Immunoglobulins and uses thereof

Abstract

To provide a monoclonal antibody platform designed to be coupled to therapeutic peptides to increase the half-life of the therapeutic peptide in a subject.SOLUTION: The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2, a HCDR3, and a light chain complementarity determining region 1 (LCDR1), a LCDR2, and a LCDR3, each having a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 413,613, filed October 27, 2016. and priority based on U.S. Provisional Patent Application No. 62 / 413,586, filed October 27, 2016. The disclosures of each of these are incorporated herein by reference in their entirety. be absorbed.

[0002] FIELD OF THE INVENTION The present invention generally relates to novel antibodies or fragments thereof and to peptides for therapeutic use in vivo. The present invention relates to a method for the preparation of a therapeutic peptide for use as a carrier to which the therapeutic peptide is bound to increase its half-life. The present invention further relates to pharmaceutical compositions and methods of use thereof.

[0003] (Reference to electronically submitted sequence listing) This application is filed under the file name "PRD3459 Sequence Listing" and Date of publication: October 23, 2017, ASCII format sequence table with file size of 20kb including sequence listings submitted electronically via EFS-Web. The sequence listing submitted herewith is a part of the present specification and is incorporated herein by reference in its entirety. The information contained herein and the filename "PRD3459 Sequence" are included. Sequence listings submitted electronically via EFS-Web in the "e Listing" section can be In the event of any discrepancy regarding the structure of columns 1-27, the information herein shall prevail. [Background technology]

[0004] Peptide-based therapeutics are often proven by the many approved peptide-based treatments. Research and development is being carried out to provide specificity and selectivity, but Methods that can be used may be limited by their relatively short in vivo half-lives. There are numerous half-life extension strategies that have been evaluated and utilized with peptide-based therapeutics. The use of antibodies or antibody fragments as half-life extending moieties for pharmacologically active moieties Therefore, efforts are being made to prevent or reduce the rapid in vivo elimination of pharmacologically active moieties. .

[0005] For pharmacologically active moieties, preferably peptide-based therapeutics, a pharmacologically inactive half-life is There is a need for improved immunoglobulins that can be used as extenders.

[0006] The foregoing discussion is presented merely to provide a better understanding of the nature of the problems facing the art. is presented for the purpose of this application and should not be construed as an admission of prior art in any way. Citation of any reference herein without prior written consent is to be construed as an admission that such reference is a "prior art" of this application. This document should not be construed as an admission that any technology constitutes a "technology." Summary of the Invention [Means for solving the problem]

[0007] In one general aspect, the invention provides the nucleic acids represented by SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively. heavy chain complementarity determining region 1 (HCDR1), HCDR2, and 21 polypeptide sequences; , HCDR3, and light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 The present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

[0008] In a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention The fragment comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 12, and It comprises a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 14. Alternatively, the isolated monoclonal antibody of the present invention has the polypeptide sequence of SEQ ID NO: 13. and a light chain (LC) having the polypeptide sequence of SEQ ID NO:15.

[0009] The present invention further relates to the monoclonal antibody or antigen-binding fragment thereof, vector, (preferably an expression vector containing the nucleic acid), and a host cell containing the vector The present invention also relates to an isolated nucleic acid. Also provided is a method for producing a binding fragment, comprising: culturing the cells under conditions that produce a monoclonal antibody or an antigen-binding fragment thereof; and recovering the antibody or antigen-binding fragment thereof from the cell or culture.

[0010] An embodiment of the present invention comprises at least one pharmacologically active moiety, preferably a therapeutic peptide. The monoclonal antibody of the present invention or The monoclonal antibody or antigen-binding fragment thereof of the present invention includes an antigen-binding fragment thereof. The compound can be conjugated to any therapeutic peptide. Examples of therapeutic peptides include , oxyntomodulin, glucagon-like peptide 1 (GLP1), peptide tyrosine tyrosine Pythin (PYY), exendin (exenatide), amylin (pramlintide), α- Melanocyte-stimulating hormone (MSH), cocaine- and amphetamine-regulated transcript (CAR) T), neuropeptide Y receptor Y1 (NPY1) antagonists, neuropeptide Y Receptor Y5 (NPY5) antagonist, neurotensin S, neuropeptide B, neuropeptide W , ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK ), orexin, melanin-concentrating hormone (MCH), oxytocin, and stress-coping Examples include, but are not limited to:

[0011] Furthermore, the monoclonal antibody of the present invention conjugated to a therapeutic peptide or its antibody A method for generating a protease-binding fragment is provided, the method comprising: The introduced electrophile (preferably bromoacetamide or maleimide) is reacted with a sulfhydryl group a aryl group (preferably SEQ ID NO: 18 of the monoclonal antibody or antigen-binding fragment thereof) The peptide reacts with the sulfhydryl group of the cysteine ​​residue of the peptide, thereby forming a therapeutic peptide and forming a covalent bond between the monoclonal antibody or antigen-binding fragment thereof include.

[0012] The present invention further provides a method for directly linking at least one pharmacologically active moiety, preferably a therapeutic peptide, to a The monoclonal antibody of the present invention or its antibody conjugated via a bond or linker. The present invention relates to a pharmaceutical composition comprising the original binding fragment and a pharmaceutically acceptable carrier.

[0013] Another general aspect of the invention relates to a method for extending the half-life of a therapeutic peptide in a subject. The method further comprises the step of synthesizing therapeutic peptides as shown in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively. and heavy chain complementarity determining region 1 (HCDR1), HCDR2, having a polypeptide sequence of 21. 2, HCDR3, and light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR 3, conjugated to an isolated monoclonal antibody or antigen-binding fragment thereof. wherein the therapeutic peptide comprises a sulfhydryl group, preferably C of SEQ ID NO: 18. The sulfhydryl group of the ys residue of the monoclonal antibody or its antigen-binding fragment is conjugated to

[0014] Further aspects, features, and advantages of the present invention are set forth in the following detailed description and claims. This will be better understood by reading "The Scope of [Brief explanation of the drawings]

[0015] The above summary and the following detailed description of preferred embodiments of the present application should be taken in conjunction with the accompanying drawings. However, the present application is not intended to be a complete translation of the present invention. It should be understood that the present invention is not limited to the above. [Figure 1] 1 shows a general peptide-mAb conjugate strategy according to one embodiment of the present invention, where X represents an electrophile introduced onto the side chain of a therapeutic peptide (e.g., bromoacetamide or maleimide), which specifically reacts with the sulfhydryl group of a Cys residue incorporated into the CDR of a half-life-extending mAb, generating a covalent bond between the therapeutic peptide and the mAb. [Figure 2] Summary of CDR residues selected for substitution in PH9H5_VH (SEQ ID NO: 4) and PH9L3_VL (SEQ ID NO: 3). Residues substituted with Cys are in bold and underlined. [Figure 3] The structure of Compound 1 (SEQ ID NO: 2) is shown. [Figure 4] Pharmacokinetics of Compound 1 in DIO mice. [Figure 5] Pharmacokinetics of Compound 1 in Cynomolgus Monkeys. [Figure 6]Food intake in DIO mice treated with Compound 1:acute administration. [Figure 7] Weight loss in DIO mice treated with Compound 1: acute administration. [Figure 8] Compound 1: Food intake in DIO mice treated chronically. [Figure 9] Compound 1: Weight loss in DIO mice treated with chronic administration. [Figure 10]

[0023] Figure 1 shows a reaction scheme for producing a monoclonal antibody oxyntomodulin (mAb-OXM) compound according to one embodiment of the present invention. The top reaction is the reduction of the disulfide at Cys102 of a mAb (e.g., MSCB97). The bottom reaction is the conjugation of the reduced mAb with an OXM peptide variant. R is cysteine ​​or glutathione. Cys102 on the heavy chain of MSCB97 is shown, along with the flanking amino acids, as their single letter codes. OXM refers to the amino acid portion of the peptide variant. Aib2, Lys30, and a bromoacetylated dispersed polyethylene glycol (dPEG)12 spacer are shown. His1 of the mAb is shown as a single letter code. [Figure 11] The structure of Compound 2 (SEQ ID NO: 27) is shown. [Figure 12] 1 shows a graph demonstrating the functional stability of Compound 2 in human plasma over 168 hours. OXM peptide analog mAb conjugate Compound 2 (▲), Control 1 (previously demonstrated to be stable in human plasma ex vivo) (◆), Control 2 (previously demonstrated to be unstable in human plasma ex vivo) (● and ◯), and an additional OXM peptide analog mAb conjugate Compound 3 (conjugate of mAb with H-Aib-QGTFTSDYSKYLDERRARDFVEWLLNK-(COCH2(OCH2CH2)12NHCOCH2Br)-NH2 (SEQ ID NO: 25)).

[0016] TIFF2025131599000001.tif5128 and compound 4 (mAb and H-Aib-QGTFTSDYSKYLDERRARDFV EWLLNTK-(COCH2CH2(OCH2CH2) 12 NHCOCHBr)-N H2 (SEQ ID NO: 26))▼ over time (hr) on the X-axis. Percent remaining normalized to time zero (t=0). [Figure 13] 1 shows a graph depicting the functional stability of Compound 2 in monkey plasma over 168 hours, Compound 2 ▲, Control 1 (previously demonstrated to be stable in human plasma ex vivo) ◆, Control 2 (previously demonstrated to be unstable in human plasma ex vivo) ● and ◯, and an additional OXM peptide analog mAb conjugate Compound 3.

[0017] TIFF2025131599000002.tif5128 and compound 4▼ are plotted positively relative to time zero (t=0) over time (hr) on the X-axis. Normalized remaining percent. [Figure 14] 1 shows a graph depicting the % change in food intake in cynomolgus monkeys treated with Compound 2. [Figure 15] 1 shows a graph depicting the % change in body weight in cynomolgus monkeys treated with Compound 2. DETAILED DESCRIPTION OF THE INVENTION

[0018] Various publications, articles and patents are cited or referenced in the Background of the Invention and throughout the specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which has been included in the present specification is indicative of the principles of the present invention. Such discussion is intended to provide context. that all of the above constitute part of the prior art to any invention disclosed or claimed. This is not something that can be tolerated.

[0019] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used in this specification shall have the meanings indicated herein.

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" It should be noted that " includes multiple referents unless the context makes clear otherwise. do not have.

[0021] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein are the total In all cases, the terms "about" and "about" should be understood as being modified by the term "about." Therefore, values ​​generally include ±10% of the stated value. For example, 1 mg / m The concentration of L includes 0.9 mg / mL to 1.1 mg / mL. Similarly, 1% to 10% (w / v ) concentration range includes 0.9% (w / v) to 11% (w / v). The use of a numerical range refers to the integers and numbers within that range unless the context clearly indicates otherwise. All possible subranges, including fractions of values, explicitly include all individual numbers within the range. nothing.

[0022] Unless otherwise indicated, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the present invention. One will recognize many equivalents to the specific procedures of the invention described herein, or Such equivalents are intended to be encompassed by this invention. do.

[0023] As used herein, the terms "comprises" and "comprising" "includes," "including," "has," "having" ng}," "contains," or "containing" or any of these Any other variations are intended to include the stated integer or groups of integers, but this It does not exclude any other integer or group of integers other than these, and is non-exclusive or non-limiting. It will be understood that any combination of ingredients is contemplated. For example, compositions, mixtures, processes, etc., which comprise a list of ingredients are not contemplated. The process, method, article, or apparatus is not necessarily limited to only those elements. Other elements not expressly listed, or any such composition, mixture, process, or method Furthermore, unless expressly stated to the contrary, Unless otherwise stated, "or" refers to an inclusive "or" and not an exclusive "or." For example, a condition A or B is a condition where A is true (or exists) and B is false. (or does not exist), then A is false (or does not exist) and B is true (or exists). A is true if A is true (or exists), and B is true if A and B are both true (or exist). This is fulfilled.

[0024] As used herein, "about" refers to a size or characteristic of a component of the preferred invention. Terms such as "approximately," "generally," and "substantially" are used to indicate that the dimensions / characteristics stated are exact boundaries. It is not a bound or parameter, but rather a set of parameters that are functionally the same or similar, as will be understood by those skilled in the art. It will be understood that this does not exclude slight variations from these. Such references, including meters, are based on art-recognized mathematical and engineering principles (e.g., the smallest significant error (e.g., rounding, measurement error or other systematic error, manufacturing tolerances, etc.) Includes variations that do not change the order of magnitude.

[0025] The terms "identical" or "identity" refer to the sequence of two or more nucleic acid or polypeptide sequences (e.g., For example, cyclic PYY 3~36 Polypeptide sequence, oxyntomodulin polypeptide sequence, anti In the context of the present disclosure, methods known in the art may be used to identify the specific sequences of the target polypeptides (e.g., human light or heavy chain sequences). as determined using one of the sequence comparison algorithms, or by visual inspection. By inspection, when compared and aligned for maximum correspondence, are identical or identical Two or more sequences or subsequences having a specified percentage of amino acid residues or nucleotides that are Refers to a division sequence.

[0026] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer When the array is created, subsequence coordinates are specified as needed, and the array algorithm program parameters are The sequence comparison algorithm then runs using the designated program parameters. The percent sequence identity of the test sequence relative to the reference sequence is calculated based on the

[0027] Optimal alignment of sequences for comparison can be performed using, for example, Smith & Waterman's Local homology algorithms in Adv. Appl. Math. 2:482 (1981) , Needleman & Wunsch, J. Mol. Biol. 48:443(19 70) Homology Alignment Algorithm, Pearson & Lipman, Pro c. Nat'l. Acad. Sci. USA 85:2444 (1988) similarity search Methods, computerized implementation of these algorithms (Wisconsin Genetic cs Software Package(Genetics Computer Gr oup, 575 Science Dr., Madison, WI) GAP, BES TFIT, FASTA, and TFASTA), visual inspection (generally, Current P rotocols in Molecular Biology, FMAsube l et al., eds.,Current Protocols(Greene P Publishing Associates, Inc. and John Wiley & S ons, Inc. Joint Venture (1995 Supplement) (Aus This can be done by the following:

[0028] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is BL AST and BLAST 2.0 algorithms, which are Altschul et al. l et al. (1990) J. Mol. Biol. 215:403~410 and Al tschul et al. (1997) Nucleic Acids Res.25: The software for performing BLAST analysis is described in the National Biosciences Library of Japan. It is publicly available through the National Institute of Technology Information.

[0029] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. Therefore, the polypeptide Typically, for example, the first polypeptide is substantially identical to the second polypeptide, where the two polypeptides The peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical. The hybridization occurs when two molecules hybridize to each other under stringent conditions, as described below. And so.

[0030] As used herein, a "subject" refers to any animal, preferably a mammal, most preferably a As used herein, the term "mammal" refers to any animal or mammal. Examples of mammals include, but are not limited to, cows, , horse, sheep, pig, cat, dog, mouse, rat, rabbit, guinea pig, monkey, human and more preferably, humans.

[0031] The term "administration" in the method of the present invention refers to administering a conjugate of the present invention, or a formulation or composition thereof. The use of a composition or agent to treat a syndrome, disorder, or condition described herein means a method for therapeutically or prophylactically preventing, treating or alleviating a disease. Such methods include administering an effective amount of the conjugate, conjugate formulation, composition, or This includes administering the agents at different times during the course of treatment or simultaneously in a combination form. The disclosed methods are understood to encompass all known therapeutic treatment regimens. .

[0032] The term "effective amount" refers to the amount of a substance that is being sought by a researcher, veterinarian, physician, or other clinician in a tissue-based , biological or medical response (such as the syndrome, disorder, or disease being treated) in animals or humans, or to prevent, treat, or ameliorate the symptoms of the syndrome, disorder, or disease being treated. "Amount of active conjugate or drug that elicits a therapeutic effect" refers to the amount of active conjugate or drug that elicits a therapeutic effect (including the amount of active conjugate or drug that elicits a therapeutic effect).

[0033] As used herein, the term "composition" refers to a product containing specified ingredients in specified amounts; and any product resulting directly or indirectly from the combination of specific ingredients in specific amounts. This includes the following.

[0034] As used herein, the term "bonded" refers to the joining or bonding of two or more objects together. When referring to chemical or biological compounds, bond means two or more It can refer to a covalent bond between chemical or biological compounds. The antibody of the present invention can bind to a desired peptide to form an antibody-binding peptide. Antibody-binding peptides are specific peptides designed to conjugate antibodies to peptides. In certain embodiments, the antibodies of the present invention can be formed by a linker. The linker can be covalently attached to the peptide of the present invention via a linker, for example, It may be covalently bound to an antibody or peptide, which in turn is covalently bound to a peptide or antibody.

[0035] As used herein, the term "linker" refers to a covalent linker that covalently attaches a peptide to an antibody. A linker is a chemical module that contains a bonded chain or a chain of atoms. Examples of linkers include peptides. Linkers, hydrocarbon linkers, polyethylene glycol (PEG) linkers, polypropylene Polypropylene glycol (PPG) linker, polysaccharide linker, polyester linker, PEG and and hybrid linkers consisting of embedded heterocycles, and hydrocarbon chains. The PEG linker may, for example, contain 2 to 24 PEG units, but is not limited to these. It is possible.

[0036] As used herein, the term "conjugate" refers to a compound that is covalently attached to a pharmaceutically active moiety. The term "conjugated" refers to an antibody or fragment thereof that is The antibody or fragment thereof may be linked, either directly or indirectly via a linker, to a pharmaceutically active moiety. (preferably a therapeutic peptide) As a non-limiting example, the antibody is a monoclonal antibody of the present invention. The pharmaceutically active moiety may be a cyclic PYY, oxyntomodulin peptide, or The therapeutic peptide may be any other therapeutic peptide of the present invention. The moiety may also be a non-peptide organic moiety (i.e., a "small molecule"). With respect to an antibody or antigen-binding fragment thereof according to one embodiment of the invention, the phrase "antibody or and an antigen-binding fragment of the compound conjugated to the compound, a pharmaceutically active moiety (or therapeutic peptide). A "conjugate comprising a pharmaceutically active moiety (or therapeutic peptide)" is a conjugate comprising a pharmaceutically active moiety (or therapeutic peptide) The term "antibody or antigen-binding fragment thereof" is used interchangeably with the term "antibody or antigen-binding fragment thereof."

[0037] The peptide sequences described herein are written according to the usual convention and include the N-terminus of the peptide. The terminal region is on the left and the C-terminal region is on the right. Isomeric forms of amino acids are known but are not shown separately. Unless explicitly stated, amino acids are L-amino acids.

[0038] antibody In one general aspect, the invention provides a method for the preparation of a pharmaceutical composition comprising: therapeutically active moieties (e.g., therapeutic peptides (e.g., cyclic PYY peptides, oxyntomodulin The peptides (e.g., nucleotides or variant peptides) are chemically conjugated (i.e., and novel antibodies containing cysteine ​​residues that can be used for binding (i.e., binding), This allows the antibody-conjugated peptide to have a prolonged / increased half-life compared to the peptide alone. As used herein, the term "non-targeting" in the context of antibodies refers to As used herein, "target" refers to an antibody that does not specifically bind to any target in vivo. An antibody that "specifically binds" to a target antigen has a KD of 1 x 10 -7 M or less, preferred 1×10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5 x10 -10 M or less, or 1 x 10 -10 The term "KD" refers to an antibody that binds at or below M. refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as the molar concentration The KD value of an antibody can be calculated using methods known in the art in light of the present disclosure. For example, the KD of an antibody can be measured using, for example, Biacore (registered trademark). By using a biosensor system such as the Octet Surface plates can be visualized by using biolayer interferometry such as the RED96 system. The smaller the KD of an antibody, the greater the binding affinity. , the antibody has a high affinity for binding to the target antigen.

[0039] Monoclonal antibodies (whole or fragments thereof) are used as half-life extending moieties Monoclonal antibodies are available and characterized for in vivo use. It is a well-studied protein that has been shown to be highly effective in prolonged in vivo The mechanisms that achieve half-life and for clearance in vivo are well understood. Furthermore, the spatial separation and presentation of the two "arms" of the monoclonal antibody allows for the therapeutic This may be advantageous for the effective bivalency of a molecule (i.e., a therapeutic peptide) to a toxin or other small molecule. Therapeutic drugs have been developed in which molecular drugs are chemically linked to monoclonal antibodies; Typically, antibody-drug conjugates bind to specific antigens and deliver the antibody-drug conjugate to tissues / cells of interest (which is These utilize monoclonal antibodies that target the target antigen (preferentially expressing the target antigen), typically The drug / small molecule is attached to the antibody in a manner that does not affect antigen binding of the antibody.

