EA041709B1 - IMMUNOGLOBULINS AND THEIR USE - Google Patents

Description

Scope of the invention

The present invention pertains essentially to a novel antibody or fragments thereof and their use as carriers that bind to a therapeutic peptide to increase the in vivo half-life of the therapeutic peptide. The invention also relates to pharmaceutical compositions and methods for their use.

Cross-references to related applications

This application claims priority over U.S. Provisional Application No. 62/413,613, filed October 27, 2016, and U.S. Provisional Application No. 62/413,586, filed October 27, 2016. Each description is incorporated herein by reference in its entirety.

Link to sequence listing filed electronically

This application contains a sequence listing electronically submitted via EFS-Web as an ASCII sequence listing with file name PRD3459 Sequence Listing and creation date October 23, 2017 and is 20 kb in size. The sequence listing provided by EFS-Web is part of the description and is incorporated herein by reference in its entirety. In the event of any inconsistency regarding the structures for SEQ ID NOs: 1-27 between the information described in this document and the sequence listing provided electronically via EFS Web with the file name PRD3459 Sequence Listing, the information given in this document.

Prerequisites for the creation of the invention

Peptide-based therapeutics are often studied and developed for their ability to provide specificity and selectivity as evidenced by many approved peptide-based therapies, however, their relatively short in vivo half-lives place limitations on their methods of application. Many strategies for extending the half-life of peptide-based therapeutics have been evaluated and are being used. Antibodies or antibody fragments have been used as groups that extend the half-life of pharmacologically active groups to prevent or slow down the rapid degradation of pharmacologically active groups in vivo.

There is a need for an improved immunoglobulin that can be used as a pharmacologically inactive half-life prolonging group for a pharmacologically active group, preferably in peptide-based therapeutics.

The foregoing description is provided solely for a better understanding of the nature of the problems facing the art and should not be construed in any way as an admission of prior art; nor is the citation of any reference herein to be construed as an admission that such reference is prior art as compared to the present application.

Brief summary of the invention

In one general aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 16, 17, 18, 19, 20 and 21, respectively.

In a preferred embodiment, an isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 12 and a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 14. The isolated monoclonal the antibody of the invention more preferably comprises a heavy chain (HC) having the polypeptide sequence of SEQ ID NO: 13 and a light chain (LC) having the polypeptide sequence of SEQ ID NO: 15.

The invention also relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment of the invention;

a vector, preferably an expression vector, containing the nucleic acid; and a host cell containing the vector.

In addition, a method is provided for obtaining an isolated monoclonal antibody or an antigen-binding fragment of the invention, the method comprising the steps of cultivating a host cell of the invention under conditions allowing the production of a monoclonal antibody or an antigen-binding fragment thereof; and recovering the antibody or antigen-binding fragment thereof from the cell or culture.

Embodiments of the invention include a monoclonal antibody or an antigen-binding fragment of the invention conjugated to at least one pharmacologically active group, preferably a therapeutic peptide, either directly or via a linker. The monoclonal antibody or antigen-binding fragment thereof of the invention may be conjugated to

- 1 041709 any therapeutic peptide. Examples of a therapeutic peptide include, without limitation, oxyntomodulin, glucagon-like peptide 1 (GLP1), tyrosine-tyrosine peptide (PYY), exendin (exenatide), amylin (pramlintide), alpha-melanocyte-stimulating hormone (MSH), cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y1 receptor antagonists (NPY1), neuropeptide Y5 receptor antagonists (NPY5), neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin, melanin-concentrating hormone ( MCH), oxytocin and stresscopin.

In addition, a method is provided for obtaining a monoclonal antibody or antigen-binding fragment of the invention, conjugated to a therapeutic peptide, comprising the reaction of an electrophile, preferably bromoacetamide or maleimide, introduced into the side chain of a therapeutic peptide, with a sulfhydryl group, preferably a sulfhydryl group of a cysteine residue of SEQ ID NO: 18, a monoclonal antibody or antigen-binding fragment thereof, thereby forming a covalent bond between the therapeutic peptide and the monoclonal antibody or antigen-binding fragment.

The invention also relates to a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment of the invention conjugated to at least one pharmacologically active group, preferably a therapeutic peptide, directly or via a linker, and a pharmaceutically acceptable carrier.

Another general aspect of the invention relates to a method for increasing the half-life of a therapeutic peptide in a subject, the method comprising the steps of conjugating the therapeutic peptide to a monoclonal antibody or antigen-binding fragment thereof containing complementarity determining region 1 of the heavy chain (HCDR1), HCDR2, HCDR3 and light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively, wherein the therapeutic peptide is conjugated to a monoclonal antibody or antigen-binding fragment thereof in a sulfhydryl group, preferably sulfhydryl group of a cysteine residue of SEQ ID NO: 18.

Additional aspects, features and advantages of the present invention will be better understood upon reading the following detailed description of the invention and the claims.

Brief description of graphic materials

The above brief description, as well as the following detailed description of the preferred embodiments of the present invention, will be better understood when studied in conjunction with the attached drawings. However, it should be understood that the application is not limited to the exact embodiments shown in the drawings.

In FIG. 1 shows a general strategy for conjugating a peptide to a mAb in accordance with an embodiment of the invention. X is an electrophile introduced into the side chain of a therapeutic peptide, such as bromoacetamide or maleimide, which reacts site-specifically with the sulfhydryl group of a cysteine residue artificially introduced into the CDR of the half-life-extending mAb to form a covalent bond between the therapeutic peptide and the mAb.

In FIG. 2 shows a summary of the CDR residues selected for replacement in PH9H5_VH (SEQ ID NO: 4) and PH9L3_VL (SEQ ID NO: 3). Residues substituted with cysteine are in bold and underlined.

In FIG. 3 shows the structure of compound 1 (SEQ ID NO: 2).

In FIG. 4 shows the pharmacokinetics of Compound 1 in DIO mice.

In FIG. 5 shows the pharmacokinetics of compound 1 in cynomolgus monkeys.

In FIG. 6 shows the food intake of DIO mice treated with compound 1: acute dosing.

In FIG. 7 shows weight loss in DIO mice treated with compound 1: acute dosing.

In FIG. 8 shows the food intake of DIO mice treated with compound 1: chronic dosing.

In FIG. 9 shows weight loss in DIO mice treated with compound 1: chronic dosing.

In FIG. 10 shows a reaction scheme for preparing an oxyntomodulin monoclonal antibody (mAb-OXM) compound according to an embodiment of the invention. The upper reaction is the reduction of disulfide in Cys102 mAb (eg MSCB97). The bottom reaction is the conjugation of the reduced mAb to the OXM peptide variant. R is cysteine or glutathione. Cys102 in the MSCB97 heavy chain is shown along with the flanking amino acids as single letter codes. OXM refers to the amino acid portion of a variant peptide. Aib2, Lys30 and a bromoacetylated discrete polyethylene glycol (dPEG) 12 spacer are shown. His1 mAb is shown as a single letter code.

In FIG. 11 shows the structure of compound 2 (SEQ ID NO: 27).

In FIG. 12 is a graph showing the functional stability of compound 2 in plasma.

- 2 041709 human for 168 hours. Percentages normalized to time point zero (t=0) of compound 2, which is a conjugate of OXM peptide analog with mAbA, control 1, which, as previously shown to be stable in human plasma υ ex vivo, control 2, which was previously shown to be unstable in human plasma λ and □ ex vivo, and compound 3 (mAb conjugated to H-AibQGTFTSDYSKYLDERRARDFVEWLLNK-(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) ₁₂ NHCOCH ₂ Br)-NH ₂ (SEQ ID NO: 25)) T and Compound 4 (mAb conjugated to H-Aib-QGTFTSDYSKYLDERRARDFVEWLLNTK(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) ₁₂ NHCOCH ₂ Br)-NH ₂ ( SEQ ID NO: 26))^, which are conjugates of additional OXM peptide analogue with mAb, depending on time in hours (h) along the X axis.

In FIG. 13 is a graph showing the functional stability of compound 2 in monkey plasma for 168 hours. Percentages normalized to time point zero (t=0) of compound 2A, control 1, which was previously shown to be stable in human υ plasma in ex vivo conditions, control 2, which, as previously shown, is unstable in human plasma λ and □ under ex vivo conditions of compound 3 and compound 4 ▼, which are conjugates of additional OXM peptide analogue with mAb, depending on time in hours ( h) along the X axis.

In FIG. 14 is a graph showing the % change in food intake in cynomolgus monkeys treated with Compound 2.

In FIG. 15 is a graph showing the % change in body weight of cynomolgus monkeys treated with Compound 2.

Detailed description of the invention

In the Background section of the invention and in the text of this application, citations or descriptions of various publications, articles and patents are given, and each of these references is fully incorporated into the present description by reference. Discussion of documents, acts, materials, devices, products, etc., which have been included in the present description, is given as a context for the present invention. Such discussion is not an admission that any or all such sources are part of the prior state of knowledge with respect to any of the inventions described or claimed.

All technical and scientific terms in this document, unless otherwise indicated, have the generally accepted meaning understood by any person skilled in the field to which this invention pertains. Otherwise, certain terms in this document have the meanings established in the present description.

It should be noted that in the present document and in the appended claims, the singular form includes objects in the plural, unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values such as concentration or range of concentrations described herein are to be understood as modified in all instances by the term about. Thus, the numerical value typically includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes 0.9 to 1.1 mg/ml. Similarly, the concentration range from 1 to 10% (w/v) includes from 0.9 to 11% (w/v). In the context of this document, the use of a numeric range explicitly includes all possible subranges, all individual numeric values within that range, including integers within such ranges, and fractional values, unless the context clearly dictates otherwise.

Unless otherwise indicated, a term at least preceding a series of elements should be understood to refer to each element in that series. Those skilled in the art will be able to determine, or with only standard experimentation will be able to establish many equivalents of specific embodiments of the invention described in this document. It is understood that such equivalents are specified in the invention.

As used herein, the terms contains, containing, includes, including, has, having, contains, or containing, or any other variation thereof, are intended to include the specified integer or group of integers, but not to exclude from it any other integer or group of integers, and are non-exclusive or non-limiting. For example, a composition, mixture, process, method, article, or device that contains a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or device. Also, unless explicitly stated otherwise, the conjunction or refers to an inclusive or, not an exclusive or. For example, condition A or B is true in any one of the following situations: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present) and both A and B are true (or present).

It should also be understood that the terms about, approximately, mostly, mainly and like terms used herein when referring to the size or characteristic of a component of the preferred invention indicate that the described size/characteristic is not a strict limit or parameter and does not exclude minor deviations from them that are functionally the same or similar, as will be understood by the ordinary specialist

- 3 041709 sheet in this area. At a minimum, such references containing a numerical parameter will include deviations that, using mathematical and industrial principles accepted in the art (for example, rounding, measurement or other systematic errors, manufacturing tolerances, etc.), will not change the smallest significant digit.

