibody fragment contains a cysteine ​​residue at a position selected from the group consisting of D70 of the antibody light chain relative to the reference sequence (SEQ ID NO: 7), E276 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8), and T363 of the antibody heavy chain relative to the reference sequence (SEQ ID NO: 8).

Images