[0040] For therapeutic peptide-mAb conjugates, half-life extended monoclonal antibodies Antigen-specific binding by a specific target is not desirable. Unexpected heavy chain (HC) and light chain (LC) variable (V) domain pairs are conjugable in the present invention. The non-targeting monoclonal antibodies can be used to prepare non-targeting monoclonal antibodies. To obtain a monoclonal antibody, the complementarity determining region (CDR) of a selected non-targeting antibody is cloned. One of these is engineered to incorporate a cysteine ​​residue. The peptide / compound contains the appropriate chemical moieties, which allow the pharmaceutically active moiety and the non-labeled This allows for conjugation of targeted monoclonal antibodies with engineered cysteine ​​residues. A typical conjugate of a peptide-monoclonal antibody according to one embodiment of the present invention is as follows: The dugete strategy is shown in Figure 1.

[0041] As used herein, the term "antibody" has a broad meaning and includes antibodies against non-human (e.g., mouse) (Serpentine, rat) monoclonal antibodies, human monoclonal antibodies, human-adapted monoclonal antibodies monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies, antibody fragments antibodies, bispecific or multispecific antibodies, dimeric antibodies, tetrameric antibodies, or multimeric antibodies, and includes single chain antibodies.

[0042] Antibody light chains of any vertebrate species are classified into two groups based on the amino acid sequence of their constant domains. are assigned to one of two distinct types: kappa (κ) and lambda (λ). Thus, the antibodies of the present invention can be prepared by incorporating a κ or λ light chain constant domain. According to certain embodiments, the antibodies of the invention can be, for example, murine or human antibodies. In addition to the heavy and light chain constant domains, antibodies also contain heavy and / or light chain constant regions derived from , an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, It consists of three domains (i.e., complementarity determining regions 1 to 3 (CDR1, CDR2, and CDR3)). The light chain variable region domain alternatively comprises LCDR1, LCDR2, and LCDR3. The heavy chain variable region domain is alternatively referred to as HCDR1, HCDR2, and HCDR3. It is called R3.

[0043] Immunoglobulins are divided into five major classes depending on the amino acid sequence of the heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM are classified into five types. It is the most stable immunoglobulin of the class, with a serum half-life of approximately 23 days in humans. IgA and IgG are classified into isotypes IgA1, IgA2, IgG1, IgG2, and Ig Each of the four IgG subclasses is further subdivided into IgG1, IgG2, IgG3, and IgG4. These effector functions are: Generally, they are activated by interaction with Fc receptors (FcγR) or by binding of C1q and fixation of complement. Binding to FcγR leads to antibody-dependent cell-mediated cytolysis. While binding to complement factors can result in complement-mediated cell lysis. The antibodies of the invention utilized for their ability to extend the half-life of the antibody may have no effector function or has minimal effector function but retains the ability to bind to FcRn, and this binding , antibodies can be a primary means of having extended in vivo half-lives.

[0044] In one embodiment, the present invention provides a light chain variable region comprising a fully human Ig germline V gene sequence. and a complete human Ig germline V gene excluding HCDR3 having the amino acid sequence of SEQ ID NO: 18. and a heavy chain variable region having a polypeptide sequence. In regard to this, this antibody or its antigen-binding fragment specifically binds to any human antigen in vivo. In a specific embodiment, the present invention provides a method for producing a human Ig antibody having a complete human Ig germline V gene sequence. and a fully human IgA1 gene except for HCDR3 having the amino acid sequence of SEQ ID NO: 18. and a heavy chain variable region having a germline V gene sequence. With respect to the fragment, the antibody or antigen-binding fragment thereof binds to any human antibody in vivo. The isolated antibody or antigen-binding fragment thereof does not specifically bind to an antigen, and is not intended for pharmaceutical use. Active moieties (e.g., cyclic PYY peptides, oxyntomodulin peptides, and / or The therapeutic peptide of the invention is attached to the

[0045] As used herein, the term "antigen-binding fragment" refers to, for example, a bispecific antibody. Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment monospecific (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), dis Disulfide-stabilized bispecific antibodies (ds diabodies), single-chain antibody molecules (scFv), single Single domain antibodies (sdab), scFv dimers (bivalent bispecific antibodies), one or more A polyspecific antibody formed from a portion of an antibody containing the above CDRs, a camelized monoclonal antibody Single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or antigen-binding antibody fragments, such as any other antibody fragment that binds to a target molecule but does not contain the complete antibody structure. An antigen-binding fragment is a fragment that binds to the same antigen as the parent antibody or parent antibody fragment. According to certain embodiments, the antigen-binding fragment is capable of binding to a light chain variable region. the heavy chain constant region, the light chain constant region, and the Fd segment (i.e., the heavy chain constant region contained in the Fab fragment) According to other particular embodiments, the antigen-binding fragment comprises Fab and F Includes (ab').

[0046] As used herein, the term "single chain antibody" refers to a short antibody of about 15 to about 20 amino acids. Conventional single chain peptides comprising heavy and light chain variable regions joined by a peptide are known in the art. As used herein, the term "single domain antibody" refers to a heavy chain variable region antibody. and heavy chain constant region, or only heavy chain variable region. Refers to the main antibody.

[0047] The phrase "isolated antibodies or antibody fragments" refers to antibodies or antibody fragments having different antigen specificities. It refers to an antibody or antibody fragment that is substantially free of other antibodies that target a certain target antigen (e.g., An isolated antibody that specifically binds to a target antigen substantially eliminates antibodies that do not specifically bind to its target antigen. Furthermore, an isolated antibody or antibody fragment may be isolated from other cellular material and / or May be substantially free of chemicals.

[0048] The antibody variable region consists of a "framework" region separated by three "antigen binding sites". The antigen-binding site is defined using various terms: (i) complementarity-determining region (CD) R), which has three HCDRs (HCDR1, HCDR2, and HCDR R) in VH based on sequence specificity. 3) and there are three LCDRs (LCDR1, LCDR2, LCDR3) in VL (Wu and d Kabat, J Exp Med 132:211-50, 1970, Kabat et al. , Sequences of Proteins of Immunology Interest, 5th Edition, Public Health Service,Natio nal Institutes of Health,Bethesda,Md.,19 91). (ii) “hypervariable regions,” “HVRs,” or “HVs,” three of which are located within VH (H1, H 2, H3) and three in VL (L1, L2, L3) are from Chothia and Les k(Chothia and Lesk,Mol Biol 196:901~17,1 refers to the region of an antibody variable domain that is hypervariable in structure as defined by Other terms include "IMGT-CDR" (Lefranc et al., Dev. Co mparat Immunol 27:55~77, 2003) and “Specificity-determining residue usage”. (SDRU) (Almagro Mol Recognit 17:132~43, 2004) International ImmunoGeneTics ( The IMGT database (http: / / www_mgt_org) provides information on antigen-binding sites. It provides standard numbers and definitions for the correspondence between CDR, HV and IMGT descriptions. For more information, see Lefranc et al., Dev.Comparat.Immunol. It is described in 2003.

[0049] The "framework" or "framework sequence" is defined as the antigen-binding site. The antigen-binding site is the remaining sequence of the variable region, excluding the The exact amino acid sequence of the framework can be determined by how the antigen-binding site is defined. Depends on how it is defined.

[0050] In one embodiment of the invention, the isolated antibody or antigen-binding fragment thereof is LCDR1 and LCDR2 of the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 and a light chain variable region having LCDR3, and SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. A heavy chain variable region having HCDR1, HCDR2, and HCDR3 of the amino acid sequence of SEQ ID NO: 18. and the area.

[0051] In another embodiment, the isolated antibody further comprises an Fc region derived from a human IgG4 Fc region. Compared to other IgG subtypes, the human IgG4 Fc region has a higher affinity for FcγR and complement receptors. Preferably, the Fc region has a reduced ability to bind to the factor. The isolated antibody comprises a human IgG4 Fc region with substitutions corresponding to the residues Substitution of glutamic acid at 233 with proline, and phenylalanine at residue 234 substitution of leucine at residue 235 with alanine or valine; and a modified human IgG4 Fc region containing one or more amino acid substitutions. It further contains an Fc region (EU numbering, Kabat, EA et al. ( 1991)Sequences of Proteins of Immunology cal Interest, 5th Ed.USDept.of Health a nd Human Services, Bethesda, Md., NIH Publi cation no. 91-3242). Substitution of Asn at residue 297 with Ala By doing so, removing the N-linked glycosylation sites within the IgG4 Fc region reduces the residual This is another way to ensure that effector activity is eliminated.

[0052] Preferably, the antibodies of the present invention are held together by disulfide bonds and various non-covalent interactions. Thus, the Fc portion useful in the antibodies of the present invention is Human IgG containing a substitution such as serine to proline at position 228 (EU numbering) 4 Fc region, which stabilizes heavy chain dimer formation and prevents the formation of half IgG4 Fc chains. Prevent.

[0053] In another embodiment, the C-terminal Lys residue of the heavy chain is a recombinantly produced monoclonal antibody. As is common with monoclonal antibodies, the IgG antibody is removed.

[0054] A "human antibody" is an antibody in which both the framework and antigen-binding sites are derived from sequences of human origin. When an antibody contains a constant region, it refers to an antibody having a heavy chain variable region and a light chain variable region. The regions are also derived from sequences of human origin.

[0055] Human antibodies are antibodies whose variable regions are derived from human germline immunoglobulins or rearranged immunoglobulins. Heavy chain variable regions "derived" from sequences of human origin when obtained from systems using purine genes Such a system involves the use of human immunoglobulins displayed on phage, which contain either the main or light chain variable regions. and a library of human immunoglobulin genes carrying the human immunoglobulin loci described herein. "Human antibodies" include those derived from naturally occurring non-human animals, e.g., mice. somatic mutations present or deliberate substitutions introduced into the framework or antigen-binding site insertion results in amino acid sequences that are identical to those of the human germline or rearranged immunoglobulin sequences. Typically, human antibodies contain human germline or is at least approximately identical to the amino acid sequence encoded by the rearranged immunoglobulin gene. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some cases, "human antibodies" are described, for example, by Knappik et al. J Mol Biol 296:57-86, 2000). Consensus framework sequences obtained from framework sequence analysis, or those described, for example, by Shi et al. Mol Biol 397:385-96, 2010 and International Publication No. 2009 / 085 phage-displayed human immunoglobulin gene library described in (Ill. No. 462). Antibodies whose antigen-binding site is derived from a non-human species may contain a synthetic HCDR3 incorporated therein. are not included in the definition of "human antibody."

[0056] The isolated antibody according to embodiments of the present invention may be synthetic. derived from globulin sequences but incorporating synthetic CDRs and / or synthetic frames displayed on a phage These antibodies can be generated using systems such as immunoglobulins or in vitro to improve antibody properties. can undergo in vitro mutagenesis and generate natural products within the human antibody germline repertoire in vivo. This results in antibodies that do not exist in nature.

[0057] As used herein, the term "recombinant antibody" refers to an antibody that has been engineered using human immunoglobulin gene transcription factors. Transgenic or transchromosomal animals (e.g., mice) or hybrids prepared therefrom Antibodies isolated from a plasmid, antibodies isolated from a host cell transformed to express the antibody antibodies isolated from recombinant combinatorial antibody libraries, as well as antibodies isolated from human immunodeficiency or by any other means involving splicing globulin gene sequences to other DNA sequences. antibodies prepared, expressed, generated, or isolated by in vitro Fab arm exchange. Any antibody prepared, expressed, produced, or isolated by recombinant means, including antibodies produced in vitro. Including the body.

[0058] As used herein, the term "monoclonal antibody" refers to an antibody molecule of single molecular composition. A monoclonal antibody composition refers to a preparation of antibodies that bind to a single antibody to a particular epitope. exhibit specificity and affinity, or in the case of bispecific monoclonal antibodies, two distinct The monoclonal antibodies of the present invention exhibit dual binding specificity for epitopes. Therapeutic methods, phage display technology, single lymphocyte gene cloning technology, or recombinant Monoclonal antibodies can be produced by DNA techniques. For example, monoclonal antibodies can be produced by introducing human heavy chains. a transgenic non-human animal (e.g., a mouse) having a genome comprising a gene and a light chain transgene; hybridomas containing B cells obtained from a mouse or rat (e.g., a transgenic mouse or rat) It can be produced by

[0059] In certain embodiments, the term "mAb" refers to a monoclonal antibody. The mAb comprises a heavy chain variable region (VH) sequence comprising SEQ ID NO: 12 and a light chain variable region (VH) sequence comprising SEQ ID NO: 14. and a variable region (VL) sequence. In a specific embodiment, the mAb comprises SEQ ID NO: 13. A fully human monoclonal antibody having a heavy chain (HC) sequence and a light chain (LC) sequence comprising SEQ ID NO: 15. In a specific embodiment, the lysine residue at position 446 of SEQ ID NO: 13 is is optionally missing.

[0060] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is The variable regions of both the light and heavy chains are often , mammals (e.g., mice, rats, rabbits) with desired specificity, affinity, and capacity. The variable region of an antibody derived from one of the following species is used to avoid eliciting an immune response in that species: To do this, the constant regions are matched to antibody sequences from another mammalian species (e.g., human). It is supported.

[0061] As used herein, the term "multispecific antibody" refers to an antibody that binds to multiple immunoglobulin variable domains. The term "antibody" refers to an antibody comprising a plurality of main immunoglobulin variable domain sequences, the first of which is , having binding specificity for a first epitope, or lacking any known binding specificity. a second immunoglobulin variable domain sequence of the plurality comprising a germline sequence; a germline antigen having binding specificity for two epitopes or lacking any known binding specificity and the first and / or second immunoglobulin variable domains optionally comprise a sequence of and a conjugated pharmaceutically active moiety (e.g., a therapeutic peptide). In some embodiments, the first and second epitopes are located on the same antigen, e.g., the same protein (or multiplex). In one embodiment, the first and second epitopes are located on a subunit of a human protein. In one embodiment, the first and second epitopes are overlapping or substantially overlapping. The groups are non-overlapping or substantially non-overlapping. The two epitopes may be on different antigens, e.g., different proteins (or multimeric proteins). In one embodiment, the first and second immunoglobulin variable domains are In one embodiment, the first and second pharmaceutically active moieties are the same. and the second immunoglobulin variable domain comprise different pharmaceutically active moieties. and wherein only the first immunoglobulin variable domain contains a conjugated pharmaceutically active moiety. In one embodiment, only the second immunoglobulin variable domain is conjugated In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin. In one embodiment, the multispecific antibody comprises an immunoglobulin variable domain. molecule, triabody, or tetrabody molecule.

[0062] As used herein, the term "bispecific antibody" refers to an antibody that binds to two or fewer epitopes or binds to no more than two antigens and / or contains two conjugated pharmaceutically active moieties ( For example, a bispecific antibody refers to a multispecific antibody that contains multiple pharmaceutically active moieties (e.g., the same or different pharmaceutically active moieties). The antibody may have binding specificity for a first epitope or may have any known binding specificity. a first immunoglobulin variable domain sequence comprising a germline sequence lacking a second epitope; The germline sequences have binding specificity for the target polypeptide or lack any known binding specificity. and a second immunoglobulin variable domain sequence comprising the first and / or second immunoglobulin variable domain sequences. or the second immunoglobulin variable domain is optionally a conjugated pharmaceutical In one embodiment, the first and second epitopes are located on the same antigen, e.g., On the same protein (or subunit of a multimeric protein). The first and second epitopes overlap or substantially overlap. In other words, the first and second epitopes are located on different antigens, e.g., different proteins (or In one embodiment, the first and second immunization groups are located on different subunits of the immune protein. The globulin variable domains contain the same conjugated pharmaceutically active moiety. In some embodiments, the first and second immunoglobulin variable domains comprise different pharmaceutically active moieties. In one embodiment, only the first immunoglobulin variable domain is conjugated to the In one embodiment, only the second immunoglobulin variable domain comprises a conjugated immunoglobulin variable portion. In one embodiment, the bispecific antibody comprises a first heavy chain comprising a pharmaceutically active moiety conjugated to the first heavy chain. The variable domain sequence and the light chain variable domain sequence (which determine the binding specificity for the first epitope) a germline sequence that has a heterologous structure or lacks any known binding specificity), and a second The heavy chain variable domain sequence and the light chain variable domain sequence (which are directed against a second epitope) (including germline sequences with any known binding specificity or lacking any known binding specificity) wherein the first and / or second heavy chain variable domains optionally comprise a conjugate In one embodiment, the first and second heavy chain variable domains comprise a pharmaceutically active moiety. In one embodiment, the first and second heavy chains comprise a single conjugated pharmaceutically active moiety. The variable domains comprise different conjugated pharmaceutically active moieties. In one embodiment, Only the first heavy chain variable domain contains a conjugated pharmaceutically active moiety. In this embodiment, only the second heavy chain variable domain comprises a conjugated pharmaceutically active moiety.

[0063] As used herein, a "full length antibody" refers to an antibody that contains two full length antibody heavy chains and two full length antibody heavy chains. A full-length antibody heavy chain (HC) is an antibody having a heavy chain variable region (V The full-length antibody light chain (LH) consists of the constant domains (CH1, CH2, and CH3). C) consists of a light chain variable region (VL) and a constant domain (CL). Either one or both heavy chains may lack the C-terminal lysine (K).

[0064] The term "Fab arm" or "half molecule" refers to a heavy-light chain pair.

[0065] Full-length bispecific antibodies can be synthesized, for example, in a cell-free environment in vitro or using co-expression. to favor heterodimer formation of two antibody half molecules with different specificities. By introducing substitutions into the heavy chain CH3 interface in each half molecule, two monospecific bivalent Fab arms can be generated using Fab arm exchange (or half molecule exchange) between antibodies. The exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. A free cysteine ​​occurring on one of the parent monospecific antibodies is linked to a cysteine ​​of the second parent monospecific antibody molecule. The CH3 domain of the parent antibody forms an intra-heavy chain disulfide bond with the TE residue. The CH3 domain of the Fab arm forms a homodimer. The resulting products may be engineered to favor heterodimer formation. Bispecific antibodies having two Fab arms or half molecules capable of binding to different epitopes. It is the body.

[0066] As used herein with respect to antibodies, "homodimerization" refers to the formation of dimers between antibodies with the same CH3 amino acid sequence. As used herein with respect to antibodies, the term "antibody" refers to the interaction of two heavy chains having the same amino acid sequence. A "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0067] As used herein with respect to antibodies, "heterodimerization" refers to the formation of heterodimers in which non-identical CH3 The term "antibody" refers to the interaction of two heavy chains having the amino acid sequences When a "heterodimer" is formed, it has two heavy chains with non-identical CH3 amino acid sequences. Refers to antibodies.

[0068] "Knob-in-hole" strategies (e.g., WO 2006 / 02893 6) can be used to generate full-length bispecific antibodies. Therefore, the selected amino acids that form the interface of the CH3 domain in human IgG are heterodimers. Mutations at positions affecting CH3 domain interactions to promote dimerization The amino acid with a small side chain (hole) can be used to form an antibody that specifically binds to the first antigen. The heavy chain of the antibody is introduced with an amino acid with a large side chain (knob) that specifically binds to the second antigen. After co-expression of the two antibodies, the heterodimer is called the "Humanoid" It is formed as a result of the preferential interaction of heavy chains with "knobs" and heavy chains with "holes." Exemplary CH3 substitution pairs that form knobs and holes are T366Y / F405A, T3 66W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T3 66S_L368A_Y407V (modification in the first CH3 domain of the first heavy chain) (expressed as the altered position / altered position in the second CH3 domain of the second heavy chain).

[0069] Other strategies, such as combining positively charged residues on one CH3 surface and Heavy chain heterodimerization using electrostatic interactions by replacing negatively charged residues in The promotion of somatization is described in U.S. Patent Application Publication Nos. 2010 / 0015133 and 2009 / 018 2127, 2010 / 028637, or 2011 / 0123532 In another strategy, heterodimerization may be used as described in U.S. Pat. As described in No. 2012 / 0149876 or No. 2013 / 0195849 Replaces: L351Y_F405A_Y407V / T394W, T366I_K 392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L35 1Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K39 2L_T394W (modified position in the first CH3 domain of the first heavy chain / modified position in the second heavy chain This can be facilitated by the addition of a second CH3 domain (represented as an altered position in the second CH3 domain).