The terms identical or percent identity in the context of two or more nucleic acids or polypeptide sequences (e.g., PYY3-3 6 cyclic polypeptide sequences) refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum match as measured using one of the sequence comparison algorithms or by visual inspection using methods known in the art related to the present description.

For sequence comparisons, typically one sequence acts as a reference sequence against which the test sequence is compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the subsequence are determined and the parameters of the sequence algorithm program are determined. Subsequently, the sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) with respect to the reference sequence based on the specified program parameters.

Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), using the homology region alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci., USA, 85:2444 (1988), using computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575, Science Dr., Madison, WI, USA) or by visual inspection (see mainly Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (Supplement, 1995) (Ausubel)).

Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990), J. Mol. Biol., 215:403-410; and Altschul et al. (1997), Nucleic Acids Res., 25: 3389-3402 respectively. BLAST analysis software is publicly available through the National Center for Biotechnology Information.

An additional indication of the substantive identity of two nucleotide sequences or two polypeptides is the immunological cross-reactivity of the polypeptide encoded by the first nucleic acid with the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, when the two peptides differ only in conservative substitutions. Another sign of essentially identical two nucleic acid sequences is the hybridization of these two molecules with each other under stringent conditions, as described below.

As used herein, the term subject means any animal, preferably a mammal, most preferably a human. As used herein, the term mammal encompasses any mammal. Examples of mammals include, without limitation, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like. and more preferably human.

The term administration in relation to the methods of the invention means a method of therapeutic or prophylactic prevention, treatment or amelioration of a syndrome, disorder or disease, as described herein, through the use of a conjugate of the invention or its form, composition or drug. Such methods include administering an effective amount of said conjugate, conjugate form, composition, or drug at different times during the course of treatment, or simultaneously with other conjugates in combination form. The methods of the invention are to be understood as including all known therapeutic regimens.

The term "effective amount" means that amount of the active conjugate or pharmaceutical agent that elicits the biological or medical response of a tissue system, animal or human being sought by a researcher, veterinarian, physician, or other professional, which includes the prevention, treatment, or alleviation of a syndrome, disorder, or disease, to which treatment is directed, or symptoms of the syndrome, disorder or disease to which treatment is directed.

As used herein, the term composition is intended to cover a product containing specified ingredients in specified amounts, as well as any product that can be obtained directly or indirectly from combinations of specified ingredients in specified amounts.

- 4 041709

As used herein, the term related refers to the union or connection of two or more objects with each other. When applied to chemical or biological compounds, the term bound can mean a covalent bond between two or more chemical or biological compounds. As a non-limiting example, an antibody of the invention can be coupled to a peptide of interest to form an antibody-bound peptide. An antibody-bound peptide can be formed by conducting specific chemical reactions designed to conjugate an antibody to a peptide. In certain embodiments, an antibody of the invention may be covalently linked to a peptide of the invention via a linker. The linker can be, for example, first covalently attached to the antibody or peptide, and subsequently covalently attached to the peptide or antibody.

As used herein, the term linker refers to a chemical module containing a covalent or atomic chain that covalently links a peptide to an antibody. The linker may, for example, include, without limitation, a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a PEG-embedded heterocycle hybrid linker, and a hydrocarbon chain. PEG linkers may, for example, contain 2-24 PEG units.

As used herein, the term conjugate refers to an antibody or fragment thereof covalently linked to a pharmaceutically active group. The term "conjugated with" refers to an antibody or fragment of the invention covalently linked or covalently linked to a pharmaceutically active group, preferably a therapeutic peptide, either directly or via a linker. As a non-limiting example, the antibody may be a monoclonal antibody of the invention, and the pharmaceutically active group may be a therapeutic peptide such as cyclic PYY, an oxyntomodulin peptide, or any other therapeutic peptide of interest. The pharmaceutically active group may also be a non-peptide organic group (ie, a small molecule). As used herein, with respect to an antibody or antigen-binding fragment thereof, in accordance with an embodiment of the invention, the phrase a conjugate comprising the antibody or antigen-binding fragment thereof and a pharmaceutically active group (or therapeutic peptide) conjugated thereto is used interchangeably with the phrase antibody or antigen-binding fragment thereof, conjugated to a pharmaceutically active group (or therapeutic peptide).

The peptide sequences described herein are written according to the usual procedure, where the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, they are the L-form of the present amino acid, unless explicitly stated otherwise.

Antibodies.

In one general aspect, the invention relates to a novel antibody that has been designed to be untargeted and containing a cysteine residue, having the potential to be used for chemical conjugation (i.e., linking) to a pharmaceutically active group, such as a therapeutic peptide (e.g., PYY cyclic peptide, oxyntomodulin or a peptide variant, etc.), in a site-specific manner such that the antibody-bound peptide has an extended/increased half-life compared to the unconjugated peptide. As used herein, the term untargeted in the context of an antibody refers to an antibody that does not specifically bind to any target under in vivo conditions. As used herein, an antibody that specifically binds to a target means an antibody that binds to a target antigen with a KD of 1 x 10' ⁷ or less, preferably 1 x 10' ⁸ M or less, more preferably 5 x 10' ⁹ M or less, 1 x 10' ⁹ M or less, 5x10'1° M or less, or ^1x10'10 M or less. The term KD means the dissociation constant, which is the ratio of Kd to Ka (ie Kd/Ka) and is expressed in molar concentration (M). KD values for antibodies can be determined using the methods of the art related to the present description. For example, the KD of an antibody can be determined using surface plasmon resonance, such as a biosensor system, such as the Biacore® system, or using biolayer interferometry technology, such as the Octet RED96 system. The lower the KD value of an antibody, the higher the affinity with which the antibody binds to the target antigen.

Monoclonal antibodies (total or fragments thereof) can be used as a group that increases the half-life. Monoclonal antibodies are well-characterized proteins that are used and described for use in vivo, and therefore the mechanisms that provide their long in vivo half-life and the mechanisms of their degradation in vivo are well known. In addition, the spatial separation and presentation of the two arms of the monoclonal antibody may be advantageous for efficient bivalent presentation of the therapeutic moiety (ie, therapeutic peptide). Therapeutic agents have been developed in which toxins or other small molecule drugs are chemically bonded

- 5 041709 with a monoclonal antibody, but they typically use a monoclonal antibody that binds to a specific antigen and targets the antibody drug conjugate to the tissue/cell of interest that preferentially expresses the antigen, and typically the drug/small the molecule is attached to the antibody in a manner that does not affect the binding of the antigen to the antibody.

For therapeutic peptide-mAb conjugates, antigen-specific binding by a monoclonal antibody that increases half-life is undesirable. In this regard, a pair of heavy chain (HC) and light chain (LC) variable (V) domains, which should not specifically bind to any target, are used to produce a binding, non-targeted monoclonal antibody of the invention. To obtain a binding, untargeted monoclonal antibody, a cysteine residue is inserted into one of the complementarity determining regions (CDRs) of the selected untargeted antibody.

The pharmaceutically active group (eg, therapeutic peptide/compound) may contain a suitable chemical group to allow conjugation of the pharmaceutically active group to the inserted cysteine residue of a non-targeted monoclonal antibody. The general strategy for conjugating a peptide to a monoclonal antibody according to an embodiment of the invention is shown in FIG. 1.

The term antibodies is used herein in a broad sense and includes non-human (e.g., murine, rat), human, human-adapted, humanized, and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, and single chain antibodies. antibodies.

The light chains of antibodies of any vertebrate species can be assigned, depending on the amino acid sequences of their constant domains, to one of two distinct types, namely kappa (k) and lambda (λ). Accordingly, the antibodies of the invention may comprise a kappa or lambda light chain constant domain. In accordance with specific embodiments, the antibodies of the invention comprise heavy and/or light chain constant regions, such as murine or human antibodies. In addition to the heavy and light chain constant domains, antibodies contain an antigen-binding region that consists of a light chain variable region and a heavy chain variable region, each containing three domains (i.e. complementarity determining regions 1-3; (CDR1, CDR2 and CDR3)). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2 and LCRD3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2 and HCDR3.

Immunoglobulins can fall into five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgG is the most stable of the five immunoglobulin types, with a serum half-life of about 23 days in humans. IgA and IgG are further classified as IgAi, IgA ₂ , IgG1, IgG ₂ , IgG ₃ and IgG ₄ isotypes. Each of the four subclasses of IgG has different biological functions, known as effector functions. These effector functions are usually mediated through interaction with the Fc receptor (FcyR) or through C1q binding and complement fixation. Binding to FcyR can lead to antibody-dependent cell-mediated cytolysis, while binding to complement factors can lead to complement-mediated cell lysis. The antibody of the invention, used in connection with its ability to increase the half-life of a therapeutic peptide, has no or minimal effector function, but retains the ability to bind to FcRn, binding to which may be the main method by which antibodies increase half-life in vivo.

In one embodiment, the invention provides an isolated antibody, or antigen-binding fragment thereof, comprising a light chain variable region having fully human Ig germline V gene sequences and a heavy chain variable region having fully human Ig V germline gene sequences, excluding HCDR3 having the amino acid sequence of SEQ ID NO: 18, wherein the antibody or antigen-binding fragment thereof does not specifically bind to any human antigen under in vivo conditions. In certain embodiments, the invention provides an isolated antibody, or an antigen-binding fragment thereof, comprising a light chain variable region having fully human Ig germline V gene sequences and a heavy chain variable region having fully human Ig V germline gene sequences, excluding HCDR3 having the amino acid sequence of SEQ ID NO: 18, wherein the antibody or antigen-binding fragment thereof does not specifically bind to any human antigen under in vivo conditions, and the isolated antibody or antigen-binding fragment thereof is associated with a pharmaceutically active group (e.g., cyclic peptide PYY, peptide oxyntomodulin and/or therapeutic peptide of the invention).

As used herein, the term antigen-binding fragment refers to

- 6 041709 an antibody fragment, such as, for example, diatelo, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv '), disulfide-stabilized diabody (ds-diabody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv-dimer (bivalent antibody), multispecific antibody formed from an antibody portion containing one or more CDRs, camelid single-domain an antibody, nanobody, domain antibody, divalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. The antigen-binding fragment has the ability to bind to the same antigen to which the original antibody or the original antibody fragment binds. In accordance with specific embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (ie, a heavy chain region that is included in the Fab fragment). According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab').

As used herein, the term single chain antibody refers to a standard single chain antibody in the art that contains a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term single domain antibody refers to a standard single domain antibody in the art that contains a heavy chain variable region and a heavy chain constant region, or that contains only a heavy chain variable region.