[0070] In addition to the methods described above, bispecific antibodies can also be prepared using methods described in WO 2011 / 131746. According to the described method, two monospecific homodimers were isolated in a cell-free environment in vitro. Asymmetric mutations were introduced into the CH3 region of the dimeric antibody, resulting in reduction of the disulfide bond isomerization. Under original conditions, two parent monospecific homodimeric antibodies are converted into a bispecific heterodimeric antibody. In this method, a first monospecific bivalent antibody may be produced by forming The first and second monospecific bivalent antibodies contain specific substitutions that promote heterodimer stability. These antibodies were engineered to have three domains, with a systemic linker in the hinge region. Incubation of the two under reducing conditions sufficient to isomerize the disulfide bonds Incubation is continued until bispecific antibodies are generated by Fab arm exchange. The reducing conditions can optimally be reverted to non-reducing conditions. Exemplary reducing agents that can be used are 2- Mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol Dimethicone (DTE), glutathione, tris(2-carboxyethyl)phosphine (TC EP), L-cysteine, and β-mercaptoethanol, preferably 2-mercaptoethanol. Captoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphite For example, at a temperature of at least 20° C. , in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreate In the presence of ethanol, at a pH of 5 to 8, for example, at a pH of 7.0 or 7.4, A 90 minute incubation may be used.

[0071] Throughout this specification, the numbering of amino acid residues in the constant region of an antibody is not specifically indicated. Unless otherwise specified, Kabat et al., Sequences of Proteins eins of Immunological Interest,5th Ed.Pu blic Health Service,National Institutes of Health, Bethesda, Md. (1991) It is carried out in accordance with the rules.

[0072] Conjugates In another general aspect, the invention provides a method for treating a pharmaceutically active moiety (e.g., a synthetic therapeutic peptide). (e.g., a cyclic PYY peptide or an oxyntomodulin variant peptide) Conjugates comprising antibodies of the invention covalently conjugated in a specific manner This allows the antibody-bound peptide to have a prolonged / increased half-life compared to the peptide alone. The conjugates may be used to prevent, treat, or prevent a disease or disorder disclosed herein. The present invention further relates to pharmaceutical compositions, methods for their preparation, and uses. Regarding usage.

[0073] In certain embodiments, the antibodies of the invention may be conjugated to a pharmaceutically active moiety. and modified to contain at least one cysteine ​​residue substitution that can be used to The half-life of the active moiety can be extended / increased. In certain embodiments, this at least Another cysteine ​​residue substitution is in a complementarity determining region of the antibody. , wherein the at least one cysteine ​​residue substitution is in a heavy chain complementarity determining region (HCDR). In certain embodiments, the at least one cysteine ​​residue substitution is in HCDR3. , wherein the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In certain embodiments, An antibody comprising an HCDR3 comprising the amino acid sequence of No. 18, conjugated to a pharmaceutically active moiety. The amino acid sequence has at least one additional cysteine ​​substitution that can be made.

[0074] In certain embodiments, the pharmaceutically active moiety may include a linker. The linker may be chemically modified to allow for conjugation of the antibody to a functionally active moiety. Examples of linkers include peptide linkers, hydrocarbon linkers, polyethylene glycol ( PEG linker, polypropylene glycol (PPG) linker, polysaccharide linker, poly ester linkers, hybrid linkers consisting of PEG and embedded heterocycles, and carbon PEG linkers include, but are not limited to, hydroxyl groups. It can contain 2 to 24 PEG units.

[0075] In certain embodiments, the monoclonal antibodies of the invention comprise one, two, three, or more of the target molecules. conjugated to four, five, or six pharmaceutically active moieties (e.g., therapeutic peptides) In a preferred embodiment, the non-targeting monoclonal antibody binds to two pharmaceutical compounds of interest. Conjugated to at least two pharmaceutically active moieties of interest. In certain embodiments where a monoclonal antibody is conjugated, the pharmaceutically active moiety of interest The moieties may be the same pharmaceutically active moiety or may be different pharmaceutically active moieties.

[0076] Methods for conjugating the antibodies of the invention to the pharmaceutically active moieties of the invention are known in the art. Briefly, the antibodies of the present invention are reacted with a reducing agent (e.g., TCEP (Trichloroethylene Propylene glycol)). Tris(2-carboxyethyl)phosphine) and purified (e.g., protein by adsorption or gel filtration), can be conjugated with a pharmaceutically active moiety (e.g., providing lyophilized peptide to reduced antibody under conditions that allow conjugation) After the conjugation reaction, the conjugate was purified by ion exchange chromatography. Protein A adsorption by chromatography or hydrophobic interaction chromatography (HIC) In certain embodiments, the antibodies of the present invention can be purified by a final purification step such as After preparation, the conjugate can be reduced using the HIC method. For further explanation, see, for example, Examples 3 and 7, and Dennler et al., A See Antibodies 4:197-224(2015).

[0077] As used herein with respect to conjugates, "homodimerization" refers to the formation of two identical As used herein with respect to a conjugate, the term "antibody" refers to the interaction between a pharmaceutically active moiety and an antibody. When used herein, "homodimer" refers to an antibody bound to two identical pharmaceutically active moieties.

[0078] As used herein with respect to conjugates, "heterodimerization" refers to the formation of two The term "conjugate" refers to the interaction of a different pharmaceutically active moiety with an antibody. As used herein, "heterodimer" refers to an antibody conjugated to two different pharmaceutically active moieties. vinegar.

[0079] Cyclic PYY peptides PYY 3~36 is a distal intestinal agonist that acts as a Y2 receptor agonist and inhibits food intake It is an endogenous hormone secreted by L cells in the brain. It plays a role in the regulation of appetite and food intake. Considering the role of riboflavin in the gastrointestinal tract of mammals, as well as its antisecretory and absorption-promoting effects and PYY 3~36 is effective in obesity and related conditions, as well as in numerous gastrointestinal disorders. However, PYY as a therapeutic agent 3~36 Its therapeutic utility is based on its rapid PYY is limited by rapid metabolism and a short circulating half-life. 3~36 , preferably modified PYY 3~36 It can be used as a carrier for PYY 3~ 36 It increases the half-life of the peptide and reduces metabolism of the peptide in vivo.

[0080] In certain embodiments of the present invention, modified PYY 3~36 The peptide is a cyclic PYY peptide. The terms "cyclic PYY peptide" and "cyclic PYY" are used interchangeably. 3~36 Analogs," and "cyclic PYY 3~36 The terms "peptide analog" and "peptide analog" can be used interchangeably. Examples of cyclic PYY peptides that can be used are those described in U.S. Provisional Patent Application No. 62 / 413,61 No. 3 (filed October 27, 2016), and U.S. Patent Application No. ______, "Cycli c peptide tyrosine tyrosine compounds as "modulators of neuropeptide receptors" (book The same day as the application, attorney docket number PRD3411) is listed, and both of these The contents of this application are incorporated herein by reference in their entirety.

[0081] Examples of conjugates comprising antibodies of the invention and cyclic PYY peptides are described in U.S. Provisional Patent Application No. No. 62 / 413,586 (filed October 27, 2016), and U.S. Patent Application No. ____ Issue __ “Antibody coupled cyclic peptide tyro sine tyrosine compounds as modulators of neuropeptide Y receptors" (filed on the same day as this application, attorney No. PRD3436, the contents of both of which applications are incorporated herein by reference in their entirety. For example, in a cyclic PYY peptide, the N-terminal amino acid of the ring The residue is attached to a linking group via its α-amino functional group, which is further The lysine residue is attached to the side chain residue of the amino acid at position 31 of the peptide. hPYY to provide a conventional functional group handle for somatization 3~36 Various positions in the sequence Lysine residues can be incorporated into monoclonal antibodies either directly or indirectly. For indirect binding to a monoclonal antibody, the lysine residue can be modified to The group may comprise a linker that allows the cyclic PYY peptide to be attached to a monoclonal antibody. Those skilled in the art will recognize that related orthologs can also be used effectively in this manner. It will be understood that this is contemplated herein.

[0082] Oxyntomodulin peptide Oxyntomodulin (OXM) is a 37-amino acid that is secreted from enteroendocrine L cells in the intestine. It is a long acidic peptide. It binds to the GLP-1 receptor (GLP1R) and the glucagon receptor (GCGR). Through its agonist activity in the β-cell stimulatory system, OXM enhances β-cell function and reduces food intake. Through these complementary mechanisms, OXM-mediated weight loss may be superior to currently available GLP1R agonists. M can also reduce plasma cholesterol and triglycerides. The half-life of OXM is very short, on the order of minutes (Schjoldager et al. al.,Eur.J.Clin.Invest.18(5):499~503(1988 )). Thus, one embodiment of the present invention is a compound of the present invention covalently bound to oxyntomodulin. and a conjugate comprising an antibody of the present invention, which is capable of dual agonism of oxyntomodulin. It provides effective anti-inflammatory properties and a half-life that is sufficiently extended to allow for once-weekly dosing.

[0083] Extending peptide half-life may reduce the susceptibility of OXM peptides to proteolysis. This is achieved by stabilizing DPP4 and reducing its plasma clearance. Proteolysis by ATP results in the replacement of serine at position 2 with aminoisobutyric acid (Aib). The helical topology of the peptides was It is stabilized by introducing a branched salt bridge and by substituting methionine 27 with leucine. The circulating life of the peptide was significantly longer than that of a monoclonal antibody ( Such antibody-drug conjugates have been shown to be effective against those due to its large size (which may reduce glomerular filtration) and re-utilization via fetal Fc receptors. By using short oligoethylene glycol monosaccharides, it is possible to obtain a prolonged plasma half-life. A spacer is inserted between the mAb and the peptide to allow the peptide to bind to GCGR and GLP1. R.

[0084] According to an embodiment of the present invention, the oxyntomodulin conjugate of the present invention comprises four (A) Dual agonist activity: This oxyntomodulin conjugate It has dual agonist effects at the GLP-1 and glucagon receptors. (B) Receptor Balance: Excessive bias towards GLP1R prior to GCGR target engagement leads to increased exposure to GL P-1-mediated gastrointestinal adverse events may occur with conjugates, and GCGR Excessive bias towards oxyntomodulin may attenuate the glycemic effect. The conjugate has no excessive potency bias towards either GLP1R or GCGR (C) Biodistribution: This oxyntomodulin conjugate exhibited peripheral With the aim of achieving targeted engagement of GCGR and the central energy intake regulator GLP1R, GL There is a moderate bias towards P-1 receptor potency (compared to oxyntomodulin alone). (D) Once-weekly administration: The oxyntomodulin conjugate is administered once weekly. It has a competitive advantage because it can be administered to subjects in need.

[0085] Other therapeutic peptides As used herein, conjugates to the monoclonal antibody platform described herein are Conjugates containing other peptides that can be gated are also provided. Examples include glucagon-like peptide 1 (GLP1), exendin (exenatide), and Mirin (pramlintide), α-melanocyte-stimulating hormone (MSH), cocaine and Amphetamine-regulated transcript (CART), a neuropeptide Y receptor Y1 (NPY1) antagonist , neuropeptide Y receptor Y5 (NPY5) antagonist, neurotensin S, neuropeptide B , neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystosin CCK, orexin, melanin-concentrating hormone (MCH), oxytocin, and and stresscopine.

[0086] Glucagon-like peptide-1 (GLP-1) regulates tissue-specific translation of the proglucagon gene GLP-1 is a 30 amino acid long peptide hormone derived from post-processing of the Produced and secreted by enteroendocrine L cells and specific neurons upon ingestion. GLP-1(1–37) is susceptible to amidation and proteolytic cleavage, which , two truncated and equipotent biologically active forms GLP-1(7-36)amide and GLP Endogenous GLP-1 is mainly produced by dipeptidyl peptidase-4 ( DPP-4 or DPP-IV) and neutral endopeptidase 24.11 (NE P 24.11) and renal clearance, resulting in rapid degradation by multiple pathways. GLP-1-based therapy has been shown to reduce weight and lower the risk of hypoglycemia. These are important in the treatment of type 2 diabetes.

[0087] Exendin (exenatide) is approved for the treatment of type 2 diabetes mellitus (T2DM). Exenatide is a GLP-1 agonist that belongs to the incretin mimetic group. , a 39 amino acid long peptide hormone that is a synthetic version of exendin-4, Exendin-4 is an insulin secretagogue with glucose-regulating effects. It shares homology and function with mammalian GLP-1, but is secreted by DPP-4 (DPP-IV). Resistance to solution allows for a longer pharmacological half-life, which may have therapeutic benefits. This biological property of exendin-4 has led to its investigation into use in the treatment of T2DM. This led to...

[0088] Amylin (islet amyloid polypeptide (IAPP)) is a protein that transports insulin from pancreatic β cells. Amylin is a 37 amino acid long peptide hormone co-secreted with the gastrointestinal tract. Amylin slows gastric emptying. It plays a role in regulating blood sugar by slowing down blood sugar levels and promoting satiety. ) is processed from an 89 amino acid long peptide. The precursor (proIAPP) is synthesized in pancreatic β cells from an 89 amino acid long peptide. After the amino acid-long signal peptide is cleaved, it is produced as a 67 amino acid long peptide. When IAPP processing is impaired, the lack of amylin (IAPP) production leads to impaired blood glucose regulation. It is believed that this can result in a lack of control, which can lead to conditions that result in type 2 diabetes. Pramlintide is an amylinomimetic drug with efficacy at least as strong as human amylin. It is a 37 amino acid long polypeptide, containing amino acids at positions 25 (alanine), 28 (amino acid), and 30 (amino acid). (serine), and by substituting the amino acid at position 29 (serine) with proline. The amino acid sequence of this proline is different from that of rat amylin. As a result of these substitutions, pramlintide has a soluble , non-adhesive, and non-aggregative, thereby achieving many of the physical properties of native human amylin. Overcoming the scientific shortcomings (Janes et al., Diabetes 45 (Supp l 2):235A (1996);Young et al.,Drug Dev.R es.37:231-48(1996b)).

[0089] α-Melanocyte-stimulating hormone (α-MSH) is an endogenous peptide of the melanocortin family. α-MSH is a hormone and neuropeptide that is responsible for the pigmentation of hair and skin in mammals. The most important step in stimulating melanogenesis, a process involved in melanocyte deposition, is the stimulation of melanocytes. α-MSH is also involved in the regulation of feeding behavior, energy homeostasis, and sex. It also plays a role in neuroprotective activity and protection from ischemia and reperfusion injury.

[0090] Cocaine- and amphetamine-regulated transcript (CART) is a CARTPT gene in humans. CART peptides, especially CA, are neuropeptide proteins encoded by the gene RT(55-102) shows that CART peptides interact with several hypothalamic appetite circuits. Therefore, it is likely to have an important function in regulating energy homeostasis. RT expression is associated with the expression of endogenous receptors involved in appetite regulation, including leptin, cholecystokinin, and ghrelin. Regulated by peripheral peptide hormones. CART and cholecystokinin are involved in appetite regulation. CART peptides have a synergistic effect on ethanol withdrawal-induced anxiety. contribute to locomotor activity, conditioned place preference, and self-administration of the psychostimulant cocaine. It is believed to regulate the effects of visual acuity; inhibit food intake; and be involved in fear and stimulant behavior. Low CART activity in the substantia nigra is associated with hyperphagia and weight gain, and CART is a natural response It is thought to play a role in the mesolimbic dopamine circuitry of opioids that regulate reward processes. can be obtained.

[0091] Neuropeptide Y (NPY) is involved in, for example, feeding behavior, cerebral cortical neural activity, cardiac activity, and It has many roles in the body, including controlling emotional regulation. NPY is also involved in the treatment of obesity, alcoholism, and other conditions. It has also been implicated in several human diseases, including alcoholism, addiction, and depression. Antisense oligodeoxyribonucleic acid (OPI) receptor antagonists Blockade of the central action of NPY using steroids reduces food intake in energy-deficient animals. Specifically, neuropeptide Y receptor Y5 (NPY5) and neuropeptide Y receptor Neurotrophic receptor Y1 (NPY1) has been shown to stimulate different feeding phases (Br J Ph armacol.2003 Aug;139(8):1433~40). Therefore, N PY5 and NPY1 antagonists are effective in treating obesity and other related metabolic disorders. obtain.

[0092] Neurotensin is involved in the regulation of luteinizing hormone and prolactin release and inhibits dopaminergic It is a 13 amino acid long neuropeptide that has a significant interaction with the agonist system. Tensin is distributed throughout the central nervous system, with highest levels found in the hypothalamus, amygdala, and nucleus accumbens. Neurotensin induces a variety of effects, including analgesia, hypothermia, and increased locomotor activity. It can be used to regulate dopamine pathways.

[0093] Neuropeptide B (NPB) is a short biologically active peptide whose precursor is NBP NPB is encoded by the neuropeptide B / W receptor 1 and 2 (NPBWR) gene. It acts through two G protein-coupled receptors called NPBWR1 and NPBWR2. Neuropeptide B regulates feeding, the neuroendocrine system, memory, learning, and afferent pain pathways. It is thought to be related to the section.

[0094] Neuropeptide W (NPW) consists of 23 (NPW23) or 30 (NPW30) amino acids. These neuropeptides exist in two forms, consisting of hydroxybenzoates and hydroxybenzoates. It binds to the receptors NPBWR1 (also known as GPR7) and NPBWR2 (also known as GPR8) NPW may act through these pathways. It suppresses food intake and body weight, and increases thermogenesis and body temperature. It has been shown that NPW increases both endogenous catabolic signaling molecules. This suggests that it functions as a child.

[0095] Ghrelin (also known as ienomorelin (INN)) is secreted by ghrelinergic cells in the gastrointestinal tract. It is a peptide hormone produced by the nervous system and functions as a neuropeptide in the central nervous system. Ghrelin regulates appetite, energy allocation and utilization, and dopamine neurons. Ghrelin plays a role in regulating reward perception in humans. It is thought to be produced by cleavage of the proghrelin precursor, and this proghrelin precursor The precursor is cleaved to produce proghrelin, which is further cleaved to produce the 28 amino acid long ghrelin. Unlike other endogenous peptides, ghrelin is unable to cross the blood-brain barrier. This provides a unique clinical opportunity for exogenous administration of ghrelin. vinegar.

[0096] Bombesin-like receptor 3 (BRS3) binds to known naturally occurring bombesin-related peptides. It is a G protein-coupled receptor that interacts with ATP with low affinity and does not have a high affinity ligand. Therefore, BRS3 is classified as an orphan receptor.

[0097] Galanin is a neuropeptide encoded by the GAL gene. Galanin is widely expressed in the brain, spinal cord, and intestine of mammals and other animals. However, galanin is known to regulate the action potential in neurons. Galanin is primarily involved in nociception, arousal and sleep regulation, cognition, and ingestion. Numerous biological processes, including but not limited to regulation of diet, mood, and blood pressure Galanin is often involved in the synthesis of acetylcholine, serotonin, It colocalizes with classical neurotransmitters such as ATP and norepinephrine, and also with neuropeptide Y, It co-localizes with neuromodulators such as phospholipid P and vasoactive intestinal peptide.

[0098] Cholecystokinin (CCK) is a gastrointestinal hormone that acts to stimulate the digestion of fats and proteins. CCK is a peptide hormone of the GI tract. It is synthesized and secreted by enteroendocrine cells in the small intestine. CCK is a steroid hormone that stimulates the release of digestive enzymes and bile from the pancreas and gallbladder. , plays a role in satiety, and anxiety.

[0099] Orexin (also known as hypocretin) is a neuropeptide that regulates arousal, wakefulness, and appetite. There are two types of orexins, orexin A and B (hypocretin-1 and -2), The orexin system is primarily involved in stimulating food intake. In addition to the above roles, orexins are thought to be involved in the regulation of energy expenditure and endocrine Regulates organ function.

[0100] Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid long hypothalamic peptide that stimulates appetite. which is involved in regulating eating behavior, mood, sleep-wake cycles, and energy balance. MCH-expressing neurons are located in the lateral hypothalamus and the zona incerta. Despite this restricted distribution, MCH neurons are widely distributed throughout the brain. It is.

[0101] Oxytocin is normally produced by the paraventricular nucleus of the hypothalamus and released by the posterior pituitary gland Oxytocin is a peptide hormone and neuropeptide that is involved in social bonding, sexual activity, and Oxytocin receptors are thought to play a role in reproduction, and during and after birth. It is a G protein-coupled receptor that requires magnesium and cholesterol, and It belongs to the rhodopsin-type (class I) group of protein-binding receptors.

[0102] Human stresscopin (h-SCP), a 40 amino acid long peptide, acts as an adrenocorticotropic hormone. It belongs to the cytokine-releasing hormone (CRH) peptide family. The biological effects of the CRH peptide family are , two seven-transmembrane G protein-coupled receptors, CRH receptor type 1 (CRHR1) and C These receptors share a high degree of sequence homology. However, different members of the CRH peptide family have different relative binding affinities, receptor activation Many of the CRH family members show marked differences in the degree of activity and selectivity of these two receptors. Unlike CRHR2, h-SCP exhibits higher selectivity for CRHR2 and is resistant to physiological stress. It acts as a mediator that assists in the processes that attenuate the initiation and maintenance of stress. In addition to its apparent role in physical stress, h-SCP exerts numerous other physiological effects. It has been reported that the endocrine system, central nervous system, cardiovascular system, pulmonary system, It affects the gastrointestinal, renal, musculoskeletal, and inflammatory systems. CRHR2 activity also affects sarcoidosis. Involved in skeletal muscle wasting diseases such as penia, exercise activity, and food intake, and plays a cardioprotective role In addition, stresscopin mimetics are known to be involved in the regulation of bronchial relaxation and anti-inflammatory activity. Useful in the treatment of medical indications mediated by thyrotropin-releasing hormone receptor 2 activity It has been identified that there are I want to be.

[0103] Half-life extension moiety In addition to the antibodies or antigen-binding fragments thereof of the present invention, the conjugates of the present invention can also be used, e.g. one or two hydroxyl groups to extend the half-life of the pharmaceutically active moiety, for example, via covalent interactions. Other half-life extending moieties may be incorporated. albumin, albumin variants, albumin binding proteins and / or domains, transferrins, These include, but are not limited to, phospholipids, and fragments and analogs thereof. Additional half-life extending moieties that can be incorporated into the conjugate include, for example, Polyethylene glycol, such as PEG 5000 or PEG 20,000, to achieve high viscosity (PEG) molecules, such as laurate, myristate, stearate ester, arachidic acid ester, behenic acid ester, oleic acid ester, arachidonic acid ester Different acids such as ester, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, and docosanedioic acid fatty acids and fatty acid esters of various chain lengths, polylysine, octane, carbohydrates (dextran, These moieties are used to code for protein scaffolds. It may be a direct fusion with the sequence and may be produced by standard cloning and expression techniques. Alternatively, the vector may be recombinantly and chemically coupled using well-known chemical coupling methods. The moiety can be attached to an environmentally produced conjugate of the invention.

[0104] Using well-known methods, a cysteine ​​residue was incorporated at the C-terminus of the molecule to incorporate the PEGylated group. PEGylated moieties can be attached to, for example, peptide molecules of the invention by attachment to a cysteine. can be added to.

[0105] The peptide molecules of the invention incorporating additional moieties can be assayed by several known assays. For example, the functional groups of the compounds can be compared by themselves or by the conjugates according to the present invention. The biological or pharmacokinetic activity of the therapeutic peptide of interest in the formulation is assessed using known in vitro Alternatively, in vivo assays can be used to assay and compare.

[0106] Pharmaceutical Composition In another general aspect, the invention provides a method for producing a conjugate of the invention in a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition comprising a suitable carrier. means a product comprising a conjugate of the present invention together with a pharmaceutically acceptable carrier. The disclosed conjugates and compositions containing them are useful for the therapeutic uses referred to herein. It is also useful in the manufacture of pharmaceuticals.

[0107] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, Buffers, stabilizers, solubilizers, oils, lipids, lipid-containing vesicles, microspheres, liposomes refers to inclusion bodies, or other materials known in the art for use in pharmaceutical formulations. It will be appreciated that the characteristics of the excipient or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or a mixture of carriers or mixtures ... refers to non-toxic materials that do not interfere with the efficacy or biological activity of the compositions according to the present invention. In view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition may be used. Any suitable carrier can be used in the present invention.

[0108] Pharmaceutically acceptable acidic / anionic salts for use in the present invention include acetate salts, Benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate Um, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate Salt, estrus, esylate, fumarate, gluceptate, gluconate, glutamate Phosphate, Glycolyl Arsanilate, Hexyl Resorcinate, Hydrabamine, Hydrogen Bromide Acid salts, hydrochlorides, hydroxynaphthoates, iodides, isethionates, lactates, lactobioses Salt, malate, maleate, mandelate, mesylate, methyl bromide, methyl Nitrate, methylsulfate, mucoate, napsylate, nitrate, pamoate, pantothenate , phosphate / diphosphate, polygalacturonate, salicylate, stearate, base Acetate, succinate, sulfate, tannate, tartrate, octyl, tosylate and and triethiodide. These include hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, and methane sulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-thiazolinone benzenesulfonic acid, cyclohexanesulfamic acid, saccharinic acid or trifluoroacetic acid These include, but are not limited to:

[0109] Pharmaceutically acceptable basic / cationic salts include aluminum, 2-amino-2 -Hydroxymethyl-propane-1,3-diol (tris(hydroxymethyl)amino methane, also known as tromethane or "TRIS"), ammonia, benzathine, t -butylamine, calcium, chloroprocaine, choline, cyclohexylamine, diethylamine Alcoholamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine , N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium Examples of suitable amines include, but are not limited to, ammonium, triethanolamine, or zinc.

[0110] In some embodiments of the present invention, from about 0.001 mg / mL to about 100 mg / mL, 0.01 mg / mL to about 50 mg / mL, or about 0.1 mg / mL to about 25 mg / mL A pharmaceutical formulation is provided comprising a conjugate of the invention in an amount of about 3.0 to about 4.0 mg / kg. The formulation may have a pH of about 10, for example, from about 3 to about 7, or from about 5 to about 9. The formulation may further comprise a buffer system , preservative(s), isotonicity agent(s), chelating agent(s), stabilizer(s) and and / or at least one component selected from the group consisting of surfactant(s). .

[0111] Formulations of pharmaceutically active ingredients with pharmaceutically acceptable carriers are described, for example, in Remingto n:The Science and Practice of Pharmacy (e.g. For example, in the 21st edition (2005) and any subsequent revisions , which are known in the art. Non-limiting examples of additional components include buffers, diluents, , solvents, tonicity adjusting agents, preservatives, stabilizers, and chelating agents. The following pharmaceutically acceptable carriers may be used in formulating the pharmaceutical compositions of the present invention.

[0112] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is a water-soluble Liquid formulations include solutions, suspensions, emulsions, emulsions, etc. These formulations may include microemulsions, gels, etc. Aqueous formulations typically contain at least 50% w / w water or at least 60%, 70%, 75%, 80%, 85%, 90%, or less Contains at least 95% w / w water.

[0113] In one embodiment, the pharmaceutical composition can be infused via a syringe or an infusion pump. It can be formulated as an injectable preparation. Injection can be, for example, subcutaneous, intramuscular, intraperitoneal, or intravenous. can be delivered to

[0114] In another embodiment, the pharmaceutical composition is a solid formulation, e.g., a freeze-dried or spray-dried composition. which may be used as is or may be diluted with solvents and / or Solid dosage forms include tablets, such as compressed tablets, and / or coated tablets. This includes coated tablets, as well as capsules (e.g., hard or soft gelatin capsules). The pharmaceutical compositions can also be presented in the form of, for example, sachets, dragees, powders, granules, trolistes, etc. It may be in the form of a powder for reconstitution.

[0115] The dosage form may be immediate release (in which case it may include a water soluble or dispersible carrier); or delayed-release, sustained-release, or modified-release (in these cases, dissolution of the dosage form in the gastrointestinal tract) The rate-controlling polymer may be included.

[0116] In other embodiments, the pharmaceutical compositions may be delivered nasally, bucally, or sublingually.

[0117] The pH of the aqueous formulation may be between pH 3 and pH 10. In one embodiment of the present invention, the formulation In another embodiment of the present invention, the pH of the formulation is about 3. 0 to approximately 7.0.

[0118] In another embodiment of the present invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffering agents include: The ingredients include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, Malic acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, vinegar Sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinic acid salts, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and The buffering agents may be present individually or in total in a concentration of from about 0.01 mg / mL to about 50 mg / mL. It may be present at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each of these specific buffering agents constitute alternative embodiments of the invention.

[0119] In another embodiment of the present invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of buffering agents include: Benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4- Hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, Lorfenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate hydroxybenzoate, imidourea, methyl 4-hydroxybenzoate, phenol, 2- Phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate Preservatives include sodium dehydroacetate, thimerosal, and mixtures thereof. , individually or in total, from about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL Each of these specific preservatives may be present in a concentration of about 20 mg / mL to about 20 mg / mL. Pharmaceutical compositions constitute an alternative embodiment of the invention.

[0120] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonicity agent. Non-limiting examples of this embodiment These include salts (such as sodium chloride), amino acids (glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), Diols (glycerol, 1,2-propanediol, propylene glycol, 1,3-propanediol) propanediol, and 1,3-butanediol), polyethylene glycol (e.g. PEG 400), and mixtures thereof. Other examples of isotonicity agents include sugars. Non-limiting examples of sugars include monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans. For example, fructose, glucose, mannose, sorbose, xylose, Maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonic agent is sugar. The term "sugar alcohol" refers to a C(4) alcohol having at least one -OH group. 8) carbohydrates. Non-limiting examples of sugar alcohols include mannitol. , sorbitol, inositol, galactitol, dulcitol, xylitol, and alginate. Pharmaceutical compositions containing each of the isotonicity agents listed in this paragraph are also suitable for use in the present invention. The isotonicity agents, individually or in total, may be present in a concentration of from about 0.01 mg / mL to about 50 mg / mL. It may be present at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each of these specific isotonic agents constitute alternative embodiments of the invention.

[0121] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Examples include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agents may be present individually or in total in an amount of about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each of these specific chelating agents may be used in alternative embodiments of the present invention. Form is formed.

[0122] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include: For example, one or more aggregation inhibitors, one or more oxidation inhibitors, one or more These may include the above surfactants and / or one or more protease inhibitors.

[0123] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is a carboxy / Hydroxycellulose and its derivatives (HPC, HPC-SL, HPC-L and HPM C), cyclodextrin, 2-methylthioethanol, polyethylene glycol (PE G 3350, etc.), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salt (salt sodium chloride), sulfur-containing substances (such as monothioglycerol), or thioglycol The stabilizers may be present individually or in total in a concentration of from about 0.01 mg / mL to about 50 mg / mL, e.g. For example, it may be present at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each of the stabilizers constitute alternative embodiments of the invention.

[0124] In a further embodiment of the invention, the pharmaceutical composition comprises one or more surfactants, preferably or one surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any surfactant that consists of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactants include, for example, anionic surfactants, cationic surfactants, surfactants, nonionic surfactants, and / or zwitterionic surfactants The surfactants can be selected individually or in total at a concentration of about 0.1 mg / mL to about 20 Pharmaceutical compositions containing each of these specific surfactants may be present in a concentration of 0.1 mg / mL. The articles constitute alternative embodiments of the invention.

[0125] In a further embodiment of the invention, the pharmaceutical composition may comprise, for example, EDTA (ethylenediaminetetraacetic acid). acetic acid), and / or benzamidine hydrochloride (HCl), The protease inhibitors may be present individually or in total at a concentration of about 0.1 mg / mL to about 2 Each of these specific protease inhibitors may be present at a concentration of 0 mg / mL. Pharmaceutical compositions comprising the compound constitute an alternative embodiment of the invention.

[0126] The pharmaceutical compositions of the present invention are useful in reducing aggregate formation of the polypeptide during storage of the composition. The term "amino acid base" refers to one or more amino acid bases. Acids (methionine, histidine, imidazole, arginine, lysine, isoleucine, Any of the amino acids (e.g., tryptophan, threonine, etc.) or their analogs. The amino acid may be present in either its free base form or its salt form. Any stereoisomer of amine may be present (i.e., L, D, or a mixture thereof). The amino acid bases, individually or in combination with other amino acid bases, are present in a concentration of about 0.01 mg / mL to about 5 It may be present at a concentration of from about 0 mg / mL, for example from about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each of these specific amino acid bases constitute alternative embodiments of the present invention. Complete.

[0127] Furthermore, the therapeutically effective amount of the conjugate of the present invention or the pharmaceutical composition thereof may be adjusted depending on the desired effect. It is obvious to those skilled in the art that the dosage may vary depending on the patient. Therefore, the optimum dosage can be easily determined by those skilled in the art. The specific conjugate used, the method of administration, the strength of the preparation, and The dosage may vary depending on the progression of the disease, including the age, weight, diet, and time of administration of the subject. Factors related to the specific subject being treated will need to be adjusted to obtain an appropriate therapeutic concentration. It is what gives rise to sexuality.

[0128] For all indications, the conjugates of the invention are preferably administered in a single or multiple doses per day. It is administered peripherally in divided doses of about 1 μg to about 5 mg (e.g., a single dose administered 2, 3, or 4, 5, 6, 7, 8, 9, or 10 smaller doses), or administration About 0.01 μg / kg to about 500 μg / kg, more preferably about 0.05 μg / kg and is administered peripherally at a dose of about 250 μg / kg, most preferably less than about 50 μg / kg. Dosages within these ranges will, of course, vary with the potency of each agonist and may be determined by those skilled in the art. Therefore, the dosages given above are examples of the average case. However, there can be individual instances where higher or lower dosage ranges are effective. It is within the scope of the present invention.

[0129] In certain embodiments, the conjugates of the invention are administered in doses of about 1 μg to about 5 mg, or about Doses of 0.01 μg / kg to about 500 μg / kg, more preferably about 0.05 μg / kg is administered at a dose of about 250 μg / kg, most preferably at a dose of less than about 50 μg / kg; The dose of the associated second therapeutic agent (e.g., liraglutide) is about 1 μg to about 5 mg; or a dose of about 0.01 μg / kg to about 500 μg / kg, more preferably about 0.05 μg / kg to about 250 μg / kg, most preferably at a dose of less than about 50 μg / kg .

[0130] Pharmaceutically acceptable salts of the conjugates of the present invention include inorganic or organic acids or salts. These include conventional non-toxic salts or quaternary ammonium salts formed from such groups. Examples of acid addition salts include acetates, adipates, benzoates, benzenesulfonates, chlorates, and the like. Enate, camphorate, dodecyl sulfate, hydrochloride, hydrobromide, lactate, maleate, Methanesulfonate, nitrate, oxalate, pivalate, propionate, succinate, The basic salts include ammonium salts, sodium salts, and and alkaline metal salts such as potassium salts, alkaline earth salts such as calcium salts and magnesium salts. salts with organic bases such as arginine, dicyclohexylamino salts, and salts with arginine, etc. Furthermore, the basic nitrogen-containing group can be converted into a salt with, for example, an alkyl halide. Therefore, it may be quaternized.

[0131] The pharmaceutical compositions of the present invention may be administered by any means that achieves their intended use. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral or intraocular administration. Administration may be by oral route. Suitable formulations for parenteral administration include: For example, water-soluble salts, acidic solutions, alkaline solutions, aqueous dextrose solutions, isotonic carbohydrates solutions, and aqueous solutions of the active conjugate in water-soluble form, such as a cyclodextrin inclusion complex Examples include:

[0132] The present invention also provides a method for the preparation of a pharmaceutical composition comprising mixing a pharmaceutically acceptable carrier with any of the conjugates of the present invention. Additionally, the present invention also encompasses a method for producing a pharmaceutical composition, comprising: by mixing a pharmaceutically acceptable carrier of The pharmaceutical compositions produced are encompassed.

[0133] Furthermore, the conjugates of the present invention may have one or more crystalline polymorphs or amorphous crystalline forms. Therefore, these forms are also intended to be included within the scope of the present invention. The conjugates may be solvated, for example, with water (i.e., hydrates, etc.) or common organic solvents. As used herein, the term "solvate" refers to a compound of the present invention. This refers to the physical association of the gate with one or more solvent molecules. In certain cases, e.g., ionic and covalent bonding, For example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvent The term "solvate" refers to both solution-phase solvates and fractionated solvates. Non-limiting examples of suitable solvates include , ethanolate, methanolate, etc.

[0134] The scope of the present invention includes crystalline polymorphs and solvates of the conjugates of the present invention. Therefore, the term "administration" in the treatment method of the present invention includes administration of the conjugate of the present invention, Alternatively, various other embodiments may be included within the scope of the present invention, even if not specifically disclosed. The compound or a solvate thereof may be used to treat, ameliorate, or prevent the syndromes, disorders, or diseases described herein. includes preventative measures.

[0135] In another embodiment, the present invention provides a conjugate of the present invention for use as a medicament. Regarding.

[0136] The present invention includes within its scope prodrugs of the conjugates of the present invention. Such prodrugs are compounds that are readily convertible in vivo into the desired conjugate. Therefore, the term "administration" in the treatment method of the present invention is a functional derivative of benzodiazepine. The present invention relates to the treatment of various disorders described herein with the specifically disclosed conjugates. or, even if not specifically disclosed, certain conjugates may be formed in vivo after administration to a patient. This includes treatment with conjugates that are converted into prodrugs. Conventional procedures for the selection and preparation of protease derivatives are described, for example, in "Design of Protease Derivatives" "The Journal of Clinical Chemistry and Medicine, Vol. 1, No. 1, pp. 1985-1989, 2000" (Ed. H. Bundgaard, Elsevier, 1985) It is being done.

[0137] Furthermore, within the scope of the present invention, any element, particularly those mentioned in relation to the conjugates of the present invention, may be used. Any element in the sample may be naturally occurring or synthetically produced, with a natural abundance. It includes all isotopes and isotopic mixtures of the element in question, either in isotopically enriched form or in isotopically enriched form. For example, hydrogen is intended to be 1 H, 2H(D), and Beauty 3 Similarly, for carbon and oxygen, 12C, 13 C and 14 C, and 16 O and 18 These isotopes are radioactive. The radiolabeled compounds of the present invention may be radioactive isotopes or non-radioactive isotopes. Jugate is 3 H, 11 C. 18 F, 122 I, 123 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, and 82 Br Preferably, the radioisotope is 3 H, 11 C, and 18 Selected from the group consisting of F will be done.

[0138] Some conjugates of the present invention may exist as atropisomers. A cyclobutane isomer is a stereoisomer resulting from hindered rotation around a single bond. The steric interference with the conformational isomers is sufficiently high to allow for the isolation of conformers. It is understood that all such conformers and mixtures thereof are encompassed within the scope of the present invention. .

[0139] When the conjugate according to the invention has at least one stereocenter, the resulting They may exist as enantiomers or diastereomers. Such isomers and mixtures thereof are All combinations are understood to be encompassed within the scope of the present invention.

[0140] If the process for preparing the conjugate according to the invention results in a mixture of stereoisomers, These isomers can be separated by conventional methods such as preparative chromatography. Conjugates may be prepared as racemates or the individual enantiomers may be enantiomerically separated. The conjugates can be prepared either by selective synthesis or by resolution. For example, (-)-di-p-toluoyl-D-tartaric acid and / or (+)-di-p-toluoyl Diastereomeric pairs can be obtained by forming salts with optically active acids such as .beta.-L-tartaric acid. After formation of the compound, the compound can be purified by standard methods such as fractional crystallization and regeneration of the free base. The conjugates can also be separated into diastereomers. Formation of esters or amides of the mers followed by chromatographic separation allows for the formation of chiral auxiliary Alternatively, the conjugate can be resolved by removing the cleavage group. Separation is performed using a chiral column via chromatography (HPLC) or SFC. In some cases, complex multiplets and The rotational mismatch of the conjugate, observable by 1H NMR, results in the confluence of the peaks. Sexual bodies can exist.

[0141] In any process for preparing the conjugates of the present invention, it is possible to It may be necessary and / or desirable to protect sensitive or reactive groups in any This is the Protective Groups in Organic Chemistry stry, ed. JFWMcOmie, Plenum Press, 1973 and TWGreene & PGMWuts,Protective Group s in Organic Synthesis,John Wiley & Sons , 1991 (each of which is incorporated herein by reference in its entirety). This can be achieved by conventional protecting groups, such as those listed in the accompanying text. At a convenient stage, it can be removed using methods well known in the art.

[0142] How to use The present invention also provides a method for identifying a disorder, disease, or condition, or a method for identifying said disorder, disease, or condition. and (ii) preventing, treating, delaying the onset of, or preventing one or more of the following symptoms in a subject in need thereof: provides a method for ameliorating the symptoms of rheumatoid arthritis, the method comprising administering an effective amount of a conjugate or pharmaceutical composition of the present invention. The method comprises administering the pharmaceutical composition to a subject in need thereof.