The phrase isolated antibody or antibody fragment means an antibody or antibody fragment substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a target antigen is substantially free of antibodies that do not specifically bind to the target antigen). Moreover, the isolated antibody or antibody fragment may be substantially free of other cellular material and/or chemicals.

The variable region of an antibody consists of a framework region separated by three antigen-binding sites. Antigen binding sites are defined using the following different terms:

(i) complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), based on sequence variability (Wu and Kabat, J. Exp. Med., 132: 211-50, 1970; Kabat et al., Sequences of Proteins of Immunological interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991);

(ii) hypervariable regions, HVR or HV - three in VH (H1, H2, H3) and three in VL (L1, L2, L3) denote regions of antibody variable domains that are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk Mol. Biol., 196:901-17, 1987).

Other terms include IMGT-CDR (Lefranc et al., Dev. Comparat. Immunol., 27:55-77, 2003) and the use of specificity determining residues (SDRUs) (Almagro Mol. Recognit., 17:132-43, 2004 ). The international database ImMunoGeneTics (IMGT) (http://www_mgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between the CDR, HV and IMGT delimitations is described in Lefranc et al., Dev. Comparat. Immunol., 27:55-77, 2003.

The terms framework or framework sequences are residual variable region sequences that differ from those defined as antigen-binding sites. Since antigen-binding sites, as described above, may be defined by various terms, the exact amino acid sequence of the framework depends on the definition of antigen-binding site.

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

In another embodiment, the isolated antibody further comprises an Fc region derived from the Fc region of human IgG4. The Fc region of human IgG4 has a reduced ability to bind to FcyR and complement factors compared to other IgG subtypes. The Fc region preferably contains a human IgG4 Fc region having substitutions that exclude effector function. Thus, the isolated antibody further comprises an Fc region having a modified human IgG4 Fc region containing one or more of the following substitutions: proline to glutamate at position 2-33, alanine or valine to phenylalanine at position 234, and alanine or glutamate to leucine at position 235 (EC numbering, Kabat E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Dept. of Health and Human Services, Bethesda, Md., NIH Publication No. 91-3242). Removal of the N-linked glycosylation site in the Fc region of IgG4 by replacing alanine with asparagine at position 297 (EC numbering) is another way to eliminate residual effector activity.

The antibody of the invention preferably exists as dimers joined together by disul

- 7 041709 fiddle bridges and various non-covalent interactions. Thus, the Fc region used for an antibody of the invention may be a human IgG4 Fc region containing a substitution, such as a serine to proline substitution at position 228 (EC numbering), which stabilizes the formation of the heavy chain dimer and prevents the formation of half Fc- chains of IgG4.

In another embodiment, the C-terminal lysine residue in the heavy chain has been removed, as often seen in recombinant monoclonal antibodies.

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

A human antibody contains heavy or light chain variable regions that are derived from sequences of human origin if the antibody variable regions are derived from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include phage-displayed human immunoglobulin gene libraries and non-human transgenic animals such as mice harboring human immunoglobulin loci as described herein. The human antibody may contain amino acid differences from the human germline or rearranged immunoglobulin sequences due, for example, to naturally occurring somatic mutations or intentional substitutions at scaffold or antigen binding sites. Typically, a human antibody is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence encoded by the human germline gene or rearranged immunoglobulin gene. In some instances, the human antibody may contain consensus framework sequences derived from human framework sequence assays, for example, as described in Knappik et al., J. Mol. Biol., 296:57-86, 2000, or synthetic HCDR3s included in phage-displayed human immunoglobulin gene libraries, for example, as described in Shi et al., J. Mol. Biol., 397:385-96, 2010 and International Patent Publication No. WO 2009/085462). Antibodies in which the antigen-binding sites are derived from a species other than a human do not fit the definition of a human antibody.

Isolated antibodies according to an embodiment of the invention may be synthetic. Antibodies, although derived from human immunoglobulin sequences, may be generated using systems such as phage display, including synthetic CDRs and/or synthetic scaffolds, or may be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that are in vivo are not part of the repertoire of human germline antibodies in vivo.

The term recombinant antibody as used herein includes all antibodies produced, expressed, generated, or isolated by recombinant means, such as antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or from a hybridoma derived therefrom, antibodies, isolated from a host cell transformed to express an antibody, antibodies isolated from a recombinant combinatorial antibody library, and antibodies produced, expressed, generated, or isolated by any other means that involve splicing human immunoglobulin genes with other DNA sequences, or antibodies generated in vitro using Fab shoulder exchange.

The term monoclonal antibody as used herein refers to a preparation of antibody molecules of a single molecule composition. The composition of a monoclonal antibody displays single binding specificity and affinity for a particular epitope, or in the case of a bispecific monoclonal antibody, dual binding specificity for two separate epitopes. The monoclonal antibodies of the invention can be produced using the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be generated using a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse or rat, having a genome containing a human heavy chain transgene and a light chain transgene.

In certain embodiments, the term mAb refers to a monoclonal antibody. In one embodiment, the mAb has a heavy chain variable region (VH) sequence comprising SEQ ID NO: 12 and a light chain variable region (VL) sequence comprising SEQ ID NO: 14. In certain embodiments, the mAb is a fully human monoclonal antibody having a heavy chain (HC) sequence comprising SEQ ID NO: 13 and a light chain (LC) sequence comprising SEQ ID NO: 15. In certain embodiments, a lysine residue at position 446 in SEQ ID NO: 13 is optionally absent.

As used herein, the term chimeric antibody refers to an antibody, in

- 8 041709 wherein the amino acid sequence of an immunoglobulin molecule is derived from two or more species. The variable region of the light and heavy chains often corresponds to the variable region of an antibody derived from a single mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of the antibody, derived from another species of mammal (eg, human), to prevent the occurrence of an immune response in this species.

As used herein, the term multispecific antibody refers to an antibody that contains a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the plurality has a binding specificity for a first epitope or contains germline sequences having no known binding specificity, and a second immunoglobulin variable domain sequence of the plurality has a binding specificity for a second epitope or contains germline sequences lacking any known binding specificity, and wherein the first and/or second immunoglobulin variable domains optionally include a conjugated pharmaceutically active group (e.g., a therapeutic peptide ). In an embodiment, the first and second epitopes are located on the same antigen, eg on the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or do not substantially overlap. In an embodiment, the first and second epitopes are located on different antigens, eg on different proteins (or different subunits of a multimeric protein). In an embodiment, the first and second immunoglobulin variable domains comprise the same conjugated pharmaceutically active group. In an embodiment, the first and second immunoglobulin variable domains comprise different pharmaceutically active groups. In an embodiment, only the first immunoglobulin variable domain includes a conjugated pharmaceutically active group. In an embodiment, only the second immunoglobulin variable domain includes a conjugated pharmaceutically active group. In an embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, the multispecific antibody is a bispecific antibody, trispecific antibody, or tetraspecific antibody molecule.

As used herein, the term bispecific antibody refers to a multispecific antibody that binds no more than two epitopes or two antigens and/or contains two conjugated pharmaceutically active groups (eg, the same or different pharmaceutically active groups). A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope or contains germline sequences without any known binding specificity, and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope or contains germline sequences having no known binding specificity, wherein the first and/or second immunoglobulin variable domains optionally include a conjugated pharmaceutically active group. In an embodiment, the first and second epitopes are located on the same antigen, eg the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are located on different antigens, eg on different proteins (or different subunits of a multimeric protein). In an embodiment, the first and second immunoglobulin variable domains comprise the same conjugated pharmaceutically active group. In an embodiment, the first and second immunoglobulin variable domains comprise different pharmaceutically active groups. In an embodiment, only the first immunoglobulin variable domains comprise a conjugated pharmaceutically active group. In an embodiment, only the second immunoglobulin variable domain includes a conjugated pharmaceutically active group. In an embodiment, the bispecific antibody comprises a first heavy chain variable domain sequence and a light chain variable domain sequence that have a binding specificity for the first epitope or contain germline sequences lacking any known binding specificity, and a second heavy chain variable domain sequence and a sequence light chain variable domain that have a binding specificity for a second epitope or contain germline sequences that do not have any known binding specificity, and wherein the first and/or second heavy chain variable domains optionally include a conjugated pharmaceutically active group. In an embodiment, the first and second heavy chain variable domains comprise the same conjugated pharmaceutically active group. In an embodiment, the first and second heavy chain variable domains comprise different conjugated pharmaceutically active groups. In an embodiment

- 9 041709 only the first heavy chain variable domain includes a conjugated pharmaceutically active group. In an embodiment, only the second heavy chain variable domain includes a conjugated pharmaceutically active group.

The term full-length antibody as used herein means an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The heavy chain (HC) of a full length antibody consists of the variable region (VH) and constant domains (CH1, CH2 and CH3) of the heavy chain. The light chain of a full length antibody (LC) consists of a variable region (VL) and a constant domain (CL) of the light chain. A full-length antibody may not contain a C-terminal lysine (K) in either one or both heavy chains.

The term Fab-arm or half-molecule means one heavy chain-light chain pair.

Full-length bispecific antibodies can be generated, for example, by exchanging Fab arms (or exchanging hemimolecules) between two monospecific divalent antibodies, by introducing substitutions at the heavy chain CH3 interface in each hemimolecule that promote the formation of a heterodimer from two antibody hemimolecules having different specificities, or in vitro in a cell-free medium, or using co-expression. The Fab-arm exchange reaction is the result of disulfide isomerization and dissociation-association of CH3 domains. The heavy chain disulfide bridges in the hinge regions of the original monospecific antibodies were restored. The resulting free cysteines of one of the original monospecific antibodies form a disulfide bridge of heavy chains with cysteine residues of the second original molecule of the monospecific antibody, and simultaneously the CH3 domains of the original antibodies are released and reformed by dissociation-association. Fab arm CH3 domains can be designed to allow for heterodimerization rather than homodimerization. The resulting product is a bispecific antibody having two Fab arms or hemimolecules, each of which can bind to a different epitope.

The term homodimerization as used herein in relation to antibodies refers to the interaction of two heavy chains having identical CH3 amino acid sequences. As used herein, the term homodimer in relation to antibodies means an antibody having two heavy chains with identical CH3 amino acid sequences.

As used herein, the term heterodimerization with respect to antibodies refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. As used herein, the term heterodimer in relation to antibodies means an antibody having two heavy chains with non-identical CH3 amino acid sequences.

To obtain full-length bispecific antibodies, you can use the strategy of the protrusion in the cavity (see, for example, international publication PCT No. WO 2006/028936). In other words, selected amino acids that form an interface between the CH3 domains in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. A short side chain amino acid (trough) is inserted into the heavy chain of an antibody that specifically binds to the first antigen, and a long side chain amino acid (ledge) is inserted into the heavy chain of an antibody that specifically binds to the second antigen. Upon co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the pit heavy chain and the ridge heavy chain. Examples of pairs of substitutions in CH3 forming a ridge and a trough are (indicated as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain) T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T , T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions by introducing substitutions of positively charged residues on one CH3 surface and negatively charged residues on the other CH3 surface, as described in US Patent Publication No. US 2010/0015133; US Patent Publication No. US 2009/0182127; US Patent Publication No. US 2010/028637 or US Patent Publication No. US 2011/0123532. In other strategies, heterodimerization can be promoted by the following substitutions (indicated modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain):

L351Y_F405A_Y407V/T394W,

T366I_K392M_T394W/F405A_Y407V,

T366L_K392M_T394W/F405A_Y407V,

L351Y_Y407A/T366A_K409F,

L351Y_Y407A/T366V_K409F,

Y407A/T366A_K409F, or

T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in US Patent Publication No. US 2012/0149876 or US Patent Publication

- 10 041709 No. US 2013/0195849.

In addition to the methods described above, bispecific antibodies can be created in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and generating bispecific heterodimeric antibodies from the two original monospecific homodimeric antibodies under reducing conditions, which promotes isomerization of the disulfide bond, according to by the methods described in International Patent Application Publication No. WO 2011/131746. In these methods, the first monospecific divalent antibody and the second monospecific divalent antibody are designed to have specific substitutions in the CH3 domain that contribute to the stability of the heterodimer; the antibodies are incubated together under reducing conditions sufficient to expose the cysteines in the hinge to disulfide bond isomerization, thereby producing a bispecific antibody by exchanging Fab arms. The incubation conditions can be optimally returned to non-reducing conditions. Examples of suitable reducing agents include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris(2carboxyethyl)phosphine. For example, an incubation of at least 90 minutes at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of 5-8, such as pH 7.0 or at pH 7.4.

The numbering of amino acid residues in the constant region of the antibody in the present description is in accordance with the EC catalog, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) unless explicitly stated otherwise.

Conjugates.

In another general aspect, the invention relates to a conjugate comprising an antibody of the invention covalently conjugated to a pharmaceutically active group, such as a synthetic therapeutic peptide (e.g., a PYY cyclic peptide or an oxyntomodulin peptide variant), in a site-specific manner such that the antibody-bound peptide has an extended/enlarged half-life compared to the unconjugated peptide. The conjugates are used to prevent, treat, or alleviate the diseases or disorders described herein. The present invention also relates to pharmaceutical compositions, methods for their preparation and use.

In certain embodiments, an antibody of the invention is modified to contain at least one cysteine residue substitution that is conjugable to a pharmaceutically active group to lengthen/increase the half-life of the pharmaceutically active group. In certain embodiments, at least one cysteine residue substitution is contained within the complementarity-determining region of an antibody. In certain embodiments, at least one cysteine residue substitution is in the heavy chain complementarity determining region (HCDR). In certain embodiments, 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 HCDR3 antibody comprising the amino acid sequence of SEQ ID NO: 18 has at least one additional a cysteine substitution which is capable of being conjugated to a pharmaceutically active group.

In certain embodiments, the pharmaceutically active group may contain a linker. The linker can be chemically modified to allow the antibody to be conjugated to a pharmaceutically active group. The linker may, for example, include, without limitation, a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a PEG-embedded heterocycle hybrid linker, and a hydrocarbon chain. PEG linkers may, for example, contain 2-24 PEG units.

In certain embodiments, a monoclonal antibody of the invention is conjugated to one, two, three, four, five, or six pharmaceutically active groups of interest (eg, therapeutic peptide(s)). In preferred embodiments, the non-targeted monoclonal antibody is conjugated to two pharmaceutically active groups of interest. In certain embodiments in which the monoclonal antibody is conjugated to at least two pharmaceutically active groups of interest, the pharmaceutically active groups of interest may be the same pharmaceutically active group or may be different pharmaceutically active groups.

Methods for conjugating antibodies of the invention to pharmaceutically active groups of the invention are known in the art. In other words, the antibodies of the invention can be regenerated with a reducing agent (e.g., TCEP (tris(2-carboxyethyl)phosphine)), purified (e.g., by protein A adsorption or gel filtration) and conjugated to a pharmaceutical

- 11 041709 with a highly active group (for example, by adding a lyophilized peptide to the reconstituted antibody under conjugation conditions). Following the conjugation reaction, the conjugate may be purified by ion exchange chromatography or hydrophobic chromatography (HIC) followed by a final purification step by absorption on protein A. In certain embodiments, the antibodies of the invention may be purified prior to reconstitution using HIC methods. A more detailed description of the methods of conjugation is given, for example, in examples 3 and 7 and Dennler et al., Antibodies, 4:197-224 (2015).

The term homodimerization, as used herein in relation to conjugates, means the interaction of two identical pharmaceutically active groups with an antibody. The term homodimer, as used herein in relation to conjugates, means an antibody conjugated to two identical pharmaceutically active groups.

The term heterodimerization, as used herein in relation to conjugates, means the interaction of two different pharmaceutically active groups with an antibody. The term heterodimer, as used herein in relation to conjugates, means an antibody conjugated to two different pharmaceutically active groups.

Cyclic peptides PYY.

PYY3-36 is an endogenous hormone secreted by L cells in the distal intestine that acts as a Y2 receptor agonist to inhibit food intake. Given its role in the control of appetite and food intake, as well as its antisecretory and absorption-promoting effects in the mammalian gastrointestinal tract, PYY ₃ _ ₃₆ may be effective in the treatment of obesity and related conditions, as well as in a number of gastrointestinal disorders. disorders. However, the therapeutic utility of PYY ₃ - ₃₆ itself as a therapeutic agent is limited by its rapid metabolism and short circulating half-life. Thus, the antibodies of the invention can be used as a carrier for PYY ₃ - ₃₆ , preferably modified with PYY ₃ - ₃₆ , which increases the half-life of the PYY ₃ - ₃₆ peptide and reduces the metabolism of the peptide in vivo.

In certain embodiments, the modified PYY ₃ _ ₃₆ peptides are cyclic PYY peptides. The terms PYY cyclic peptide, PYY ₃ _ ₃₆ cyclic analog, and PYY3-36 cyclic peptide analog can be used interchangeably. Examples of PYY cyclic peptides that can be used in conjugates are described in U.S. Provisional Application No. 62/413613, filed October 27, 2016, and U.S. Patent Application No._ Cyclic peptide tyrosine tyrosine compounds as modulators of neuropeptide receptors filed on the same day as the present application with Patent Attorney File No. PRD3411, the contents of which are incorporated herein by reference in their entirety.

Examples of conjugates containing an antibody of the invention and a PYY cyclic peptide are described in U.S. Provisional Application No. 62/413,586, filed October 27, 2016, and in U.S. Patent Application No._ Antibody coupled cyclic peptide tyrosine tyrosine compounds as modulators of neuropeptide Y receptors, filed on the same day as the present application with Patent Attorney File No. PRD3436, the contents of which are incorporated herein by reference in their entirety. For example, in PYY cyclic peptides, the N-terminal amino acid residue of the cycle is linked via its functional α-amino group to a linking group, which in turn is connected to the amino acid side chain residue at position 31 of the NTSC-PYY peptide. Lysine residues can be introduced at various positions in the hPYY ₃ _ ₃₆ sequence to provide a convenient functional substring for further derivatization. Lysine residues can be modified for both direct and indirect binding to the monoclonal antibody. In indirect binding to a monoclonal antibody, the lysine residue can be modified to contain a linker that will allow the PYY cyclic peptide to be linked to the monoclonal antibody. One skilled in the art will appreciate that the corresponding orthologues discussed herein can also be effectively used as such.

Oxyntomodulin peptides.

Oxyntomodulin (OXM) is a 37 amino acid peptide secreted by enteroendocrine L cells in the intestine. Through its GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) agonistic activity, OXM enhances β-cell function, reduces food intake, and increases energy expenditure. Through these complementary mechanisms, OXM-mediated weight loss may be superior compared to currently marketed GLP1R agonists. OXM may also lower plasma cholesterol and triglyceride levels. The half-life of OXM in humans is very short, approximately a few minutes (Schjoldager et al., Eur. J. Clin. Invest., 18(5):499-503 (1988)). Thus, an embodiment of the invention relates to a conjugate containing an antibody of the invention covalently linked to oxyntomodulin to provide dual agonistic properties of oxyntomodulin and an extended half-life sufficient to implement dosing once a week.

- 12 041709

An increase in the half-life of the peptide is obtained by providing the OXM peptide with resistance to proteolysis and by reducing clearance from plasma. For example, proteolysis by DPP4 was inhibited by replacing the serine at position 2 with aminoisobutyric acid (Aib). The helical configuration of the peptide was stabilized by introducing a bifurcated salt bridge from Q20R to S16E and Q24E, and the potential susceptibility to oxidation was reduced by replacing methionine 27 with leucine. The duration of the existence of the peptide in the bloodstream was increased by covalent attachment of a monoclonal antibody (mAb). Such antibody drug conjugates are capable of exhibiting long plasma half-lives due to their size, which can reduce glomerular filtration, and due to recycling through the neonatal Fc receptor. A short oligoethylene glycol spacer was placed between the mAb and the peptide to ensure unhindered access of the peptide to GCGR and GLP1R.

According to embodiments of the invention, the oxyntomodulin conjugate of the invention has four characteristics.

(A) Dual agonism: Oxyntomodulin conjugates have dual agonism at GLP-1 and glucagon receptors.

(B) Receptor balance: Oxyntomodulin conjugates do not have excessive bias for GLP1R or GCGR activity, as excessive bias for GLP1R can result in conjugation that, with increased exposure, develops GLP-1-mediated GI adverse events before interacting with targeted GCGR, and excessive bias towards GCGR may decrease glycemic efficiency.

(C) Biodistribution: Oxyntomodulin conjugates have a modest shift in activity at the GLP-1 receptor (compared to unconjugated oxyntomodulin) to achieve targeted interactions with peripheral GCGR and central energy modulating GLP1R at similar levels of exposure.

(D) Dosing once a week: oxyntomodulin conjugates are competitive due to the possibility of their administration to a subject in need of this once a week.

Other therapeutic peptides.

Also provided herein are conjugates containing other peptides that are capable of being conjugated to the monoclonal antibody platform described herein. The peptides may, for example, be selected from the group consisting of glucagon-like peptide 1 (GLP1), exendin (exenatide), amylin (pramlintide), alpha-melanocyte-stimulating hormone (MSH), cocaine- and amphetamine-regulated transcript (CART), receptor antagonists Neuropeptide Y1 (NPY1), neuropeptide Y Y5 receptor antagonists (NPY5), neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin, melanin-concentrating hormone (MCH) , oxytocin and stresscopin.