[0143] In certain embodiments, the disorder, disease, or condition is a disease that is treated with a monoclonal antibody platform of the invention. Any disorder, disease that can be treated with a peptide or compound that can bind to the platform. In certain embodiments, the disorder, disease, or condition is obesity, Type 2 or 3 diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hyperglycemia Triglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, and atherosclerosis Arteriosclerosis, diabetic nephropathy, and other cardiovascular risk factors (such as hypertension) (e.g., steroids), and cardiovascular risks associated with uncontrolled cholesterol and / or lipid levels Risk factors, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver disease The disease is selected from the group consisting of nonalcoholic steatohepatitis (NASH), kidney disease, and / or eczema.

[0144] According to certain embodiments, a therapeutically effective amount is one, two, three, or four of the following effects: (i) The amount of treatment sufficient to achieve or exceed the therapeutic efficacy of the disease, disorder, or condition being treated. (ii) to reduce or ameliorate the severity of the condition or symptoms associated therewith; (iii) shortening the duration of a disease, disorder, or condition, or symptoms associated therewith; Preventing the progression of the disease, disorder or condition being treated or symptoms associated therewith; v) causing regression of the disease, disorder or condition being treated or the symptoms associated therewith; (v) To prevent the development or onset of the disease, disorder, or condition being treated, or symptoms associated therewith. (vi) prevent the recurrence of the disease, disorder, or condition being treated or symptoms associated therewith. (vii) the disease, disorder, or condition being treated, or symptoms associated therewith; (viii) reducing hospitalization of a subject with the disease, disorder, or condition being treated; or a reduction in the length of hospital stay for subjects with symptoms related thereto; (ix) being treated Increasing the survival rate of a subject with a disease, disorder or condition, or symptoms associated therewith; i) inhibiting or alleviating the disease, disorder, or condition being treated, or the symptoms associated therewith; and / or (xii) enhancing or improving the prophylactic or therapeutic efficacy of another treatment.

[0145] The therapeutically effective amount or dose depends on the disease, disorder or condition being treated, the means of administration, the target site, and the subject's Physiological status (including, for example, age, weight, and health status), whether the subject is human or animal other medications being administered, and whether the treatment is prophylactic or therapeutic, etc. Treatment doses may vary depending on a variety of factors, including: Optimally titrated.

[0146] As used herein, the terms "treat" and "treating" and "treatment" both refer to at least one treatment associated with a disease, disorder, or condition. refers to the improvement or reversal of one measurable physical parameter in a subject. It may be discernible in the subject, but not necessarily recognized. "Treat," "treating," and "treatment" also refer to the treatment of a disease, Cause regression of the disorder or condition, prevent its progression, or at least slow its progression. In certain embodiments, the term "treat" or "treament" may also refer to "treating" and "treatment" refer to a single action related to a disease, disorder, or condition. It refers to the alleviation, prevention of the onset or progression of two or more symptoms, or the shortening of their duration. In the embodiments, "treat," "treating," and "tre" are used interchangeably. "Treatment" refers to the prevention of recurrence of a disease, disorder, or condition. "Treat," "treating," and "treatment" are used to describe the treatment of a disease, disorder, or In certain embodiments, "treating" refers to improving the survival rate of a subject with a disease or condition. "eat," "treating," and "treatment" refer to the treatment of a disease in a subject. It refers to the disappearance of a condition, disorder, or disease.

[0147] In one embodiment, the present invention provides a method for treating obesity, or any one or more of the following conditions associated with obesity, in a subject in need thereof: A method for preventing, treating, delaying the onset of, or ameliorating one or more symptoms of a disease is provided. The method comprises administering an effective amount of the conjugate or pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the subject's weight is determined by administering a dose of 100 mg ... administering any of the conjugates, pharmaceutical compositions, dosage forms, or medicaments of the present invention or the conjugates of the invention, pharmaceutical compositions, compared to a control subject not receiving any of the pharmaceutical compositions, dosage forms, or drugs, e.g. For example, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1 % to about 5%, about 2% to about 3%, about 5% to about 10%, about 10% to about 15%, about 15% to about 2 0%, approx. 20% to approx. 25%, approx. 25% to approx. 30%, approx. 30% to approx. 35%, approx. 35% to approx. 4 The decrease is 0%, about 40% to about 45%, or about 45% to about 50%.

[0148] In some embodiments, this weight loss occurs within, for example, about 1 week, about 2 weeks, about 3 weeks, , about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months Months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years , about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years 1 year, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 15 years, or approximately 20 years This is maintained for a period of time.

[0149] The present invention relates to a method for treating a syndrome, disorder or disease, or a method for treating said syndrome, in a subject in need thereof. , prevent, treat, delay the onset of, or treat one or more symptoms of a disorder or disease and a method for ameliorating a syndrome, disorder, or disease, wherein the syndrome, disorder, or disease is obesity, type 1, or type 2 obesity. Diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, Glucose intolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia Hyperlipidemia, dyslipidemia, atherosclerosis, diabetic nephropathy, and other conditions Cardiovascular risk factors (such as high blood pressure) and uncontrolled cholesterol and / or lipids Cardiovascular risk factors associated with blood cholesterol levels, osteoporosis, inflammation, and nonalcoholic steatohepatitis (NA SH), kidney disease, and eczema, and the method comprises administering an effective amount of the composition of the present invention. The method includes administering the conjugate or pharmaceutical composition to a subject in need thereof.

[0150] As used herein, metabolic syndrome refers to hyperglycemia (e.g., hyperglycemia due to fasting). blood sugar), high blood pressure, abnormal cholesterol levels (e.g., low HDL levels), triglycerides Abnormal blood sugar levels (e.g., high triglycerides), large waist circumference (i.e., large waist circumference) , abdominal fat gain, insulin resistance, glucose intolerance, C-reactive protein levels High (i.e., pro-inflammatory state), and plasma plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 High fibrinogen levels (i.e., prothrombotic conditions) Point to the target.

[0151] The present invention provides a method of reducing food intake in a subject in need thereof, the method comprising: By administering an effective amount of the conjugate or pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the subject's food intake comprises: Administering any of the conjugates, compositions, dosage forms, drugs, or combinations thereof or the food intake of a subject prior to administration of the conjugates of the invention described herein. are receiving any of the following: For example, about 0.01% to about 0.1%, about 0.1% to about 0.5%, Approximately 0.5% to approximately 1%, approximately 1% to approximately 5%, approximately 2% to approximately 3%, approximately 5% to approximately 10%, approximately 10% or more Approximately 15%, approximately 15% to approximately 20%, approximately 20% to approximately 25%, approximately 25% to approximately 30%, approximately 30% or more Reduced by about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% do.

[0152] In some embodiments, this reduction in food intake lasts for, for example, about 1 week, about 2 weeks, about 3 weeks, or week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 year, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 15 years, or approximately 2 years It will be maintained for 0 years.

[0153] The present invention provides a method for reducing glycated hemoglobin (A1C) in a subject in need thereof. The method comprises administering an effective amount of the conjugate or pharmaceutical composition of the present invention to a person in need thereof. In some embodiments, the subject's A1C is administered to a subject as described herein. The conjugates, compositions, dosage forms, medicaments, or combinations thereof of the invention described or the A1C of the subject before administration of any of the compounds described herein. Any of the conjugates, compositions, dosage forms, medicaments, or combinations thereof of the invention For example, about 0.001% to about 0.01%, about 0.01%, or about 0.001% of the total IL-16-18 ... 0.01% to approximately 0.1%, approximately 0.1% to approximately 0.2%, approximately 0.2% to approximately 0.3%, approximately 0.3% ~approximately 0.4%, approximately 0.4% to approximately 0.5%, approximately 0.5% to approximately 1%, approximately 1% to approximately 1.5%, approximately 1.5% to approximately 2%, approximately 2% to approximately 2.5%, approximately 2.5% to approximately 3%, approximately 3% to approximately 4%, approximately 4% about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, or about It decreases by 9% to approximately 10%.

[0154] In other embodiments, methods are provided for reducing fasting blood glucose levels in a subject in need thereof. The method comprises administering an effective amount of the conjugate or pharmaceutical composition of the present invention to a person in need thereof. The fasting blood glucose level can be measured by administering to a subject the conjugates of the invention described herein. before administering any of the medicaments, compositions, dosage forms, drugs, or combinations thereof. or the fasting blood glucose level of a subject using a conjugate of the invention described herein. have not received any of the following: For example, about 140 to about 150 mg / dL or less, about 140 to about 130 mg / dL or less, compared to a control subject. Less than 130 to 120 mg / dL, Less than 120 to 110 mg / dL less than about 110 to about 100 mg / dL, about 100 to about 90 mg / dL, or about 90 can be reduced to less than about 80 mg / dL.

[0155] The present invention provides a method for modulating Y2 receptor activity in a subject in need thereof, The method comprises administering an effective amount of a conjugate or pharmaceutical composition of the present invention to a subject in need thereof. As used herein, "modulate" includes increasing or inhibiting receptor activity. It means to reduce.

[0156] In some embodiments, an effective amount of a conjugate of the present invention or a formulation, composition, or The drug is administered once daily, twice daily, three times daily, four times daily, or once a day to a subject in need thereof. In other embodiments, an effective amount of the compound of the present invention is administered 5 times a day, 6 times a day, 7 times a day, or 8 times a day. The disclosed conjugate or its dosage form, composition or medicament is administered to a subject in need thereof for 1 Once every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, twice a month It is administered once a month, three times a month, or four times a month.

[0157] Another embodiment of the present invention is directed to the treatment of a disease, disorder, or syndrome, or or one or more symptoms of such disease, disorder, or syndrome; The method includes a method for delaying or ameliorating the symptoms of rheumatoid arthritis, which comprises administering to a subject in need thereof an effective amount of This includes administering the conjugates or pharmaceutical compositions of the invention in combination therapy. In certain embodiments, the combination therapy is a second therapeutic agent. , surgical treatment.

[0158] As used herein, the term "combined" refers to the administration of two or more therapeutic agents to a subject. In relation to the use of multiple treatments.

[0159] As used herein, combination therapy refers to administering to a subject in need thereof an effective amount of a conjugate of the present invention. or a dosage form, composition or drug thereof in combination with one or more additional therapeutic agents. It refers to administering a therapeutic drug or applying one or more surgical procedures. In an embodiment, the one or more additional therapeutic or surgical therapies are administered in an effective amount of the conjugates of the present invention. In other embodiments, one or more additional The therapeutic or surgical treatment is administered in the same week or month as an effective amount of a conjugate of the invention. It is possible.

[0160] In certain embodiments, the disease or disorder is obesity, type 2 diabetes, metabolic syndrome , insulin resistance, and dyslipidemia, and the second therapeutic agent is an antidiabetic agent. In certain embodiments, the antidiabetic agent may be a glucagon-like peptide-1 (G LP-1) receptor modulators.

[0161] The present invention further provides a method for treating a disease, disorder, syndrome, or condition described herein in a subject in need thereof. or to prevent, treat, delay the onset of, or ameliorate any of the symptoms with a combination therapy. This combination treatment involves administering to a subject in need thereof a conjugate or pharmaceutical composition of the present invention. The present invention relates to a method for treating rheumatoid arthritis, including administering an effective amount of a composition in combination with one or more of the following therapeutic agents: Dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., sitagliptin, GLP-1 receptor agonists; sagliptin, linagliptin, alogliptin, etc. (e.g., short-acting GLP-1 receptor agonists such as exenatide and lixisenatide) intermediate-acting GLP-1 receptor agonists such as liraglutide; extended-release exenatide long-acting GLP-1 receptor agonists such as albiglutide and dulaglutide sodium-glucose cotransporter-2 (SGLT-2) inhibitors (e.g., canagliflozin) canaglifozin, dapagliflozin, empagliflozin glifozin, etc.); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor antagonists nitrates (e.g., bromocriptine rapid release); biguanides (e.g., metformin, etc.) ); insulin; oxyntomodulin; sulfonylureas (e.g., chlorpropamide, Glimepiride, glipizide, glyburide, glibenclamide, glibornuride, glisoxin Sepid, glyclopyramide, tolazamide, tolbutamide, acetohexamide, carbutamide and thiazolidinediones (e.g., pioglitazone, rosiglitazone, robegu) Ritazon, ciglitazone, darglitazone, englitazone, netoglitazone, rivogritazon In some embodiments, the use of additional therapeutic agent(s) The amount is reduced when given in combination with a conjugate of the present invention. In embodiments, when used in combination with a conjugate of the invention, the additional therapeutic agent is , may be used in lower doses than when each is used alone.

[0162] In certain embodiments, the disease or disorder is obesity, type 1 or type 2 diabetes, metabolic syndrome, syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, congenital hyperinsulinism Hypoglycemia, dyslipidemia, atherosclerosis, and diabetic neuropathy due to CHI phlebopathy and other cardiovascular risk factors (such as high blood pressure) and uncontrolled cholesterol Cardiovascular risk factors associated with sterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and eczema, and the second therapeutic agent may be liraglutide.

[0163] The present invention relates to a method for treating a disease, disorder, or syndrome described herein in a subject in need thereof. or any of the symptoms thereof, by combination therapy. This combination treatment involves administering to a subject in need thereof a conjugate or comprises administering an effective amount of the pharmaceutical composition in combination with a surgical treatment. This surgical treatment is different from bariatric surgery (e.g., gastric bypass surgery such as Roux-en-Y gastric bypass). Bypass surgery; Sleeve gastrectomy; Adjustable gastric band surgery; Biliopancreatic with duodenal switch gastric diversion; intragastric balloon; gastric plication, and combinations thereof).

[0164] One or more additional therapeutic agents are administered on the same day as an effective amount of a conjugate of the invention. In certain embodiments, the conjugate of the invention is administered before, after, or simultaneously with an additional therapeutic agent. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. For example, a first treatment (e.g., a composition described herein) may be administered to a subject in combination with a second Prior to administration of treatment (e.g., 5 min, 15 min, 30 min, 45 min, 1 hour, 2 hours, 4 hours, 6 hours) Hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks , 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with administration, or After administration (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 1 hour) 2 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks , 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

[0165] Embodiment The present invention further provides the following non-limiting embodiments.

[0166] Embodiment 1 comprises a light chain variable region having a fully human Ig germline V gene sequence and a nucleotide sequence as set forth in SEQ ID NO: Contains the complete human Ig germline V gene sequence, excluding HCDR3, which contains 18 amino acids. and a heavy chain variable region comprising: The antigen-binding fragment does not specifically bind to any human antigen in vivo.

[0167] Embodiment 2 is an isolated antibody or antigen-binding fragment thereof, each of which is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2. Heavy chain complementarity determining regions having polypeptide sequences 6, 17, 18, 19, 20, and 21 HCDR1, HCDR2, HCDR3, and light chain complementarity determining region 1 (LCDR1 ), LCDR2, and LCDR3. It is an antibody or an antigen-binding fragment thereof.

[0168] Embodiment 3 is a method for producing an isolated monoclonal antibody comprising administering to a subject the method of the present invention ... a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 14; and a light chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 14. Embodiment 3 is an isolated monoclonal antibody of embodiment 2, comprising a variable domain (VL). The antibody is an antibody or an antigen-binding fragment thereof.

[0169] Embodiment 4 is the isolated antibody of any one of embodiments 1 to 3, further comprising an Fc portion. It is a monoclonal antibody.

[0170] Embodiment 5 is a method for producing an antibody comprising administering to a subject the method of the present invention, wherein the Fc portion further comprises an Fc region derived from a human IgG4 Fc region. The isolated monoclonal antibody of embodiment 4.

[0171] Embodiment 6 is a method for producing a human IgG4 Fc region comprising: 1. The isolated monoclonal antibody of embodiment 5.

[0172] Embodiment 7 is a method for treating a monoclonal antibody comprising the step of: substitution of phenylalanine at residue 234 with alanine or valine; Substitution of leucine at residue 5 with alanine or glutamic acid, and alanine at residue 297 substitution of paragine with alanine, 7. The isolated monoclonal antibody of embodiment 6, further comprising a modified human IgG4 Fc region. It is an antibody.

[0173] Embodiment 8 is a method for producing a human IgG4 Fc region comprising: 8. The isolated monoclonal antibody of embodiment 6 or 7, comprising a substitution.

[0174] Embodiment 9 is a combination of a heavy chain (HC) having the polypeptide sequence of SEQ ID NO: 13 and a polypeptide of SEQ ID NO: 1 and a light chain (LC) having the polypeptide sequence of The isolated monoclonal antibody is described.

[0175] Embodiment 10 is a monoclonal antibody or its derivative according to any one of embodiments 1 to 9. An isolated nucleic acid encoding an antigen-binding fragment.

[0176] Embodiment 11 is a vector comprising the isolated nucleic acid of embodiment 10.

[0177] Embodiment 12 is a host cell comprising the vector of embodiment 11.

[0178] Embodiment 13 is a method for producing an isolated monoclonal antibody or antigen-binding fragment thereof. 13. A method of treating a host cell of embodiment 12 with a monoclonal antibody or culturing the antibody or antigen-binding fragment thereof under conditions to produce the antibody or antigen-binding fragment thereof; and recovering the fragment from the cell or culture.

[0179] Embodiment 14 further comprises at least one pharmacologically active moiety conjugated thereto. The isolated monoclonal antibody or its composition according to any one of embodiments 1 to 9, It is an antigen-binding fragment.

[0180] Embodiment 15 is a method for treating a psoriasis according to embodiment 14, wherein the pharmacologically active moiety is a therapeutic peptide. The antibody is an isolated monoclonal antibody or antigen-binding fragment thereof.

[0181] Embodiment 16 is a method for treating a cancer, wherein the therapeutic peptide is conjugated to a cysteine ​​residue of SEQ ID NO: 18. 16. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 15, It is a nt.

[0182] Embodiment 17 is a method for administering a therapeutic peptide to an antibody or an antigen-binding fragment thereof via a linker. 17. The isolated monoclonal antibody of embodiment 15 or 16, conjugated to a The antibody is an antibody or an antigen-binding fragment thereof.

[0183] Embodiment 18 is a method for preparing a hydroxylase comprising administering to a subject a subject, the subject being ... Polyethylene glycol (PEG) linker, polypropylene glycol (PPG) linker, polysaccharide linker linker, polyester linker, or hybrid linker consisting of PEG and an embedded heterocycle. 18. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 17, comprising a It is a fragment.

[0184] Embodiment 19 is a method for treating a rheumatoid arthritis, ... or rheumatoid arthritis, wherein the therapeutic peptide is oxyntomodulin, glucagon-like peptide 1, or hydroxybenzoates. (GLP1), peptide tyrosine tyrosine (PYY), exendin (exenatide), Amylin (pramlintide), α-melanocyte-stimulating hormone (MSH), cocaine and and amphetamine-regulated transcript (CART), neuropeptide Y receptor Y1 (NPY1) antagonist Drug, Neuropeptide Y receptor Y5 (NPY5) antagonist, Neurotensin S, Neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystine CCK, orexin, melanin-concentrating hormone (MCH), oxytocin, and stresscopine. The present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof.

[0185] Embodiment 20 is a method for treating a cancer, comprising administering to a patient a therapeutic peptide comprising the polypeptide sequence of SEQ ID NO: 24. 20. The isolated monoclonal antibody or its antigen of embodiment 19, which is tomodulin. It is a combined fragment.

[0186] Embodiment 21 is directed to a monoclonal antibody conjugated to an oxyntomodulin therapeutic peptide. a conjugate comprising the antibody or antigen-binding fragment thereof, the conjugate being shown in FIG. 1, in which the mAb has the structure of SEQ ID NO: 27 shown in any one of embodiments 1 to 9.

[0023] represents the monoclonal antibody or antigen-binding fragment thereof according to

[0024] , wherein 2 is an oxine 1 shows the tomodulin therapeutic peptide covalently conjugated to the mAb.

[0187] Embodiment 22 is an isolated monoclonal antibody according to any one of embodiments 15 to 21. a method for producing a human antibody or antigen-binding fragment thereof, the method comprising administering to a subject a therapeutic peptide The electrophile (preferably bromoacetamide or maleimide) introduced onto the side chain is The cysteine ​​residue of SEQ ID NO: 18 of the monoclonal antibody or antigen-binding fragment thereof The peptide reacts with the hydroxyl group, thereby forming a therapeutic peptide and a monoclonal antibody or its This involves forming a covalent bond between the antigen-binding fragment and the

[0188] Embodiment 23 is an isolated monoclonal antibody according to any one of embodiments 14 to 21. a pharmaceutical composition comprising a human antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier; is.

[0189] Embodiment 24 is a monoclonal antibody according to any one of embodiments 14 to 21, or A method for producing a pharmaceutical composition comprising the antigen-binding fragment thereof, the method comprising: a human clonal antibody or antigen-binding fragment thereof in combination with a pharmaceutically acceptable carrier, obtaining a pharmaceutical composition.