Glucagon-like peptide 1 (GLP-1) is a 30 amino acid long peptide hormone derived from tissue-specific post-translational processing of the proglucagon gene. The production and secretion of GLP-1 is carried out by some intestinal enteroendocrine L-cells and some neurons during food intake. The original product of GLP-1 (1-37) is susceptible to amidation and proteolytic cleavage, resulting in two truncated forms with the same biological activity: amide GLP-1 (7-36) and GLP-1 (7-37). Endogenous GLP-1 is rapidly degraded through a variety of pathways, mainly by dipeptidyl peptidase 4 (DPP-4 or DPP-IV), but also by neutral endopeptidase 24.11 (NEP 24.11) and via renal clearance, resulting in a half-life of approximately 2 minutes. GLP-1 based treatments are associated with weight loss and reduced risks of hypoglycemia, which is important in the treatment of type II diabetes.

Exendin (exenatide) is a GLP-1 agonist that belongs to a group of incretin mimetics that have been approved for the treatment of type II diabetes mellitus (T2DM). Exenatide is a synthetic version of exendin-4 (a 39-amino acid peptide hormone), which is an insulin secretagogue with glucoregulatory effects. Exendin-4 has extensive homology and similar functions to mammalian GLP-1, but has a therapeutic advantage in that it is resistant to degradation by DPP-4 (DPP-IV), allowing for a longer pharmacological half-life. Due to the biological characteristics of exendin-4, it is being considered as a treatment for T2DM.

Amylin (island amyloid polypeptide (IAPP)) is a 37-amino acid peptide hormone co-secreted with insulin from pancreatic β-cells. Amylin has an effect on the regulation of glycemia by slowing down gastric emptying and stimulating satiety. Amylin (IAPP) is formed from an 89 amino acid peptide. The islet amyloid polypeptide precursor (proIAPP) is formed in pancreatic β-cells from an 89-amino acid peptide and, after cleavage of the 22-amino acid signal peptide, is a 67-amino acid peptide. It is believed that disruption of proIAPP processing may lead to the conditions that trigger type II diabetes because the lack of amylin production

- 13 041709 (IAPP) may contribute to the disappearance of glucose control. Pramlintide is an amylinomimetic agent and has at least the same activity as human amylin. It is a 37 amino acid polypeptide and differs from the amino acid sequence of human amylin by the substitution of proline amino acids at positions 25 (alanine), 28 (serine) and 29 (serine). Prolines are naturally occurring variations found in rat amylin. As a result of these substitutions, pramlintide is soluble, non-adhesive and non-aggregating, thus overcoming a number of physicochemical disadvantages of native human amylin (Janes et al., Diabetes 45(Suppl 2):235A (1996); Young et al., Drug Dev. Res. ., 37:231-48 (1996)).

α-melanocyte-stimulating hormone (α-MSH) is an endogenous peptide hormone and neuropeptide of the melanocortin family. α-MSH is the most important of the melanocyte-stimulating hormones (MSH) in stimulating melanogenesis, which is the mammalian process responsible for hair and skin pigmentation. α-MSH also plays a role in feeding behavior, energy homeostasis, sexual activity, and protection against ischemic and reperfusion injury.

Cocaine and amphetamine-regulated transcript (CART) is a neuropeptide encoded by the CARTPT gene in humans. CART peptides, in particular CART (55-102), are believed to have an important function in regulating energy homeostasis, as CART peptides interact with several hypothalamic appetite circuits. CART expression is regulated by peripheral peptide hormones involved in appetite regulation, which include leptin, cholecystokinin, and ghrelin. CART and cholecystokinin have a synergistic effect on appetite regulation. CART peptides are thought to have an effect on anxiety behavior induced by ethanol withdrawal;

modulation of locomotor effects, conditioned reflexes to the place and the effect of self-administration of psychostimulants cocaine;

suppress food intake; and are involved in anxious and hypereclectic behavior.

Decreased CART activity in the hypothalamus is associated with hyperphagia and weight gain, and CART is thought to be involved in the opioid mesolimbic dopamine circuit that modulates natural reward processes.

Neuropeptide Y (NPY) plays many roles in the body, which include, for example, control of eating behavior, cortical neuronal activity, cardiac activity, and emotional regulation. NPY has also been linked to several human diseases, including obesity, alcoholism, and depression. In addition, blockade of the central actions of NPY by anti-NPY antibodies, anti-NPY antisense oligodeoxynucleotides, and NPY receptor antagonists results in reduced food intake in energized animals. In particular, neuropeptide Y Y5 receptor (NPY5) and neuropeptide Y Y1 receptor (NPY1) have been shown to stimulate different feeding phases (Br. J. Pharmacol., 2003 Aug, 139(8):1433-40). Thus, NPY5 and NPY1 antagonists may be effective in the treatment of obesity and other related metabolic diseases.

Neurotensin is a 13-amino acid neuropeptide that is involved in the regulation of luteinizing hormone and prolactin release and has a strong relationship with the dopaminergic system. Neurotensin is distributed throughout the central nervous system, with the highest levels found in the hypothalamus, amygdala, and nucleus accumbens. Neurotensin is able to induce various effects, including analgesia, hypothermia, increased locomotor activity, and is involved in the regulation of dopamine pathways.

Neuropeptide B (NPB) is a short biologically active peptide whose precursor is encoded by the NBP gene. NPB can act through two G protein-coupled receptors called neuropeptide B/W receptors 1 and 2 (NPBWR1 and NPBWR2). Neuropeptide B is thought to be involved in the regulation of feeding, the neuroendocrine system, memory, learning, and the afferent pain pathway.

Neuropeptide W (NPW) exists in two forms, consisting of 23 (NPW23) or 30 (NPW30) amino acids. These neuropeptides bind and act through two G protein-coupled receptors NPBWR1 (also known as GPR7) and NPBWR2 (also known as GPR8). NPW has been shown to suppress food intake and body weight and increase both heat production and body temperature, indicating the functioning of NPW as an endogenous catabolic signaling molecule.

Ghrelin (also known as ienomorelin (INN)) is a peptide hormone produced by ghrelinergic cells in the gastrointestinal tract that functions as a neuropeptide in the central nervous system. Ghrelin plays a role in regulating appetite, regulating the distribution and rate of energy use, and regulating reward perception in dopamine neurons. Ghrelin is encoded by the GHRL gene and is believed to be produced by the cleavage of preprogrelin, which is cleaved to produce progrelin, which is cleaved to form the 28-14 041709 amino acid ghrelin. Unlike other endogenous peptides, ghrelin is able to penetrate the blood-brain barrier, due to which exogenously administered ghrelin acquires a unique clinical potential.

Bombesin-like receptor 3 (BRS3) is a G protein-coupled receptor that only interacts with low affinity naturally occurring bombesin peptides, and because it does not have a high affinity ligand, BRS3 is classified as an orphan receptor.

Galanin is a neuropeptide encoded by the GAL gene. Galanin is extensively expressed in the brain, spinal cord, and intestines of humans as well as other animals. The functions of galanin are not yet fully understood; however, galanin is mainly involved in the modulation and inhibition of action potentials in neurons. Galanin has been implicated in many biologically diverse functions including, but not limited to, nociception, regulation of arousal and sleep, cognitive function, feeding, mood regulation, and blood pressure regulation. Galanin is often co-localized with classical neurotransmitters such as acetylcholine, serotonin, and norepinephrine, as well as with neuromodulators such as neuropeptide Y, substance P, and vasoactive intestinal peptide.

Cholecystokinin (CCK) is a peptide hormone of the gastrointestinal system responsible for stimulating the breakdown of fat and protein. CCK is synthesized and secreted by enteroendocrine cells in the small intestine, and its presence causes the release of digestive enzymes and bile from the pancreas and gallbladder. CCK is responsible for digestion, satiety and anxiety.

Orexin (also called hypocretin) is a neuropeptide that regulates activation, wakefulness, and appetite. There are two types of orexin, orexin A and B (hypocretin-1 and -2), which are 33 and 28 amino acids long, respectively. The orexin system was first thought to be involved in stimulating food intake, and in addition to the functions described above, orexins regulate energy expenditure and modulate visceral function.

Melanin concentrating hormone (MCH) is a cyclic 19-amino acid orexigenic hypothalamic peptide thought to be involved in the regulation of eating behavior, mood, sleep-wake cycles, and energy balance. MCH-expressing neurons are located within the lateral hypothalamus and indeterminate zone, and despite this limited localization, MCH neurons have an extensive network of processes throughout the brain.

Oxytocin is a peptide hormone and neuropeptide that is normally produced by the paraventricular nucleus of the hypothalamus and released by the posterior pituitary gland. Oxytocin is believed to play a role in social bonding, sexual reproduction, and during and after childbirth. The oxytocin receptor is a G-protein-coupled receptor that requires magnesium and cholesterol and belongs to the group of G-protein-coupled rhodopsin-type (class I) receptors.

Human stresscopin (h-SCP) is a 40-amino acid peptide that is a member of the corticotropin-releasing hormone (CRH) family. The biological action of the CRH family peptides is due to two 7-transmembrane G-protein-coupled receptors, the CRH type 1 receptor (CRHR1) and the CRH type 2 receptor (CRHR2). Despite the fact that the receptors are characterized by high sequence homology, various CRH family peptides show significant differences in the relative values of binding affinity, degree of receptor activation and selectivity for these two receptors. Unlike many CRH family peptides, h-SCP exhibits greater selectivity for CRHR2 and acts as a mediator that helps attenuate developmental processes and maintain physiological stress. In addition to the apparent influence on the formation of physiological stress, there is also evidence that a number of other physiological actions occur due to h-SCP. It has effects on the endocrine, central nervous, cardiovascular, pulmonary, gastrointestinal, renal, musculoskeletal, and inflammatory systems. CRHR2 also plays a role in the development of diseases associated with skeletal muscle wasting such as sarcopenia, influences locomotor activity and food intake, induces a cardioprotective effect, and exhibits bronchorelaxant and anti-inflammatory activities. In addition, stresscopin mimetics have been identified that are used to treat diseases mediated by the activity of corticotropin-releasing hormone receptor 2; see, for example, US Patent Application No. 20100130424.

Fragments that prolong the half-life.

In addition to the antibody of the present invention, or an antigen-binding fragment thereof, the conjugates of the invention may include one or more other groups to increase the half-life of the pharmaceutically active group, for example, through covalent interaction. Examples of other half-life prolonging groups include, without limitation, albumin, albumin variants, albumin-binding proteins and/or domains, transferrin, and fragments and analogs thereof. Complement

- 15 041709 Significant half-life increasing groups that can be incorporated into the conjugates of the invention include, for example, polyethylene glycol (PEG) molecules such as PEG5000 or PEG20000, fatty acids and fatty acid esters of various chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedicarboxylic acid, tetradecanedicarboxylic acid, octadecanedicarboxylic acid, docosanedicarboxylic acid and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) to obtain the desired properties. These fragments may be direct fusions to scaffold-encoding sequences and may be generated using standard cloning and expression techniques. In an alternative embodiment, well-known chemical coupling methods can be used to attach groups to the recombinantly or chemically modified conjugates of the invention.