[0190] Embodiment 25 is a monoclonal antibody according to any one of embodiments 1 to 9 and 14 to 21. and a kit comprising a human antibody or antigen-binding fragment thereof.

[0191] Embodiment 26 is a method of extending the half-life of a therapeutic peptide in a subject, the method comprising: The therapeutic peptides were synthesized using the peptides of SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively. Heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, HCDR7, HCDR8, HCDR9, HCDR10, HCDR11, HCDR12, HCDR13, HCDR14, HCDR15, HCDR16, HCDR17, HCDR18, HCDR19, HCDR11, HCDR11, HC 3, and a single fragment containing light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 and conjugating the antibody to an isolated monoclonal antibody or antigen-binding fragment thereof. The therapeutic peptide is a monoclonal antibody or its antigen-binding fragment at the Cys residue of SEQ ID NO: 18. The antibody is conjugated to a binding fragment.

[0192] Embodiment 27 is a method for treating a rheumatoid arthritis, ... or rheumatoid arthritis, wherein the therapeutic peptide is oxyntomodulin, glucagon-like peptide 1, or hydroxybenzoates. (GLP1), peptide tyrosine tyrosine (PYY), exendin (exenatide), Amylin (pramlintide), α-melanocyte-stimulating hormone (MSH), cocaine and and amphetamine-regulated transcript (CART), neuropeptide Y receptor Y1 (NPY1) antagonist Drugs (antagnoists), neuropeptide Y receptor Y5 (NPY5) antagonists, neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), Galanin, cholecystokinin (CCK), orexin, melanin-concentrating hormone (MCH) oxytocin, and stresscopin, as described in embodiment 26. This is the method.

[0193] Embodiment 28 is the method of embodiment 27, wherein the therapeutic peptide is oxyntomodulin. This is the method.

[0194] Embodiment 29 is a method for treating oxyntomodulin comprising administering to a subject the method of claim 1, wherein the oxyntomodulin has the polypeptide sequence of SEQ ID NO: 24. 29. The method of claim 28.

[0195] Embodiment 30 is a method for producing a monoclonal antibody comprising administering to a subject a subject the ability to produce a polypeptide having the polypeptide sequence of SEQ ID NO: 12. 30. The method according to any one of embodiments 25 to 29, wherein the VH comprises a chain variable domain (VH). .

[0196] Embodiment 31 is a method for producing a monoclonal antibody comprising administering to a subject a subject the ability to produce a polypeptide having the polypeptide sequence of SEQ ID NO: 13. 31. The method according to any one of embodiments 25 to 30, comprising the chain (HC).

[0197] Embodiment 32 is a method for treating a cancer, wherein the monoclonal antibody is a light chain reaction product having the polypeptide sequence of SEQ ID NO: 14. 32. The method according to any one of embodiments 25 to 31, wherein the VL chain variable domain (VL) is .

[0198] Embodiment 33 is a method for treating a cancer, wherein the monoclonal antibody is a light antibody having the polypeptide sequence of SEQ ID NO: 15. 33. The method according to any one of embodiments 25 to 32, comprising a chain (LC).

[0199] Embodiment 34 is an isolated monoclonal antibody comprising a heavy chain complementarity determining region 3 comprising SEQ ID NO: 18. an isolated monoclonal antibody or an antigen-binding fragment thereof; The antigen-binding fragment is capable of binding to a therapeutic peptide. [Example]

[0200] Example 1: Identification and production of mAb MSCB97 PH9L3 VL and PH9H5 VH were selected as the starting V regions for engineering. The antibody light chain variable region (VL) designated PH9L3 (SEQ ID NO: 3) (Teplyako v et al., “Structural diversity in a huma n antibody germline library,” mAbs Aug-Se p 8(6):1045-63(2016)), and an antibody heavy chain potential designated PH9H5. Variable domain (VH) (SEQ ID NO: 4) (Teplyakov et al., "Structu ral diversity in a human antibody germli ne library,” mAbs Aug-Sep 8(6):1045~63(20 16)) as the starting variable region to engineer mAbs that allow for peptide conjugation. PH9L3 was selected because it contains the complete human Ig germline V gene sequence. Sequence differentiation by an in vivo affinity maturation process that can result in high affinity antigen-specific binding It contains no mutations. The CDR3 of PH9H5 contains a human germline V gene sequence in its VH. The CDR3 of PH9H5 (SEQ ID NO: 5) is the only segment that does not bind to the anti-human CCL 2 antibody, CNTO 888, a neutralizing CCL2 antibody, is identical to the CDR3 of the antibody, e.g., U.S. Pat. See Patent Application Publication No. 20100074886(A1) (CNTO 888 (The relevant disclosures of which are incorporated herein by reference in their entireties.) A Fab containing the L3 VH / VL pair was generated.

[0201] PH9L3, PH9H5, and the human Ig germline V and J regions to which they are most similar Sequences were aligned to determine sequence identity or similarity to the germline sequence. is the human Ig germline gene IGHV3-23 * 01(PubMed ID:M9966 0) (SEQ ID NO: 7) and human IGHJ1 * 01(PubMed ID:J00256)( The PH9H5 amino acid sequence was aligned to the concatenation of the PH9H5 amino acid sequence (SEQ ID NO: 8) and the concatenation of the PH9H5 amino acid sequence (SEQ ID NO: 6). Human IGHV3-23 * 01-IGHJ1 * The only difference between the VH CDR3 and 01 sequences is This became SEQ ID NO:5 of PH9H5.

[0202] PH9L3 is a human Ig germline gene, IGKV3-11 * 01(PubMed ID :X01668) (SEQ ID NO: 10) and IGKJ1 * 01(PubMed ID:J00 242) (SEQ ID NO: 11) to the concatenation (SEQ ID NO: 9), and the only difference is the V gene The only deviation was in the / J gene combination.

[0203] Design and generation of Cys-substituted mutants of PH9H5 and PH9L3 Contains single Cys substitutions at selected CDR residues across all three CDRs of the V regions Design and generate PH9H5 VH variants containing the complete human IgG1 constant region. The heavy chain was cloned into a mammalian host expression vector. The b structure was used to provide a substitution that would make it more accessible for conjugation. In some variants, an additional glycine (Gl y) residues on either side of the introduced Cys residue to provide a conjugate We designed a similar mutant of PH9L3 VL, potentially increasing the accessibility of Cys. However, these were cloned into an expression vector as complete light chains with a human kappa constant region. Twenty-four expression constructs of single Cys mutants of PH9H5 and PH9L3 were cloned. 22 expression constructs of single Cys mutants were generated. and the residues selected for substitution within PH9L3_VL (SEQ ID NO: 3) are summarized in Figure 2. .

[0204] The generated expression constructs were used to transfect each PH9H gene with the wild-type PH9L3 LC construct. By transient co-transfection of 5-based HC Cys mutant constructs, or each PH9L3-based LC Cys mutant with the wild-type PH9H5 HC construct. The Cys mutants were expressed by cotransfection of the constructs. Experimental transfections were performed using HEK-derived Expi293 as the expression host. The majority of both HC and LC Cys mutants were isolated from the culture supernatant. Based on the quantification of mutant proteins by et al., they were well expressed.

[0205] The five initial HC Cys mutants, MSCB33-MSCB37, were cultured in a 750 mL scale. The mutant proteins were expressed in Expi293 and purified. The yield and quality characteristics were very similar, and the purified There was enough protein to use.

[0206] Evaluation of peptide conjugates to PH9H5-based HC Cys mutants The analytical mass determination of MSCB33 protein and other mutant proteins was performed using 2 mAb per mAb. One shows the presence of a cysteine ​​adduct at the Cys engineered for conjugation. , as well as the HC C-terminal Lys residue commonly found in recombinantly produced mAbs To prepare mutant mAbs for conjugation, we investigated the removal of natural mAbs from the mAb. The adducts were removed by a reduction process developed to preserve the natural disulfide bonds. (See Example 3.) Human oxyntomodulin (OXM) peptide analogs (GCG A ib2, Gly16,24, Arg20, Leu27, Lys30(PEG 12 )-NH 2) Initial test conjugates with maleimides on all five HC Cys mutant mAbs Conjugation efficiency varied among the mAb variants, which The results are qualitatively estimated by the conjugated reaction products and their relative percentages. The highest efficiency (measured by the highest proportion of homodimer products) was achieved by flanking This was observed in MSCB33 compared to other I102C variants containing Gly residues. Little or no conjugation was observed with the 3C mutants MSCB35 or MSCB37. It wasn't done.

[0207] Various CDRs (T28C, S30C, and and S54C substitutions, and S30C and S9 in PHpL3_VL (SEQ ID NO: 128) Several other HC and LC Cys with engineered cysteine ​​substitutions in The mutants were expressed on a large scale in Expi293. Removal of Cys adducts from purified proteins is difficult, and these mutants were not pursued further. The most common issues observed with Cys variants were the I102C PH9H5 variant. The good initial conjugation efficiency observed with the antibody mAb, MSCB33, led to the conclusion that this particular Mutant-focused process development and further engineering efforts were undertaken.

[0208] Fc manipulation of MSCB33 MSCB33 silences human IgG4 to reduce Fc function in vivo. Human IgG4_PAA was re-engineered to contain human IgG4 antigen. The mutant G4m(a) has the mutations S228P / F234A / L235A (I IGHG4 defined by MGT * 01 allele). IgG4_PAA Fc An expression construct with the VH of MSCB33 fused to it was generated and used for MSCB33 expression. Used with the same LC expression construct to produce the IgG4_PAA variant of MSCB33 This is designated MSCB97. The amino acids of MSCB97 VH, HC, VL, and LC are The sequences are provided by SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0209] Test expression of MSCB97 was transiently expressed in Expi293 cells at a 20 mL scale. MSCB97 was used in large-scale experiments and this mAb variant was successfully expressed. The purified yield of MSCB97 was 264.53 mg / L. The quality was determined to be 85% monomer species. Subsequent larger-scale expression experiments and refinement The production yields and quality were similar or better, demonstrating the consistency with which this mAb can be produced. Ta.

[0210] Evaluation of peptide conjugates to MSCB97 and scalability of the conjugation reaction Ability The LC-HC disulfide bond differs between IgG1 and IgG4 isotypes; To test the reduced and maleimide conjugates, the OXM-maleimide test peptides described above were used. from IgG1 mAb MSCB33 using TCEP reduction and conjugation It was confirmed that IgG4_PAA mAb can be transferred to MSCB97. The bond resulting from the hydroxyl conjugate is known to be potentially reversible, however. Therefore, we adopted bromoacetamide conjugate chemistry, which provides a more stable bond. It was successfully carried out at a 10 mg scale based on development MSCB97.

[0211] OXM analogue GCG Aib2, Glu16 generated by bromoacetamide chemistry , 24, Arg20, Leu27, Lys30-ε-(PEG 12 )-NH2 MSCB 97 conjugate (Compound 2—"Compound 2" and "Conjugate 2" are used herein can be used interchangeably) (MSCB97 is H-Aib-QGTFTSDYS KYLDERRARDFVEWLLNTK-(COCH2CH2(OCH2CH2) 12 NHCOCHBr)-NH (SEQ ID NO: 24) to produce Compound 2 (Figure 11 , forming SEQ ID NO: 27) was assayed to detect GLP-1R and GCGR in vitro. The efficacy of the conjugates was determined. The potency was reasonable, and the conjugates made with the same peptide as MSCB33 (IgG1) This indicates that the isotype is MSCB97 (IgG4 The single difference between the PAA and MSCB33 (IgG1) is that the In addition, these data demonstrate that the efficacy of Bromoacetamide chemistry produces bonds with desirable in vitro potency and in vivo stability. We have shown that the peptide-mAb conjugates produced by other methods can retain the ATP. XM analogs were also conjugated to MSCB97, and the in vitro efficacy of these conjugates was These conjugates were shown to be effective against various peptides while retaining peptide potency. It has the ability to conjugate various peptides to MSCB97, and has the same GLP activity as compound 2. -1R and GCGR efficacy.

[0212] Evaluation of peptide-MSCB97 conjugates binding to human CCL2 MSCB97 was selected and engineered for its lack of specific antigen binding, and this mAb The antigen most likely to bind, if any, is human CC based on the origin of the VH CDR3. The two peptide-MSCB97 conjugates were then combined with the OXM peptide analog. (Compound 2), or a PYY peptide analog (Compound 1—"Compound 1" and "Conjugate "Iron 1" may be used interchangeably herein) to It was assessed whether the antibodies exhibited any specific CCL2 binding.

[0213] Potential CCL2 binding was measured directly by surface plasmon resonance (SPR), and this For surface plasmon resonance (SPR), the conjugate is surface-immobilized using an anti-Fc capture method. A commercially available anti-CCL2 mouse mAb was used as a positive control, and two nonspecific human antibodies, CNT O9412 and HH3B33 served as negative controls. All controls were surface-immobilized in the same manner. Recombinant human CCL2 was flowed over the immobilized conjugate and controlled at concentrations up to 400 nM. Based on pre-established assay criteria, CCL2 accumulation indicative of specific antigen binding is considered positive. This was observed in the control but not in the negative control, and neither peptide-MSCB97 conjugate This was not observed in the peptide-mAb conjugates (compounds 1 and 2). In a relevant therapeutic form of adjuvant, the lack of human CCL2 binding by MSCB97 Confirmed.

[0214] SPR binding method: The ProteOn XPR36 system (BioRad) was used to Binding measurements were performed using surface plasmon resonance (SPR). Amine coupling chemistry Anti-human IgG Fc (Jackson catalog) was used using the manufacturer's instructions. No. 109-005-098) and anti-mouse IgG Fc (Jackson Cat. No. 3 The mixture was transferred onto a GLC chip (BioRad, Cat. No. 17 The biosensor surface was then coupled to a modified alginate polymer layer (6-5011). Approximately 5700 RU (response units) of mAb were immobilized. Binding experiments were performed by running The assay was performed at 25°C in buffer (DPBS; 0.01% P20; 100 μg / mL BSA To perform binding kinetics experiments, samples (compound 2, compound 1, and control mAb positive) were The antibodies were then captured using recombinant human CCL2 (positive and negative) at five concentrations (four-fold serial dilutions). Thermo Scientific, catalog number RMCP120) was injected at 50 μL / min for 3 minutes. After monitoring the dissociation step, the buffer was run for 5 minutes (dissociation step). H3PO4 (Sigma, Cat. No. 7961) was injected twice for 18 seconds each time to coat the chip surface. The surface was regenerated.

[0215] The collected data was processed using ProteOn Manager software. First, the data were corrected for background using Interspot. Double reference subtraction of data was performed using buffer injection for the precipitate injection. Compound 2, Compound 1, and the positive control were analyzed for CCL. Pre-established assay criteria for determining specific detectable binding to 2 include: Concentrations requiring a dose-proportional response with a signal >10 RU, and a negative control response signal <10 RU Based on the assay criteria, the results for each sample were calculated as a dose to human CCL2. Response binding was reported as "yes" or "no."

[0216] Example 2: Expression and purification of mAb Fully human monoclonal antibodies (mAbs) are recombinantly expressed in mammalian expression hosts. and can be purified from cell culture supernatant using standard methods known in the art. For example, the light chain (LC) and heavy chain (HC) of a mAb (with appropriate secretion-enabling signals) can be cDNA sequences encoding the nucleotide sequences (each containing a nucleotide sequence) were extracted using standard molecular biology methods. cloning into separate mammalian expression vectors or into a single expression vector using The expression vectors used are pEE12.4, pcDNA™ 3.1 (+ ), or commercially available products such as pIRESpuro3, or any custom products with similar functions. In such vectors, transcription of the heavy and light chains of the mAb is carried out by It is driven by one of the known effective promoters, such as the hCMV-MIE promoter. Transfection-grade plasmid DNA is available from Qiagen Plasmid M Separate LC and HC expression constructs, or LC, can be generated using standard methods, such as the idi Kit. A single construct expressing both HC and HC is prepared.

[0217] Purified plasmid DNA was used in a Freestyle™ Max transfection assay. Prepared for transfection with lipid-based transfection reagents such as drugs (following the manufacturer's instructions) and then a standard The mAb LC and HC are expressed separately in a suitable mammalian expression host cell line. If the construct encodes the nucleotide sequence, the two constructs are transfected simultaneously. Before and after transfection, mammalian cells were cultured for maintenance or for mAb expression. The cells were cultured according to standard cell culture methods, so that the cell density was within the range for maintenance. The culture medium used, and subsequent other cell culture conditions, will depend on the specific mammalian host utilized. These parameters are typically determined by the supplier from whom the cell line was obtained. For example, CHO-S cells are grown in suspension. The cells were maintained in medium CHO Freestyle™ medium and maintained at 37°C and 8% CO2. Shake at 125 RPM in a humidified incubator until the cell concentration reaches 1.5-2.0 x 10 6 pieces / mL.

[0218] Cell culture supernatant from transiently transfected mammalian cells expressing mAb , harvested several days after transfection, clarified by centrifugation, and filtered. The duration of expression in -S cells is typically 4 days but can be adjusted and varied depending on the mammalian host. For mammalian host cell lines, this may vary. The mAb is concentrated 10-fold using a concentrator such as Centramate. Protein A affinity column (e.g., HiTrap MabSelect Sure) is used. The mAb was bound to Protein A resin, the resin was washed, and the mAb was then titrated using a low pH buffer. Proteins are purified from the clarified supernatant using standard methods for eluting proteins. The protein fraction was immediately neutralized by elution into a test tube containing pH 7 buffer. The peak fractions were pooled, filtered, and incubated overnight in phosphate-buffered saline (PBS), pH 7.2. After dialysis, the mAb was filtered again (0.2 μm filter) to obtain the protein. The concentration is measured by absorbance at 280 nm. The quality of the purified mAb protein is For SDS-PAGE (polyacrylamide gel electrophoresis) and analytical size-exclusion HPLC Endotoxin concentrations were assessed by Limulus amebocyte lysate (LAL). The purified mAb is stored at 4°C.

[0219] Expression and purification of MSCB97 from transiently transfected CHO cells MSCB97 was cultured in ExpiCHO-S™ cells (ThermoFisher Sc MSCs were cultured in a medium containing 100% MSCs (Centific, Waltham, MA, Cat. No. A29127). Purified plasmid DNA of the B97 expression construct was used to perform transient transfection according to the manufacturer's recommendations. Briefly, expression was achieved by transfection into ExpiCHO-S™ cells. Expiry was performed in a shaking incubator set at 37°C, 8% CO2 and 125 RPM. CHO™ Expression Medium (ThermoFisher Scientific, catalog The cells were maintained in suspension in PBS (product number A29100). On the day of administration, 6.0 x 10 per mL 6 The cells can be diluted to 98% Transient transfection was performed using ExpiFectam ine™ CHO Transfection Kit (ThermoFisher Science) Transfection was performed using a recombinant vector (Cat. No. A29131). Use 1 microgram of plasmid DNA for every 1 mL of diluted cells and OptiPR Dilute in O™ SFM complexed medium. ExpiFectamine™ CHO test The drugs were used in a 1:3 ratio (v / v, DNA:reagent), also using OptiPRO™ The diluted DNA and transfection reagent are combined and left for 1 minute. NA / lipid complex formation was allowed to occur and then added to the cells. After overnight incubation, Ex piCHO™ Feed and ExpiFectamine™ CHO Enhancer The cells were cultured at 32°C for 5 days with shaking, and the culture supernatant was collected.

[0220] Culture supernatant from transiently transfected ExpiCHO-S™ cells The solution was clarified by centrifugation (30 min, 6000 rpm) and then filtered (0.2 μm P First, Pall Centramate (ES membrane, Corning) was used for collection. Use the Tangential Flow Filtration system to For large scale transfections (5-20 liters), use 10x concentrated 10x Dalbedo. Add dichloromethane phosphate-buffered saline (DPBS, pH 7.2) to this supernatant to a final concentration of 1x. Then, using an AKTA FPLC chromatography system, Equilibrated (DPBS, pH 7.2) HiTrp at a relative concentration of approximately 20 mg of protein per 100 ml of solution. ap MabSelect Sure Protein A column (GE Healthcare ;Little Chalfont, United Kingdom). After passing the protein through the column, the column was washed with 10 column volumes of DPBS (pH 7.2). Proteins were eluted with 10 column volumes of 0.1 M sodium acetate (pH 3.5). The protein fraction was prepared by eluting 20% ​​of the fraction volume with 2.0 M tris(hydroxymethyl)aminomethyl. The solution was immediately neutralized by dissolving it in a test tube containing TRIS (pH 7). Peak fractions were pooled and, if necessary, the pH was adjusted to approximately 5.5 with additional TRIS. The purified protein was filtered (0.2 μm) and its concentration was measured using a BioTek Syner The absorbance was measured at 280 nm on a gyHT™ spectrophotometer. Protein quantity was determined by SDS-PAGE and analytical size exclusion HPLC (Dionex HP Endotoxin levels were assessed by LAL assay (Pyro tell®-T, Associates of Cape Cod Measured.