For example, a pegil moiety can be added to the peptide molecules of the invention by adding a cysteine residue to the C-terminus of the molecule and attaching the pegil group to the cysteine using well known methods.

Peptide molecules of the invention, including additional groups, can be compared for functionality using several known assays. For example, the biological or pharmacokinetic activity of an unconjugated or conjugated therapeutic peptide of interest can be analyzed using known in vitro or in vivo assays and compared.

pharmaceutical compositions.

In another general aspect, the invention relates to a pharmaceutical composition comprising the conjugates of the invention and a pharmaceutically acceptable carrier. The term pharmaceutical composition in the context of this document means a product containing the conjugate of the invention, together with a pharmaceutically acceptable carrier. The conjugates of the invention and compositions containing them can also be used to produce a medicament for the therapeutic applications mentioned herein.

As used herein, the term carrier refers to any excipient, diluent, excipient, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use. in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. In the context of this document, the term pharmaceutically acceptable carrier refers to a non-toxic material that does not adversely affect the effectiveness of the composition in accordance with the present invention or the biological activity of the composition in accordance with the present invention. In accordance with specific embodiments, in light of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody-based pharmaceutical composition may be used in the present invention.

Pharmaceutically acceptable acid/anionic salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, estholate, fumarate, glyceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsilate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, tosylate, and triethiodide. Organic or inorganic acids also include, without limitation, hydroiodic, perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, saccharic or trifluoroacetic acid.

Pharmaceutically acceptable base/cationic salts include, without limitation, aluminum salts, 2-amino-2-hydroxymethylpropane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethane or TRIS), ammonium salts, benzathine, t-butylamine, salts calcium, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium salts, L-lysine, magnesium salts, meglumine, N-methylD-glucamine, piperidine, potassium salts, procaine, quinine, sodium salts, triethinolamine or zinc salts.

In some embodiments, the invention provides pharmaceutical compositions containing the conjugates of the invention in an amount of from about 0.001 mg/ml to about 100 mg/ml, from about 0.01 mg/ml to about 50 mg/ml, or from about 0.1 mg/ml up to about 25 mg/ml. Pharmaceutical formulations may have a pH of about 3.0 to about 10, such as about 3 to about 7 or about 5 to about 9. The composition may further contain at least one ingredient selected from the group consisting of a buffer system, a preservative( s), agent(s), tonicity regulator(s), chelating agent(s), stabilizer(s), and surfactant(s).

A formulation containing pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, in Remington: The Science and Practice of Pharmacy (eg, edition 21 (2005) and any subsequent editions). Not having a restrictive ha

Examples of additional ingredients include buffer solutions, diluents, solvents, tonicity agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used in formulating the pharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is a liquid formulation.

A preferred example of a liquid formulation is an aqueous formulation, i. composition containing water. The liquid formulation may contain a solution, a suspension, an emulsion, a microemulsion, a gel, and the like. The aqueous composition usually contains at least 50% wt./wt. water, or at least 60, 70, 75, 80, 85, 90% wt./wt., or at least 95% wt./wt. water.

In one embodiment, the pharmaceutical composition can be formulated as an injectable preparation that can be administered, for example, via a syringe or infusion pump. The injection may, for example, be delivered subcutaneously, intramuscularly, intraperitoneally or intravenously.

In another embodiment, the pharmaceutical composition is a solid formulation, such as a lyophilized or spray-dried composition, that can be used as is or to which diluents and/or diluents are added by a physician or patient prior to use. Solid dosage forms may include tablets, such as compressed tablets and/or coated tablets, and capsules (eg, hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, sachets, dragees, powders, granules, lozenges or dissolving powders.

Dosage forms may be immediate release forms, in which case they may contain a water-soluble or dispersible carrier, or they may be delayed release, sustained release, or modified release, in which case they may contain water-insoluble polymers, which regulate the rate of dissolution of the dosage form in the gastrointestinal tract.

In other embodiments, the implementation of the pharmaceutical composition can be delivered intranasally, intraperitoneally or sublingually.

The pH value in the aqueous composition may range from pH 3 to pH 10. In one embodiment of the invention, the pH of the composition is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the composition is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical composition contains a buffer solution. Non-limiting examples of buffer solutions include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine and tris(hydroxymethyl)-aminomethane and mixtures thereof. The buffer solution may be present alone or as part of an aggregate at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific buffer solutions are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a preservative. Non-limiting examples of buffer solutions include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorcresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate , imidourea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomersal, and mixtures thereof. The preservative may be present alone or as part of an aggregate at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific preservatives are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains an isotonic agent. Non-limiting examples of this embodiment include a salt (such as sodium chloride), an amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditol (such as glycerol, 1,2-propanediol, propylene glycol, 1,3-propanediol, 1,3-butanediol), polyethylene glycol (eg PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars may be mono-, di-, or polysaccharides or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, where the term sugar alcohol is defined as a C(4-8) hydrocarbon having at least one hydroxyl group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. Pharmaceutical compositions containing

- 17 041709 each isotonic agent specified in this paragraph are alternative embodiments of the invention. The isotonic agent may be present alone or as part of an aggregate at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific isotonic agents are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof.

The chelating agent may be present alone or as part of an aggregate at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific chelating agents are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In another embodiment, the pharmaceutical composition contains a stabilizer, said stabilizer being carboxy/hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (such as PEG 3350 ), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur compounds such as monothioglycerol), or thioglycolic acid. The stabilizer may be present alone or as part of an aggregate at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific stabilizers are alternative embodiments of the invention.

In additional embodiments, the pharmaceutical composition contains one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term surfactant refers to any molecules or ions that are formed from a water soluble (hydrophilic) portion and a fat soluble (lipophilic) portion. The surfactant may, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants and/or zwitterionic surfactants. The surfactant may be present alone or as part of an aggregate at a concentration of from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific surfactants are alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical composition contains one or more protease inhibitors such as, for example, EDTA (ethylenediaminetetraacetic acid) and/or benzamidine hydrochloride (HCl). The protease inhibitor may be present alone or as part of an aggregate at a concentration of from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific protease inhibitors are alternative embodiments of the invention.

The pharmaceutical composition of the invention may contain an amount of amino acid base sufficient to reduce the formation of polypeptide aggregates during storage of the composition. The term amino acid base refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine) or their analogs. Any amino acid may be present either in free base form or in salt form. Any stereoisomer (ie, L, D, or mixture thereof) of an amino acid base may be present. The amino acid base may be present alone or in combination with other amino acid bases at a concentration of about 0.01 mg/mL to about 50 mg/mL, such as about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific amino acid bases are alternative embodiments of the invention.

The person skilled in the art will also appreciate that the therapeutically effective dose of the conjugates of the present invention or pharmaceutical composition thereof will vary depending on the desired effect. Therefore, one of ordinary skill in the art can readily determine the optimal doses to be administered, and these will vary depending on the particular conjugate used, route of administration, drug concentration, and degree of progression of the disease state. In addition, factors associated with the particular subject being treated, including the subject's age, body weight, diet, and time of administration, lead to the need to adjust the dose to the appropriate therapeutic level.

- 18 041709

For all indications, the conjugates of the invention are preferably administered peripherally at a dose of from about 1 μg to about 5 mg per day in single or divided doses (for example, a single dose can be divided into 2, 3, 4, 5, 6, 7, 8, 9 or 10 subdose) or from about 0.01 µg/kg to about 500 µg/kg, more preferably from about 0.05 µg/kg to about 250 µg/kg, most preferably below about 50 µg/kg. Dosages within these ranges will vary in activity for each agonist and can be easily determined by one of skill in the art. Therefore, the above doses are examples for the average case. Undoubtedly, there may be individual cases requiring the use of higher or lower dosage ranges, which are included in the scope of this invention.

In certain embodiments, the conjugates of the invention are administered at a dose of from about 1 μg to about 5 mg, or at a dose of from about 0.01 μg/kg to about 500 μg/kg, more preferably at a dose of from about 0.05 μg/kg to about 250 mcg/kg, most preferably at a dose below about 50 mcg/kg with a dose of the second therapeutic agent (eg, liraglutide) at a dose of about 1 mcg to about 5 mg, or at a dose of about 0.01 mcg/kg to about 500 mcg/ kg, more preferably at a dose of about 0.05 μg/kg to about 250 μg/kg, most preferably at a dose of about 50 μg/kg.

Pharmaceutically acceptable salts of the conjugates of the invention include conventional non-toxic salts or quaternary ammonium salts derived from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate, dodecyl sulfate, hydrochloride, hydrobromide, lactate, maleate, methanesulfonate, nitrate, oxalate, pivalate, propionate, succinate, sulfate, and tartrate. Basic salts include ammonium salts, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexamine salts, and amino acid salts such as arginine. In addition, basic nitrogen-containing groups can be converted to the Quaternary state using, for example, alkyl halides.

The pharmaceutical compositions of the invention may be administered by any means suitable for the intended purpose. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal or eye instillation. Administration can be by oral route. Suitable soluble formulations for parenteral administration include aqueous solutions of the active conjugates in water soluble form, eg water soluble salts, acidic solutions, alkaline solutions, solutions in water with added dextrose, isotonic carbohydrate solutions, and inclusion complexes with cyclodextrin.

The present invention also covers a method for preparing a pharmaceutical composition, which includes mixing a pharmaceutically acceptable carrier with any of the conjugates of the present invention. The present invention further includes pharmaceutical compositions obtained by mixing one or more pharmaceutically acceptable carriers with any of the conjugates of the present invention.

The conjugates of the present invention may additionally exist in one or more polymorphic or amorphous crystalline forms and are intended to be included within the scope of the invention. In addition, the conjugates can form solvates with, for example, water (ie hydrates) or common organic solvents. As used herein, the term solvate means the physical association of the conjugates of the present invention with one or more solvent molecules. Such physical bonding includes varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, the solvate has the ability to release, for example, if one or more solvent molecules are included in the crystal lattice of a crystalline solid. The term solvate is intended to encompass both solution phase solvates and releasable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

The present invention is intended to include polymorphs and solvates of the conjugates of the present invention. Thus, in the methods of treating the invention, the term administration encompasses the means of treating, alleviating, or preventing the syndrome, disorder, or disease described herein, using the conjugates of the invention, or a polymorph or solvate thereof, obviously included in the scope of the invention, although not specifically described.

In another embodiment, the invention relates to conjugates of the invention for use as a drug.