[0221] Example 3: Conjugates of mAb and Cyclic PYY Peptides Method A: Partial reduction of mAb with TCEP 10 mg / mL mAb solution in TRIS acetate buffer (20 mL, 1 mM in EDTA) The solution was treated with 3 equivalents of TCEP. The solution was adjusted to pH 6 and left at room temperature for 1 hour. Afterwards, high pressure liquid chromatography with mass spectrometry (LCMS) was used to identify the The disulfide adducts were shown to be completely reduced. The reduced mAb was then purified by Purify by protein A adsorption and elution (4 CV 100 mM acetic acid) to obtain 180 mg of reduced protein. I got Ab.

[0222] Conjugates of reduced mAb and cyclic PYY peptides Lyophilized peptide (5 equivalents relative to mAb) was added to the reduced mAb. TA was added to a final concentration of 1 mM and the pH was adjusted to 7. The concentration was adjusted to 8 mg / mL. The reaction was allowed to proceed for 16 hours at room temperature with gentle shaking. TCEP (0.01% relative to mAb) was added. 5 equivalents) was added and the reaction was allowed to proceed further with gentle shaking at room temperature for 4 hours, after which time After filtering, the high molecular weight (MW) species were reduced to less than 3%.

[0223] The reaction mixture was adjusted to pH 5.5 and subjected to ion exchange chromatography on CaptoSP resin. Purification was performed by (gradient: 100% A (100 mM TRIS acetate, pH 5.5) 100% B (100 mM TRIS acetate, pH 5.5, 0.5 M NaCl), 20 CV). Fractions containing the desired conjugate were pooled and purified along with a small amount of unreacted peptide. 140 mg of the eluted conjugate was collected. Final purification was performed by Protein A adsorption and Elution was performed with 4 CV 100 mM acetic acid. The pH of the product was adjusted to 6 and 120 mg of conjugate (60% yield) was obtained with a purity of >90% and <3% of high MW species.

[0224] Method B: Hydrophobic interaction chromatography (HIC) purification of mAb A 20 mg / mL solution of mAb in TRIS acetate buffer was applied to a hydrophobic interaction column (T The column was loaded onto an OSOH TSKgel Phenyl 7.5 x 21 cm column and eluted with a linear gradient ( 0-70% B / A, solvent A: 5% iPrOH, 1M (NH4)2SO4, 100mM Li (solvent B: 20% iPrOH, 100 mM phosphate buffer, pH 6.0; solvent B: 20% iPrOH, 100 mM phosphate buffer). The Ab monomer peak was pooled, concentrated (5–10 mg / mL), and purified by 3-(N-morpholino)- ) Dialyzed against propanesulfonic acid (MOPS) buffer (100 mM, pH 5.5) .

[0225] Partial reduction with TCEP and conjugation of reduced mAb with peptide analogues Purified mAb (27 mL, 9.28 mg / mL) was treated with 4 equivalents of TCEP, followed by EDT. A (1 mM) was added. After 2 hours at room temperature, LCMS showed the presence of a disulfide bond at position C102. The reduced mAb was then purified using a Zebra desalting spin column. (7 x 10 mL, 7K MWCO, pre-equilibrated with MOPS 100 mM pH 5.5) The combined fractions of reduced mAb (28 mL) were treated to remove free cysteine / GSH. ) to a solution of cyclic PYY peptide in Milli Q grade water (6.5 for mAb) Equivalent, 15-20 mg / mL), followed by the addition of EDTA (1 mM). The pH of the reaction was adjusted to 7.2-7.4 by dropwise addition of The reaction was allowed to proceed for 18 hours at room temperature with stirring. An additional 0.5 equivalents of TCEP was added, followed by further addition of 0.5 equivalents of TCEP. The reaction was continued for 12 hours to reduce the mAb-mAb dimers formed during the course of the reaction. The reaction mixture was converted to the desired mAb homodimer by adding 2M acetic acid. The pH was lowered to 5.5 and the crude conjugate was purified by hydrophobic interaction chromatography. Purification was performed by linear gradient elution (0–100% B / A, solvent A: 5% iPrO H, 1 M (NH4)2SO4, 100 mM phosphate buffer, pH 6.0; solvent B: 20% iPrOH, 100 mM phosphate buffer). Final purification was performed using Protein A adsorption (PBS) and The product was adjusted to pH 6 and dissolved in PBS. The final sample was obtained by dialysis against the sucrose (56%).

[0226] Alternatively, the mAb was reduced with GSH and / or Cys. Tangential flow filtration After removal of the reducing agent by TFF, optionally in the presence of 0.2-0.5 equivalents of TCEP At 100°C, an excess of peptide was added to the reduced mAb.

[0227] Example 4: In vitro testing Compound 1 (SEQ ID NO: 2) is a monoclonal antibody that binds to cyclic PYY peptides (Figure 3 ) to the human, rat, mouse, and rhesus monkey Y2 receptors, as well as human Y1, Y4, and In vitro, NP was expressed in clonal cells (HEK or CHO) expressing the Y5 receptor. PYY3-36, NPY, and PP were evaluated for their ability to activate Y receptors. was included in these assays as a test control.

[0228] cell line Stable transfectants expressing NPY receptors were prepared for use in cAMP assays. Briefly, HEK293 cell lines were cultured in Lipo The Infectamine 2000 kit (Invitrogen) was used to carry out the protocol. According to the protocol, human Y2 receptor (accession number: NM_000910.2), human Y 5 receptor (accession number: NM_006174.2), mouse Y2 receptor (accession Accession number: NM_008731), and rhesus monkey Y2 receptor (accession number An expression plasmid containing the coding sequence of :NM_001032832 was used to 48 hours after transfection, the cells were transferred to selective medium (DMEM High glucose, 10% fetal bovine serum (FBS), 50 IU penicillin, 50 μg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and After maintaining the cells in the selective medium for 2 weeks, the cells were reseeded in the medium containing 600 μg / mL G418. After that, single clones were picked using limiting dilution. , 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 1% penicillin DMEM high glucose supplemented with thrombin / streptomycin and 600 μg / mL G418 The cells were maintained by culturing in Cellgro medium.

[0229] In addition, human Y1 receptor (Cat. No. 93-0397C2) and human Y4 receptor (Cat. No. 93-0397C3) were also identified. The CHO-K1 cell line expressing the IgG1 gene (catalog number 95-0087C2) was DiscoverX cells were obtained from DiscoverX Corporation. Cultured in F12 medium (Gibco) supplemented with S under G418 selection (800 μg / mL). The rat Y2 receptor was obtained from Promega Corporation. The lo-Sensor was expressed in the CHO-K1 cell line. These cells were then transfected with the luminescent system cAM. Transduced with pGloSensor™-23F cAMP plasmid for P assay Transfected with IgG but not for use in the Perkin-Elmer LANCE cAMP assay Rat Y2 cells were cultured in 10% FBS and 800 μg / mL The cells were cultured in F12 medium (Gibco) supplemented with G418.

[0230] All cell lines were cultured in vials (4 x 10 cells). 6 Store in a cool, dry place (1 / vial) until ready to use. Store under nitrogen. The day before the assay, thaw a vial and add it to 15 mL of the appropriate medium. The cells were centrifuged at 450 x g for 5 minutes, the supernatant was aspirated, and the cells were diluted to 0.2 x 10 6 The cells were resuspended in medium without G418 at a density of 100 cells / mL. Dispense (25 μL / well) into a 384-well plate coated with PEG. The density was 5000 cells / well. The cell plate was maintained in a 5% CO2 / 90% O2 atmosphere. The plates were incubated overnight in a humidified tissue culture incubator at 37°C under 5% CO₂.

[0231] Experimental protocol The cAMP assay was the same as the various receptor assays. (Perkin Elmer Corporation; Waltham, MA) Intracellular cAMP levels were quantified in all experiments. The cells were decanted and 6 μL of peptide (2x concentration) was added to the wells. 11-point dose response (100 nM or 10 μM using 1:3 serial dilutions) in stimulation buffer The stimulation buffer was prepared as follows: HBSS (Hank's Balanced Salt Solution) containing 5 ml of M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 500 μM IBMX and 0.1% bovine serum albumin (BSA). follicular (2x, 5 μM final concentration) and LANCE cAMP antibody (1:100) After incubation at room temperature for 25 minutes, 6 μL of stimulation buffer containing 1 μL of ... 2 μL of assay detection mix was added to each well. The detection mix consisted of LANCE cAM Biotin-cAMP (1:750) and europium-W as provided in the P kit The plate was prepared by diluting 8044 (1:2250) with detection buffer. After incubation at room temperature for 2 hours, the plates were read using an Envision plate reader (excitation 320 nm). The TR-FRET assay was read at 615 nm and 665 nm (emission at 615 nm and 665 nm). Channel 1 fluorescence (relative fluorescence units at 615 nm) and channel 2 fluorescence (relative fluorescence units at 665 nm) units) along with their ratios were exported to an Excel file.

[0232] Data analysis Data from the Envision plate reader is (615nm / 665nm) x 1 The results were expressed as relative fluorescence units (RFU) calculated as 0,000. The in-house Crucabl designed by Eudean Shaw was used for the measurements. e Data were analyzed using data analysis software. The EC values ​​were interpolated from reference standards of known cAMP concentrations included on each plate. 50 , Log(EC 50 ), HillSlope(nH), maximum value, minimum value, etc. is a Non-Clinical Statistics & The results were obtained using a nonlinear weighted least squares algorithm applied within the R environment, performed by the Computing Department. The cAMP concentration values ​​were plotted against the log compound concentration fitted with the 4-P model using the obtained by downloading (open source http: / / cran.us.rp project.org / ).

[0233] [Table 1]

[0234] Example 5: Pharmacokinetics (PK) DIO Mouse PK Male DIO C57BL / 6N mice (20 weeks old, 14 weeks on a high-fat diet) were cultured at Taconi c Obtained from the Laboratory. Mice were fitted with AlphaDri bedding. House one animal per cage and place in a temperature-controlled room with a 12-hour light-dark cycle. Mice were allowed free access to water and maintained on a high-fat diet (D12492, Research The subjects were maintained on a rhesus diet.

[0235] Mice were administered 1 mg / kg of Compound 1 subcutaneously (sc), and three mice were sacrificed at each time point. Blood was collected at t = 4, 8, 24, 48, 72, 120, and 168 hours. Blood was also collected from three animals. After decapitation under gas anesthesia, approximately 300 μL of blood was collected from each animal via the jugular vein. The blood sample (approximately 300 μL) was diluted with 12 μL (4% concentration) of total protease inhibitor solution. and 3 μL (1% ratio) of DPP-IV inhibitor containing K3E(EDTA) coating. Blood was collected in a Sarstedt Microvette® tube. Samples were placed on ice and cooled to remove cells within 30 minutes of collection at each time point. Centrifuge at 10,000 rpm for approximately 4 minutes under storage conditions (approximately 5°C) to remove all available blood. The plasma was transferred to a 96-well plate. The well plate was kept in a -80°C freezer until The data are shown in Table 2 and Figure 4.

[0236] Rat PK Compound 1 was administered to male Sprague-Dawley rats (Charles River r Laboratories, Wilmington, MA) in PBS The solution was administered subcutaneously and intravenously at a dose level of approximately 500 μL / kg (pH 7.0-7.6). Blood was collected from the saphenous vein from three animals per time point (t=1, 4 post-dose). , 24, 48, 72, 96, 168, and 240 hours). Blood samples taken 336 hours after administration Samples were decapitated under gas anesthesia induced by a mixture of 70% CO2 and 30% O2. After the injection, blood samples were collected from the jugular vein. 20 μL of total protease (4%) was added to the blood. Inhibitor solution and 5 μL (1% ratio) of DPPIV inhibitor in K3E(EDTA) Collect the sample in a coated Sarstedt Microvette® tube. Blood samples were placed on ice and collected within 60 minutes at each time point to remove cells. To obtain the desired product, centrifuge at 10,000 rpm for approximately 4 minutes under refrigerated conditions (approximately 5°C) and use the All plasma was transferred to a 96-well plate. The concentration of Compound 1 was determined using the LCMS method described below. The data are shown in Table 3.

[0237] Cynomolgus monkey (Cyno)PK All animals were fasted for at least 8 hours before dosing, with blood samples taken during the first 4 hours. Three animals received a single IV dose of 1 mg / kg of Compound 1, and three animals received a single IV dose of 1 mg / kg of Compound 1. Received a single SC dose of Compound 1 at 1 mg / kg pre-dose and at 1, 6, 10, 24, and 30 days post-dose. Blood after 36, 48, 72, 120, 168, 240, 336, 432, and 504 hours An additional sample was taken 0.5 hours after administration of the IV group. Approximately 1 hour was taken from each animal. mL of blood was collected in a 4% total protease inhibitor solution and a 1% DPPIV inhibitor solution. K3E (EDTA) coated Sarstedt Microvett Blood samples were collected in ® tubes on ice and then taken at each time point. Within 30 minutes of centrifugation, the resulting plasma was divided into three portions and plated in triplicate on a 96-well plate. The well plate was kept on dry ice until it was placed in a -80°C freezer. The data are shown in Table 4 and Figure 5.

[0238] Fully quantitative assay for plasma level measurement Plasma samples were processed by immunoaffinity capture using anti-human Fc antibodies and then analyzed in triplicate. Reversed-phase LC high-resolution full-scan MS analysis was performed on a TOF (time-of-flight) mass spectrometer. MS spectra were deconvoluted to resolve the molecular weights of the components in the injected sample. The molecular ion peak of the intact conjugate was used for quantification. Standard curve and quality control Samples were prepared by spiking the reference standard in plasma and were run simultaneously with the applied samples. The PK data for DIO mice, rats, and cynomolgus monkeys were processed using the same procedure. The PK data for DIO mice and cynomolgus monkeys are shown in Figures 4 and 5, respectively. and 5.

[0239] [Table 2]

[0240] [Table 3]

[0241] [Table 4]

[0242] Example 6: In vivo efficacy testing Weight loss in diet-induced obese (DIO) mice: acute administration Compound 1 reduced food intake and body weight in male DIO C57B1 / 6 mice after a single administration. Male DIO C57BL / 6N mice (20 weeks old, high-fat diet) were evaluated for their ability to induce serotonin production. Mice were obtained from Taconic Laboratory. They were housed one per cage with aDri bedding and had a 12-hour light-dark cycle. The mice were placed in a temperature-controlled room. They were allowed free access to water and high-fat diet. Before the start of the experiment, the animals were maintained on a diet (D12492, Research Diet). The animals were allowed to acclimate to the facility for at least one week.

[0243] The day before dosing, mice were grouped into cohorts of 8 animals based on individual body weight. The next day, between 3:00 and 4:00 pm, the animals were weighed and administered the vehicle via subcutaneous (sc) administration. (dPBS, pH 7.2) and treated with 0.1, 0.3, 1.0, 3.0, or 7.5 nm Treatment with Compound 1 at a dose of 0.05 nmol / kg or dulaglutide at a dose of 0.3 nmol / kg Body weight and food intake were measured 24, 48, and 72 hours after administration. The percentage of body weight loss and reduction in food intake were calculated. Statistical analysis was performed using the Tew method in Prism. Analysis was performed using a two-way repeated measures analysis of variance with Key's post-hoc test. All data are shown as mean±SEM (FIGS. 6 and 7).

[0244] Weight loss in diet-induced obese mice: chronic administration Compound 1 was administered repeatedly to male DIO C57B1 / 6 mice over an 8-day period, and the mice were administered the compound in the diet. The ability to reduce intake and body weight and improve glucose homeostasis was assessed. Male DIO C57BL / 6N mice (20 weeks old, 14 weeks on a high-fat diet) were cultured in Taconi c Obtained from the Laboratory. Mice were fitted with AlphaDri bedding. House one animal per cage and place in a temperature-controlled room with a 12-hour light-dark cycle. Mice were allowed free access to water and maintained on a high-fat diet (D12492, Research Before the start of the experiment, the animals were allowed to acclimate to the facility for at least one week. Ta.

[0245] The day before administration, mice were grouped based on their individual body weight. Animals were weighed and food intake was assessed between 3:00-4:00 pm. Animals were administered subcutaneous The subjects were administered 0.3 nmol / kg of vehicle (dPBS, pH 7.2) or dulaglutide. or at doses of 0.1, 0.3, 1.0, or 3.0 nmol / kg administered subcutaneously every 3 days After 8 days, the mice were fasted for 5 hours and then treated with 2 g / kg of Compound 1 at t=0. After glucose loading, a glucose bolus of 1000 mg / kg was orally administered. Blood glucose was measured at t = 0, 30, and 90 minutes, and plasma insulin was measured at t = 0, 30, and 90 minutes. Blood samples were collected to measure the serotonin level. Statistical analysis was performed using Tukey's post-hoc test in Prism. All data were analyzed using one-way analysis of variance or two-way repeated measures analysis of variance. Data are presented as mean±SEM (FIGS. 8 and 9 and Tables 5 to 8).

[0246] [Table 5] Each value represents the mean ± SEM for data from 8 animals per time point per group. * p<0.05 vs. vehicle; two-way repeated measures ANOVA, Tukey's multiple comparison test

[0247] [Table 6] Each value represents the mean ± SEM for data from 8 animals per time point per group. * p<0.05 vs. vehicle; for blood glucose levels, two-way ANOVA repeated measures, Tew's Tukey's multiple comparison test; for AUC, one-way ANOVA, Tukey's multiple comparison test

[0248] [Table 7] Each value represents the mean ± SEM for data from 8 animals per time point per group. * p<0.05 vs. vehicle; for blood glucose levels, two-way ANOVA repeated measures, Tew's Tukey's multiple comparison test; for AUC, one-way ANOVA, Tukey's multiple comparison test

[0249] [Table 8] Each value represents the mean ± SEM for data from 8 animals per time point per group. * p<0.05 vs. vehicle; one-way ANOVA, Tukey's multiple comparison test

[0250] Example 7: Synthetic Strategy for the Preparation of mAb-Oxyntomodulin (mAb-OXM) Compounds Human oxyntomodulin (OXM) has beneficial pharmacology in patients with type 2 diabetes mellitus. It is a 37 amino acid long endogenous peptide (SEQ ID NO: 23) that has been shown to possess The therapeutic effects are the result of agonist action at both GLP1R and GCGR. Studies of analogs have demonstrated good glycemic control and weight loss in rodent models. The half-life of OXM in vivo is only a few minutes in humans. Therefore, further design of OXM will provide a half-life suitable for once-weekly administration. The increased half-life is believed to be a measure of the stability of the OXM peptide against proteolysis. by increasing the peptide size and by covalent attachment of monoclonal antibodies (mAbs) This was achieved by increasing the circulating half-life of the peptide-loaded TA-101 by DPP4. Protein degradation is reduced by replacing the serine at position 2 with aminoisobutyric acid (Aib). The helical topology of the peptide was determined by the branched chains of Q20R~S16E and Q24E. The introduction of a bridge stabilizes the enzyme, and the M27L mutation reduces its oxidative potential. The parent mAb, MSCB97 (described above), was designed to have low intrinsic antigen binding and Isoleucine to cysteine ​​in the HCDR3 (SEQ ID NO: 18) region of the nucleotide sequence of the amino acid ... The nucleotide sequence was selected to have a point mutation in the nucleotide sequence (which acts as an attachment point for the synthetic peptide loading). Effector functions were supported by using the IgG4 PAA isotype. A short oligoethylene glycol spacer was inserted between the mAb and the peptide. Intercalated to allow unhindered access of peptides to GLP1R and GCGR This spacer also provides good water solubility, facilitating conjugation chemistry. The binding of the peptide spacer to the mAb is achieved by reacting with the distal end of the glycol spacer. This was achieved by introducing a reactive bromoacetamide group. The OXM peptide was attached to the OXM variant via the side chain of 30. The amino acid sequences of the conjugates are provided by SEQ ID NOs: 22 and 24, respectively. was achieved by reaction with thiol functional groups of cysteine ​​point mutations in the mAb heavy chain. The amino acid sequences of the mAb heavy and light chains are provided by SEQ ID NOs: 13 and 15, respectively. do.