Included within the scope of the present invention are prodrugs of the conjugates of the present invention. In general, such prodrugs are functional derivatives of conjugates that readily convert to the desired conjugate under in vivo conditions. Thus, in the methods of treatment of the present invention, the term administration encompasses the treatment of the various disorders described using the particular conjugate described, or using a conjugate that has not been specifically described, but which converts to the established conjugate under in vivo conditions after administration to a patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. Bundgaard, Elsevier, 1985.

Moreover, it is intended that, within the scope of the invention, any element specifically referred to

- 19 041709 in relation to the conjugates of the invention, will include all isotopes and mixtures of isotopes of the specified element of natural origin or synthetic origin, with natural abundance or in isotopically enriched form. For example, reference to hydrogen also covers 1H, 211 (D) and ³ H (T). Similarly, references to carbon and oxygen encompass 12C, ^13C , and ^14C , and ^16O , and ^18O , respectively. Isotopes may be radioactive or non-radioactive. Radiolabeled conjugates of the invention may contain a radioactive isotope selected from the group consisting of ³ H, ⁿ C, ¹⁸ F, ¹²² I, ¹²³ 1, 125 I, ¹³¹ I, ⁷⁵ Br, ^{76 Br} , ⁷⁷ Br, and ⁸² Br. The radioactive isotope is preferably selected from the group consisting of ³ H, 11 C and ¹⁸ F.

Some conjugates of the present invention may exist as atropisomers. Atropisomers are stereoisomers obtained by hindered rotation around single bonds, wherein the steric barrier to rotation is high enough to allow isolation of the conformers. It should be understood that all such conformers and mixtures thereof are within the scope of the present invention.

When the conjugates according to the present invention have at least one stereocenter, they may respectively exist as enantiomers or diastereomers. It should be understood that all such isomers and mixtures thereof are within the scope of the present invention.

If mixtures of stereoisomers are formed during the methods for preparing conjugates according to the invention, these stereoisomers can be isolated using conventional techniques such as preparative chromatography. The conjugates may be prepared in racemic form, or the individual enantiomers may be prepared by enantiospecific synthesis or resolution. The conjugates can, for example, be separated into their constituent enantiomers using standard techniques such as formation of diastereomeric pairs by salt formation with an optically active acid such as (-)-di-p-toluoyl-D-tartaric acid and/or (+) -di-p-toluoyl-L-tartaric acid, followed by fractional crystallization and regeneration of the free base. Conjugates can also be separated by formation of diastereomeric esters or amides followed by chromatographic separation and removal of the chiral auxiliary. In an alternative embodiment, the conjugates can be separated using a chiral column by high performance liquid chromatography (HPLC) or SFC. In some cases there may be conjugate rotamers observed by 1H NMR leading to complex multiplets and peak merging in the 1H NMR spectrum.

In the course of any of the methods for preparing the conjugates of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules in question. For these purposes, you can use standard protecting groups, such as the groups described in publications Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is hereby incorporated by reference in its entirety for all purposes. The protecting groups can subsequently be removed at a convenient stage using methods known in the art.

Application methods.

The present invention also provides a method for preventing, treating, delaying or alleviating a disorder, disease or condition or any one or more symptoms of said disorder, disease or condition in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the conjugate or pharmaceutical composition of the invention.

According to specific embodiments, the disease, disorder, or condition may be any disease, disorder, or condition that can be treated with a peptide or compound capable of binding to a monoclonal antibody platform of the present invention. In certain embodiments, the disease disorder or condition is selected from the group consisting of obesity, type I or II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, glucose intolerance (i.e., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, congenital hyperinsulinism (CHI) hypoglycemia, dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and/or lipid levels, osteoporosis , inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and/or eczema.

According to specific embodiments, a therapeutically effective amount refers to an amount of a drug that is sufficient to provide one, two, three, four or more of the following effects:

(i) reducing or alleviating the severity of the disease, disorder or condition being treated, or an associated symptom;

(ii) shortening the duration of the disease, disorder or condition being treated

- 20 041709 nii, or a symptom associated with it;

(iii) preventing the progression of the disease, disorder or condition being treated, or associated symptom;

(iv) regression of the cause of the disease, disorder, or condition being treated, or associated symptom;

(v) preventing the development or onset of the disease, disorder or condition being treated, or a symptom associated therewith;

(vi) preventing recurrence of the disease, disorder or condition being treated, or associated symptom;

(vii) reducing the likelihood of hospitalization of a subject having a disease, disorder or condition being treated, or an associated symptom;

(viii) reducing the duration of hospitalization of a subject having a disease, disorder or condition being treated, or an associated symptom;

(ix) improving the survival of a subject with the disease, disorder or condition being treated, or a symptom associated therewith;

(xi) inhibition or suppression of the disease, disorder or condition being treated, or a symptom associated therewith, in a subject; and/or (xii) enhancing or improving the prophylactic(s) or therapeutic(s) effect(s) of the other therapy.

The therapeutically effective amount or dosage may vary depending on various factors such as the disease, disorder or condition being treated, the means of administration, the target site, the physiological state of the subject (including, for example, age, body weight, health), whether the subject is person or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages were optimally adjusted to optimize safety and efficacy.

As used herein, the terms treat, treating, and treating refer to alleviation or reversal of at least one measurable physical parameter associated with a disease, disorder, or condition that is not necessarily obvious in a subject, but may be visible in a subject. The terms treat, treating, and treating may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a specific embodiment, the terms treat, treating, and treating refer to alleviating, preventing the development or onset, or reducing the duration of one or more symptoms associated with a disease, disorder, or condition. In a specific embodiment, the terms treat, treating, and treating refer to preventing the recurrence of a disease, disorder, or condition. In a specific embodiment, the terms treat, treating, and treating refer to improving the survival of a subject having a disease, disorder, or condition. In a specific embodiment, the terms treat, treating, and treating refer to the elimination of a disease, disorder, or condition in a subject.

In one embodiment, the invention provides a method for preventing, treating, delaying or alleviating obesity or any one or more symptoms of obesity in a subject in need thereof, the method comprising administering to the subject in need an effective amount of a conjugate or pharmaceutical composition of the invention . In some embodiments, the subject's body weight is reduced, for example, in the range of about 0.01% to about 0.1%, in the range of about 0.1% to about 0.5%, in the range of about 0.5% to about 1%, in the range of about 1% to about 5%, in the range of about 2% to about 3%, in the range of about 5% to about 10%, in the range of about 10% to about 15%, in the range about 15% to about 20%, in the range of about 20% to about 25%, in the range of about 25% to about 30%, in the range of about 30% to about 35%, in the range of about 35% to about 40%, in the range of about 40% to about 45%, or in the range of about 45% to about 50%, based on the body weight of the subject prior to administration of any of the conjugates, pharmaceutical compositions, formulations, or medicaments of the invention described herein, or in comparison with control subjects not receiving any of the conjugates, compositions, forms, drugs or combinations of the invention described herein.

In some embodiments, the weight loss is maintained for, for example, about 1 week, for about 2 weeks, for about 3 weeks, for about 1 month, for about 2 months, for about 3 months, for about 4 months, for about 5 months, for about 6 months, for about 7 months, for about 8 months, for about 9 months, for about 10 months, for about 11 months, for about 1 year , for about 1.5 years, for about 2 years, for about 2.5 years, for about 3 years, for about 3.5 years, for about 4 years, for about 4.5 years , for about 5 years, for about 6 years, for about 7 years, for about 8 years, for about 9 years, for about 10 years, for about 15 years, or for

- 21 041709 for about 20 years.

The present invention provides a method for preventing, treating, delaying or alleviating a syndrome, disorder or disease or any one or more symptoms of said syndrome, disorder or disease in a subject in need thereof, said syndrome, disorder or disease being selected from the group consisting of obesity , type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (i.e., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, dyslipidemia, atherosclerosis, diabetic nephropathy, and other risk factors for the cardiovascular system, such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and/or lipid levels, osteoporosis, inflammation, non-alcoholic steatohepatitis (NASH), kidney disease, and eczema, the method comprising administering to a subject in need thereof an effective quantities a conjugate or pharmaceutical composition of the invention.

As used herein, metabolic syndrome refers to a subject having any one or more of the following: high blood sugar (eg, high fasting blood sugar), high blood pressure, abnormal cholesterol (eg, low HDL levels) , abnormal triglyceride levels (eg, high triglyceride levels), large waist (i.e., waist circumference), increased abdominal fat, insulin resistance, glucose intolerance, elevated C-reactive protein levels (i.e., a pro-inflammatory state ) and elevated levels of plasma plasminogen activator inhibitor-1 and fibrinogen (i.e., a prothrombotic state).

The present invention provides a method for reducing food intake in a subject in need thereof, the method comprising administering to the subject in need an effective amount of a conjugate or pharmaceutical composition of the invention. In some embodiments, the subject's food intake is reduced, for example, in the range of about 0.01% to about 0.1%, in the range of about 0.1% to about 0.5%, in the range of about 0.5% to about 1%, in the range of about 1% to about 5%, in the range of about 2% to about 3%, in the range of about 5% to about 10%, in the range of about 10% to about 15%, in the range about 15% to about 20%, in the range of about 20% to about 25%, in the range of about 25% to about 30%, in the range of about 30% to about 35%, in the range of about 35% to about 40%, in the range of about 40% to about 45%, or in the range of about 45% to about 50%, of the subject's food intake prior to administration of any of the conjugates, compositions, forms, drugs, or combinations of the invention described herein , or in comparison with control subjects not receiving any of the conjugates, compositions, forms, drugs, or combinations of the invention described herein.

In some embodiments, the reduction in food intake is maintained, for example, for about 1 week, for about 2 weeks, for about 3 weeks, for about 1 month, for about 2 months, for about 3 months, for about 4 months, for about 5 months, for about 6 months, for about 7 months, for about 8 months, for about 9 months, for about 10 months, for about 11 months, for about 1 year , for about 1.5 years, for about 2 years, for about 2.5 years, for about 3 years, for about 3.5 years, for about 4 years, for about 4.5 years , for about 5 years, for about 6 years, for about 7 years, for about 8 years, for about 9 years, for about 10 years, for about 15 years, or for about 20 years.

The present invention provides a method for reducing glycated hemoglobin (A1C) in a subject in need thereof, the method comprising the steps of: administering to the subject in need an effective amount of a conjugate or pharmaceutical composition of the invention. In some embodiments, the subject's A1C is reduced, for example, in the range of about 0.001% to about 0.01%, in the range of about 0.01% to about 0.1%, in the range of about 0.1% to about 0, 2%, in the range of about 0.2% to about 0.3%, in the range of about 0.3% to about 0.4%, in the range of about 0.4% to about 0.5%, in the range about 0.5% to about 1%, in the range of about 1% to about 1.5%, in the range of about 1.5% to about 2%, in the range of about 2% to about 2.5%, in the range of about 2.5% to about 3%, in the range of about 3% to about 4%, in the range of about 4% to about 5%, in the range of about 5% to about 6%, in the range of about 6% to about 7%, in the range of about 7% to about 8%, in the range of about 8% to about 9%, or in the range of about 9% to about 10%, relative to the A1C of the subject prior to administration of any of the conjugates, compositions, forms, drugs or combinations of the invention described herein, or in comparison with control subjects not receiving l any of the conjugates, compositions, formulations, drugs, or combinations of the invention described herein.