[0251] Chemical synthesis of mAb-OXM compounds The overall synthetic scheme for the production of mAb-OXM compounds is shown in FIG.

[0252] Preparation of oxyntomodulin (OXM) peptide variants: Resin-bound protected peptides were prepared using the target Typical 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids (N-terminal His, Nα-Boc-His(Boc)-OH and Lys30, Nα-Fmoc Lys(iv The PEG-1000-DDE (except for DDE-OH) was synthesized on PAL-PEG-polystyrene resin. Standard amino acid activation and Fmoc deprotection strategies were used throughout. After that, it is treated with dilute anhydrous hydrazine in N,N-dimethylformamide (DMF). The C-terminal lysine side chain was selectively deprotected by Fmoc-dPEG. 12 -CO2H Diisopropylcarbodiimide (DIC) / ethyl(hydroxyimino)-cyanoacetate Acetate activation was used to couple to the free amine. The Fmoc group was removed and the resulting amide The amine was bromoacetylated using a solution of bromoacetic anhydride in DMF.

[0253] A triflate containing phenol, water, and triisopropylsilane as a scavenger All protecting groups were simultaneously removed by treatment with trifluoroacetic acid (TFA) to give the phenyl group. The peptide was removed from the resin. The crude peptide was isolated by cold precipitation with ether and eluted. Reversed-phase high-performance liquid chromatography was performed using acetonitrile / water with 0.1% v / v TFA as the solvent. The peptide was purified by RP-HPLC. After lyophilization, the pure peptide was obtained. It was recovered as a fluffy white solid and stored at -80°C.

[0254] Reduction of monoclonal antibody MSCB97: The monoclonal antibody is a cytosolic or proliferation Disulfides on adventitious cysteine ​​or glutathione residues recovered from either the medium The engineered Cys102 residue is isolated and linked to these disulfide bonds. Preliminary reduction of the oxidase followed by removal of the unwanted cysteine ​​residues yields the OXM peptide variants. Reduction is required prior to conjugation of Protein A immobilized on resin beads. This was achieved by using a mAb containing a reducing agent (tricarboxyethylphosphine, TCEP). The column was circulated at pH 5 until reduction was complete (1 h). The advantage of TCEP as a reducing agent is that it is effective in reducing disulfide bonds at low pH. After washing away the by-products, the reaction mixture was dissolved in an acidic buffer (pH 3.5 The adsorbed proteins were eluted from the column using 1000 mg of sodium acetate. in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 5.5. The patient was dialyzed twice.

[0255] The mAb was reduced in a pH 5 solution using 2.5 equivalents of TCEP. A smaller exploratory test batch was prepared using the same procedure. The reduction reaction was carried out at room temperature for 2 hours. Small molecular by-products were removed by filtration (PD10 column), and the reduced MSCB97 was diluted to 10 Elution was performed with 10 mM MOPS, pH 5.5.

[0256] Preparation of mAb-OXM compounds: A solution of reduced MSCB97 was added to the lyophilized OXM peptide derivative. A 7.6-fold excess of the isomer was added. 1 mM EDTA solution was added to convert the reactive thiols to gold. The reaction was prevented by adding MOPS buffer (1 M, pH 8.1) to protect the enzyme from oxidation. The pH of the reaction solution was raised to 7.3. The reaction was allowed to react at room temperature with gentle shaking for 18 hours. The reaction was allowed to proceed. The reaction was quenched by adjusting the pH to 5.5 with the addition of 2M acetic acid. The crude conjugate was adsorbed onto protein A to remove excess peptide, unfolded peptide, and The crude product and by-products were removed by washing, and the product was purified by elution (sodium acetate). The mixture was dialyzed against acetate buffer (pH 5.6) and then acetate buffer (pH 5.6).

[0257] Starting with 250 mg, 500 mg, and 500 mg of MSCB97, three independent studies were performed. Synthesis was carried out and the products were combined into a single batch.

[0258] Analysis of mAb-OXM compounds The prepared mAb-OXM compounds were characterized using (i) analytical hydrophobic interaction chromatography (AHCI) (ii) liquid chromatography electrospray ionization mass spectrometry (HIC); (iii) analytical size exclusion chromatography (LC-ESIMS) for intact mass determination (iv) SDS-polyacrylamide gel electrophoresis. and analyzed.

[0259] Stability in human and monkey plasma OXM is rapidly metabolized by plasma peptidases. Enzymatic degradation in human and monkey plasma. To determine the resistance of the mAb-OXM compound ("Compound 2") (Figure 11), The ex vivo stability of Compound 2 was evaluated. Compound 2 was administered at 20 mM in fresh (not frozen) The samples were incubated with heparinized (non-heparinized) plasma at 37°C for up to 168 hours. Analysis of cAMP production in human GLP-1 receptor-transfected HEK cells This was done using a functional cell-based bioassay to measure the amount of cAMP produced. The concentration of active OXM analogue in the stability sample was directly proportional to the concentration of the OXM analogue. The concentration was determined by interpolation from a reference standard. The results of the more stable control (Control 1) and the less stable control (Control 2) previously determined The data was used to rank the relative stability, which is shown in Figures 12 and 13. Shown in Figure 13.

[0260] Example 8: In vitro studies: GLP1 and GLP2 in humans, mice, rats, and cynomolgus monkeys In vitro potency of Compound 2 at the glucagon receptor. The potency and species specificity of the target compounds, including Compound 2, were evaluated in humans, cynomolgus monkeys, rats, or mice. Mouse cells were transfected to stably express either the GLP1 or GCG receptor. The receptors were characterized in assays using HEK293 cells. The cloned cells were cultured in 384-well plates at an appropriate density of 2000–5000 cells / well. Plates were seeded with 5 mM HEPES, 0.1% BSA, and 0.5 mM IBMX. The compounds were diluted in HBSS supplemented with 100% ethanol and added to the cells. Incubate at room temperature for 5 or 10 minutes depending on the sample, and then lyse for cAMP measurement. cAMP concentrations were measured using a LANCE cAMP plate reader with an EnVision plate reader. A standard curve was prepared for each assay plate for back-calculation of cAMP concentrations. Dose-response curves were analyzed to determine the compound EC 50 Graphpad Pri Calculated using sm or Crucible. EC 50 The data are summarized in Tables 9-10.

[0261] [Table 9] Values ​​represent the mean ± SEM of 3–7 experiments.

[0262] [Table 10] Values ​​represent the mean ± SEM of 3–8 experiments.

[0263] Example 9: Efficacy of other related GPCRs The potency of compound 2 in assays using cells expressing several class B GPCRs Compound 2 was evaluated in CALCR, PTHR1, PTHR2, CRHR1, and CRHR2. 2, and VIPR1-expressing cells were used to perform six in vitro cAMP assays. The assay was performed in accordance with the manufacturer's instructions, with positive controls included for each receptor tested. Standard operating procedures were followed.

[0264] In addition, compound 2 was tested in a stable cell line expressing the human GIP receptor. Cells were seeded into 84-well plates and treated for 24 hours, followed by compound treatment for 5 minutes at room temperature. AMP concentrations were measured using the CisBio HTRF cAMP kit, and the results were compared with those of the Gra Analysis was performed using phPad Prism, and the results are shown in Tables 11-12.

[0265] [Table 11]

[0266] [Table 12]

[0267] Example 10: In vivo studies: Single dose efficacy in DIO mice Food intake, body weight, glucose tolerance, and plasma FGF21 levels were measured in DIO mice after single The results were evaluated after administration of Compound 2 or the GLP1R agonist dulaglutide at doses of 100 mg / kg / day. One day before administration, the animals were weighed and grouped according to their BW. The next morning, food was removed and a 6-hour fast was initiated. The weight of the body (FW) was recorded. Fasting blood glucose was measured and insulin was measured at 12:00 pm. After 1 hour, the mice were given glucose (1 g / kg, 20% glucose, 5mL / kg) was intraperitoneally administered. Plasma insulin was measured by tail blood sampling 10 minutes later. An additional 20 μL of blood was drawn at 10, 30, 60, and 90 minutes after glucose loading. Afterwards, blood glucose levels were measured using a blood glucose meter. Then, the mice were euthanized with CO2, and the final blood Samples were collected by cardiac puncture and the data are shown in Tables 13-19.

[0268] [Table 13] * p<0.01 vs. vehicle (18 hours after administration); 24 hours before each administration p<0.001; # Dulaglutide (0.3nmol / kg) 18 hours after administration p<0.0001 Two-way analysis of variance, Sidak's multiple comparison test Values ​​are from 8 animals per group at each time point, except for 24 hours before vehicle administration (n=7). Data represent the mean ± SEM

[0269] [Table 14] * p<0.01 vs. vehicle; p vs. dulaglutide (0.3 nmol / kg) <0.05 Two-way repeated measures ANOVA, Tukey's multiple comparison test. Data from the 18-hour time point. Show The percent weight change is calculated relative to the weight on day 0 (pre-dose). Each value represents the mean ± SEM per group for data from 8 animals.

[0270] [Table 15] Each value represents the mean ± SEM for data from 8 animals per time point per group.

[0271] [Table 16] * p<0.05 vs. vehicle; dulaglutide (0.3 nmol / kg) and JNJ p<0.05 vs. -64151789 (4.0 and 8.0 nmol / kg) One-way analysis of variance, Tukey's multiple comparison test Each value represents the mean ± SEM per group for data from 8 animals.

[0272] [Table 17] * p<0.05 vs. vehicle; p vs. dulaglutide (0.3 nmol / kg) <0.05 One-way analysis of variance, Tukey's multiple comparison test Each value represents the mean ± SEM per group for data from 8 animals.

[0273] [Table 18] Each value represents the mean ± SEM per group for data from 8 animals.

[0274] [Table 19] * p<0.0001 for all groups; all except compound 2 (2.0 nmol / kg) p<0.05 for all groups One-way analysis of variance, Tukey's multiple comparison test Each value represents the mean ± SEM per group for data from 8 animals.

[0275] Example 11: In vivo study: Repeated administration in DIO mice GLP1R / GCGR dual agonist (compound 2), and GLP1R agonist (dual The effects of raglutide in DIO mice were monitored during repeated administration over a 9-day period. To control the frequency of administration, all animals were dosed daily. The results were determined based on the PK data of dulaglutide and dulaglutide in DIO mice. Animals received the drug daily. Vehicle-treated animals received the vehicle daily. Compound 2 Animals receiving 1.0, 2.0, or 4.0 nmol / kg of this compound were given every 3 days. On days when no compound was injected, the vehicle was administered. Between 1 and 3 pm, the animals were given subcutaneous injections according to instructions. In these treatment groups, food intake and Body weight was monitored daily.

[0276] The first dose was 24 hours after the first administration, and the three groups of mice were divided into 1.0, 2.0, or 4. Pair-fed mice were pair-fed (PF) with 0 nmol / kg Compound 2-treated animals. The amount of food in the hopper was determined by the amount of food previously consumed by mice in the matched compound treatment group. All PF groups were treated with the vehicle and adjusted to match the average food consumed in a 24-hour period. On day 9 (or day 10 for the pair-fed group), the animals were dosed daily for 6 h. The rats were fasted, and their body weight and fasting blood glucose were measured. Measurements of insulin, FGF21, and plasma lipids were also performed. Blood samples were taken to measure food intake, body weight, body composition, fasting blood glucose, and fasting insulin. , fasting FGF21, and fasting plasma lipid data are shown in Tables 20 to 26.

[0277] [Table 20] * p<0.05 vs. vehicle; p vs. dulaglutide (0.3 nmol / kg) <0.05; #p<0.05 vs. Compound 2 (4.0 nmol / kg) Two-way repeated measures analysis of variance, Tukey's multiple comparison test All PF groups were given the average food intake from each treatment group (data not shown). was made. Each value represents the mean ± SEM for data from 6–10 animals per time point and group. vinegar.

[0278] [Table 21] T: treatment; V: solvent; 1: dulaglutide (0.3 nmol / kg); 2-3: compounds 2(1.0nmol / kg);4~5: Compound 2(2.0nmol / kg);6~7: Chemical Compound 2 (4.0nmol / kg) * p<0.01 vs. all other treatments; dulaglutide (0.3 nmol / kg) p<0.05 for each PF group; $JNJ-6415 p<0.05 vs. 1789 (1.0 and 2.0 nmol / kg); &JNJ-641 p<0.05 vs. 51789 (2.0 nmol / kg) Two-way ANOVA repeated measures, Tukey's multiple comparison test. The % change in body weight is calculated relative to the body weight on day 0 (pre-dose). Each value represents the mean ± SEM for data from 10 animals per time point per group.

[0279] [Table 22] * p<0.05 vs. vehicle One-way analysis of variance, Tukey's multiple comparison test Each value represents the mean ± SEM per group for data from 5 animals.

[0280] [Table 23] *p<0.05 vs. vehicle; p vs. dulaglutide (0.3 nmol / kg) <0.05; p<0 vs. #JNJ-64151789 (4.0 nmol / kg) PF .05 One-way analysis of variance, Tukey's multiple comparison test Each value represents the mean ± SEM per group for data from 10 animals.

[0281] [Table 24] * p<0.05 vs. vehicle; p<0.05 vs. Compound 2 (2.0 nmol / kg) PF 0.05 One-way analysis of variance, Tukey's multiple comparison test Each value represents the mean ± SEM per group for data from 10 animals.

[0282] [Table 25] * p<0.05 for all groups; ^Compound 2 (4.0 nmol / kg) PF p<0.05 One-way analysis of variance, Tukey's multiple comparison test Values ​​represent the mean ± SEM of data from 10 animals per group, except for Compound 2 (1 0.0 nmol / kg) and Compound 2 (4.0 nmol / kg) PF, N=9

[0283] [Table 26] * p<0.05 vs. Compound 2 (4.0 nmol / kg) PF; ^ vs. all groups p<0.05; #Compound 2 (1.0 nmol / kg) and Compound 2 (2.0 nmol / kg) g) p<0.05 for PF; all except duraglutide (0.3 nmol / kg) p<0.05 compared to the group One-way analysis of variance, Tukey's multiple comparison test Values ​​are from 10 animals per group for free fatty acids and 10 for free glycerol. Data from 8-10 animals per group, triglycerides and total cholesterol Figures represent the mean ± SEM of data from 9-10 animals per group.

[0284] Example 12: In vitro testing: Single dose efficacy in cynomolgus monkeys Biologically intact cynomolgus monkeys were monitored daily for food intake for 3 weeks prior to treatment. At the start of treatment, the animals were divided into four groups with similar mean body weights (n = 8-9, each group had a mean body weight of 1.5 kg / m²). The animals in each group received 1, 3, 5, or 7.5 nmol / kg. They received a single subcutaneous dose corresponding to 1000 mg of Compound 2. Food consumption was monitored daily for an additional 14 days. Body weight was measured on the day of administration and 4, 7, 10, 14 and 21 days after administration. Percent change in food intake was calculated by dividing the daily food intake by the average daily food intake over the 7 days prior to dosing. The results are shown in Figures 14 and 15.

[0285] Example 13: Pharmacokinetic (PK), Pharmacokinetic (PK) / Pharmacodynamic (PD) Analysis DIO Mouse PK The time course of Compound 2 was evaluated in DIO mice (Table 27). Animals (n=3 per time point) 10 nmol / kg was administered subcutaneously.

[0286] [Table 27] Each value represents the mean ± SEM for data from three animals per time point per group.

[0287] Rat PK The pharmacokinetic parameters of Compound 2 were evaluated in Sprague-Dawley rats. For determination of pharmacokinetic parameters (Table 28), the doses were measured at 0, 24, 48, 72, and 144 h after administration. Bioassays were performed on samples taken at 216, 312, 408, and 504 hours. Measured exposure was used.

[0288] [Table 28] Data are mean + / - standard deviation, exposure bioassay

[0289] Cynomolgus monkey PK The pharmacokinetic parameters of Compound 2 were evaluated in cynomolgus monkeys. The doses were administered intravenously or subcutaneously at 1 / kg. For the determination of pharmacokinetic parameters (Table 29), 0, 1, 6, 24, 48, 72, 120, 240, 336, 432, and 528 days after administration Bioassay-measured exposure was used from samples taken after hours.

[0290] [Table 29] Values ​​represent the mean ± standard deviation

[0291] Those skilled in the art may make changes to the above-described embodiments without departing from the broad inventive concept. It should be understood, therefore, that the invention is not limited to the particular embodiments disclosed. and modifications within the spirit and scope of the invention as defined herein. It is understood as intended.

[0292] All documents cited are incorporated herein by reference.

Claims

1. An isolated non-targeting antibody or antigen-binding fragment thereof, comprising: a light chain variable region (VL) having a completely human Ig germline V gene sequence; and a heavy chain variable region (VH) having a completely human Ig germline V gene sequence except for the heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO:

18.

1. An isolated non-targeting antibody or antigen-binding fragment thereof, comprising:

2. An isolated non-targeting antibody or antigen-binding fragment thereof described in claim 1, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively, and the light chain variable region comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively.

3. An isolated non-targeting antibody or antigen-binding fragment thereof described in claim 2, wherein the heavy chain variable region (VH) comprises the amino acid sequence of SEQ ID NO: 12 and the light chain variable region (VL) comprises the amino acid sequence of SEQ ID NO:

14.

4. An isolated non-targeting antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, further comprising an Fc portion.

5. An isolated nucleic acid encoding an isolated non-targeting antibody or antigen-binding fragment thereof described in any one of claims 1 to 4.

6. A vector comprising the isolated nucleic acid described in claim 5.

7. A host cell comprising the vector described in claim 6.

8. A method for producing an isolated non-targeting antibody or antigen-binding fragment thereof, comprising: The method comprises culturing the host cell of claim 7 under conditions to produce the non-targeting antibody or antigen-binding fragment thereof, and recovering the non-targeting antibody or antigen-binding fragment thereof from the cell or culture. A method comprising:

9. A conjugate comprising an isolated non-targeting antibody or antigen-binding fragment thereof described in any one of claims 1 to 4 and at least one pharmacologically active moiety conjugated thereto.

10. The conjugate of claim 9, wherein the pharmacologically active moiety is a therapeutic peptide.

11. The conjugate of claim 10, wherein the therapeutic peptide is conjugated to the non-targeting antibody or its antigen-binding fragment at a cysteine ​​residue of SEQ ID NO:

18.

12. A conjugate described in claim 10 or 11, wherein the therapeutic peptide is conjugated to the non-targeting antibody or its antigen-binding fragment via a linker.

13. The conjugate of claim 12, wherein the linker comprises a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, or a hybrid linker consisting of PEG and a heterocycle incorporated into the PEG.

14. The conjugate of any one of claims 10 to 13, wherein the therapeutic peptide is selected from the group consisting of oxyntomodulin, glucagon-like peptide 1 (GLP1), exendin, amylin, alpha-melanocyte-stimulating hormone (MSH), cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y receptor Y1 (NPY1) antagonist, neuropeptide Y receptor Y5 (NPY5) antagonist, neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin, melanin-concentrating hormone (MCH), oxytocin, and stresscopin.

15. The conjugate described in claim 14, wherein the therapeutic peptide is oxyntomodulin having the polypeptide sequence of SEQ ID NO:

24.

16. A pharmaceutical composition comprising the conjugate described in any one of claims 9 to 15 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition for use in a method for reducing food intake in a subject in need thereof, comprising: A pharmaceutical composition comprising the conjugate of claim 15 and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition for use in a method for treating a disease or disorder in a subject in need thereof, comprising:

16. A pharmaceutical composition comprising the conjugate of claim 15 and a pharmaceutically acceptable carrier, The disease or disorder may be obesity, type 2 diabetes, metabolic syndrome, insulin resistance, or the like. Resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, dyslipidemia, Atherosclerosis, diabetic nephropathy, hypertension, non-alcoholic fatty liver disease ( 1. A pharmaceutical composition comprising a pharmaceutical composition selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

19. The pharmaceutical composition described in claim 17 or 18, wherein the pharmaceutical composition is administered in combination with at least one additional therapeutic agent.

20. The pharmaceutical composition described in claim 17 or 18, wherein the pharmaceutical composition is administered in combination with liraglutide or dulaglutide.

21. The pharmaceutical composition described in claim 17 or 18, wherein the pharmaceutical composition is a solid freeze-dried composition or a solid spray-dried composition.

22. The pharmaceutical composition of claim 21, wherein the solid composition is selected from tablets, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

23. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is a compressed tablet, a coated tablet, or a capsule.

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