In other embodiments, methods are provided for lowering fasting blood glucose levels in a subject in need thereof, the methods comprising administering to the subject in need thereof an effective amount of a conjugate or pharmaceutical composition of the invention. Fasting blood glucose levels may be reduced to less than about 140 mg/dL to about 150 mg/dL, less than

- 22 041709 about 140 mg/dl to about 130 mg/dl, less than about 130 mg/dl to about 120 mg/dl, less than about 120 mg/dl to about 110 mg/dl, less than about 110 mg/dl up to about 100 mg/dl, less than about 100 mg/dl to about 90 mg/dl, or less than about 90 mg/dl to about 80 mg/dl, relative to the subject's fasting blood glucose levels prior to administration of any of the conjugates, compositions, forms, drugs or combinations of the invention described herein, or in comparison with control subjects not receiving any of the conjugates, compositions, forms, drugs or combinations of the invention described herein.

The present invention provides a method for modulating Y2 receptor activity in a subject in need thereof, the method comprising administering to the subject in need an effective amount of a conjugate or pharmaceutical composition of the invention. As used herein, the term modulation refers to an increase or decrease in receptor activity.

In some embodiments, an effective amount of a conjugate of the invention, or a form, composition, or drug thereof, is administered to a subject in need once a day, twice a day, three times a day, four times a day, five times a day, six times a day. , seven times a day or eight times a day. In other embodiments, an effective amount of a conjugate of the invention or a form, composition or drug thereof is administered to a subject in need thereof once every two days, once a week, twice a week, three times a week, four times a week, five times a a week, six times a week, twice a month, three times a month, or four times a month.

Another embodiment of the invention includes a method for preventing, treating, delaying the onset or alleviating a disease, disorder or syndrome, or one or more symptoms of any of said diseases, disorders or syndromes in a subject in need thereof, the method comprising the steps of administering to the subject in need to that subject an effective amount of the conjugate or pharmaceutical composition of the invention as a combination therapy. In certain embodiments, the combination therapy is the second therapeutic agent. In certain embodiments, the combination therapy is a surgical therapy.

As used herein, the term in combination in the context of administering two or more drugs to a subject refers to the use of more than one therapy.

As used herein, the term combination therapy refers to administering to a subject in need of one or more additional therapeutic agents, or performing to a subject in need of one or more surgical therapies concomitantly with the administration of an effective amount of a conjugate of the invention or a form, composition or drug thereof. In some embodiments, administration of one or more additional therapeutic agents or administration of one or more surgical therapies may occur on the same day as administration of an effective amount of a conjugate of the invention, and in other embodiments, administration of one or more additional therapeutic agents or administration of one or more surgical therapies can be carried out in the same week or the same month as the administration of an effective amount of the conjugate of the invention.

In certain embodiments, in which the disease or disorder is selected from the group consisting of obesity, type II diabetes, metabolic syndrome, insulin resistance, and dyslipidemia, the second therapeutic agent may be an antidiabetic agent. In certain embodiments, the antidiabetic agent may be a glucagon-like peptide-1 (GLP-1) receptor modulator.

The present invention also provides for the prevention, treatment, delay or amelioration of any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof by means of a combination therapy which comprises administering to the subject in need thereof an effective amount of a conjugate or a pharmaceutical composition. of the invention in combination with one or more of the following therapeutic agents: a dipeptidyl peptidase-4 (DPP-4) inhibitor (eg, sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP-1 receptor agonist (eg, short acting GLP-1 agonists such as exenatide and lixisenatide; intermediate acting GLP-1 receptor agonists such as liraglutide; sustained release GLP-1 receptor agonists such as sustained release exenatide, albiglutide, dulaglutide); sodium gluconate cotransporter-2 (SGLT-2) inhibitors (eg, canaglifosin, dapaglifosin, empaglifosin, etc.); bile acid sequestrants (eg, colesevelam, etc.); dopamine receptor agonists (eg, fast release brompiraptine); biguanides (eg metformin, etc.); insulin; oxyntomodulin; sulfonylurea (eg chlorpropamide, glimepiride, glipizide, glyburide, glibenclamide, glibornuride, glizoxepide, glycopyramide, tolazamide, tolbutamide, acetohexamide, carbutamide, etc.); and thiazolidinediones (eg, pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, etc.). In some embodiments, the dose of additional(s) therapeutic(s) agent(s) is reduced when administered in combination with a conjugate of the invention. IN

- 23 041709 In some embodiments, when used in combination with a conjugate of the invention, the additional therapeutic agent(s) may be used at lower doses than when used alone.

In certain embodiments, in which the disease or disorder is selected from the group consisting of obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, glucose intolerance (e.g., glucose intolerance) hyperglycemia, hyperinsulinemia, hypertriglyceridemia, congenital hyperinsulinism (CHI) hypoglycemia, dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol levels and/or lipids, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the second therapeutic agent may be liraglutide.

The present invention provides for the prevention, treatment, delay or amelioration of any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof by means of a combination therapy which comprises administering to the subject in need thereof an effective amount of a conjugate or a pharmaceutical composition of the invention in combination with surgical therapy. In certain embodiments, the surgical therapy may be bariatric surgery (e.g., gastric bypass surgery, such as gastric bypass with Py gastrojejunostomy; sleeve gastrectomy; adjustable gastric banding; duodenal biliopancreatic bypass; intragastric balloon; gastric plication; and combinations thereof ).

In embodiments in which one or more additional therapeutic agents are administered on the same day as an effective amount of the conjugate of the invention, the conjugate or compound of the invention may be administered before or simultaneously with or after the additional therapeutic agent. The use of the term in combination does not limit the order in which drugs are administered to a subject. For example, the first therapeutic agent (for example, the composition described herein) can be administered before (for example, 5, 15, 30, 45 minutes, 1, 2, 4, 6, 12, 16, 24, 48, 72, 96 hours , 1, 2, 3, 4, 5, 6, 8, or 12 weeks), after (for example, after 5, 15, 30, 45 min, 1, 2, 4, 6, 12, 16, 24, 48, 72 , 96 hours, 1, 2, 3, 4, 5, 6, 8 or 12 weeks) administration of the second therapeutic agent to a subject in need thereof or simultaneously with such administration.

Embodiments

The present invention also provides the following non-limiting embodiments.

Embodiment 1 is an isolated antibody, or an antigen-binding fragment thereof, comprising a light chain variable region having fully human Ig V germline gene sequences and a heavy chain variable region having fully human Ig V germline gene sequences, excluding HCDR3, having the amino acid sequence of SEQ ID NO: 18, and the antibody or antigen-binding fragment does not specifically bind to any human antigen under in vivo conditions.

Embodiment 2 is the isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof contains the heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3 and the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 16, 17, 18, 19, 20 and 21, respectively.

Embodiment 3 is an isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 2, wherein the isolated monoclonal antibody comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 12 and a light chain variable domain (VL) having the polypeptide the sequence of SEQ ID NO: 14.

Embodiment 4 is an isolated monoclonal antibody according to any one of Embodiments 1-3 further comprising an Fc region.

Embodiment 5 is the isolated monoclonal antibody of Embodiment 4, wherein the Fc region further comprises an Fc region derived from a human IgG4 Fc region.

Embodiment 6 is the isolated monoclonal antibody of Embodiment 5 wherein the human IgG4 Fc region has substitutions that exclude effector function.

Embodiment 7 is the isolated monoclonal antibody of Embodiment 6, wherein the isolated monoclonal antibody further comprises a modified human IgG4 Fc region containing at least one substitution selected from the group consisting of proline to glutamate at position 233, alanine, or valine for phenylalanine in polo

- 24 041709 234, alanine or glutamate for leucine at position 235, alanine for asparagine at position 297.

Embodiment 8 is the isolated monoclonal antibody of Embodiment 6 or 7 wherein the human IgG4 Fc region contains a serine to proline substitution at position 228.

Embodiment 9 is an isolated monoclonal antibody according to any one of Embodiments 4-8, comprising a heavy chain (HC) having the polypeptide sequence of SEQ ID NO: 13 and a light chain (LC) having the polypeptide sequence of SEQ ID NO: 15.

Embodiment 10 is an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1-9.

Embodiment 11 is a vector containing the isolated nucleic acid of Embodiment 10.

Embodiment 12 is a host cell containing the vector of Embodiment 11.

Embodiment 13 is a method for producing an isolated monoclonal antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell of Embodiment 12 under conditions to produce a monoclonal antibody or antigen-binding fragment thereof, and reconstituting the antibody or antigen-binding fragment thereof from a cell or culture.

Embodiment 14 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1-9, further comprising at least one pharmacologically active group conjugated thereto.

Embodiment 15 is the isolated monoclonal antibody or antigen-binding fragment thereof of Embodiment 14, wherein the pharmacologically active group is a therapeutic peptide.

Embodiment 16 is the isolated monoclonal antibody or antigen-binding fragment thereof of Embodiment 15, wherein the therapeutic peptide is conjugated to a cysteine residue of SEQ ID NO: 18.

Embodiment 17 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 15 or 16 wherein the therapeutic peptide is conjugated to the antibody or antigen-binding fragment thereof via a linker.

Embodiment 18 is the isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 17, wherein the linker is 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 from PEG and built-in heterocycle.

Embodiment 19 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 15-18, wherein the therapeutic peptide is selected from the group consisting of oxyntomodulin, glucagon-like peptide 1 (GLP1), tyrosine-tyrosine (PYY) peptide, exendin (exenatide), amylin (pramlintide), alphamelanocyte-stimulating hormone (MSH), cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y Y1 receptor antagonists (NPY1), neuropeptide Y Y5 receptor antagonists (NPY5), neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3), galanin, cholecystokinin (CCK), orexin, melanin-concentrating hormone (MCH), oxytocin, and stresscopin.

Embodiment 20 is the isolated monoclonal antibody or antigen-binding fragment thereof of Embodiment 19, wherein the therapeutic peptide is oxyntomodulin comprising the polypeptide sequence of SEQ ID NO: 24.

Embodiment 21 is a conjugate containing a monoclonal antibody, or antigen-binding fragment thereof, linked to the therapeutic peptide oxyntomodulin, the conjugate having the structure of SEQ ID NO: 27 as shown in FIG. 11, wherein the mAb is a monoclonal antibody or an antigen-binding fragment thereof according to any one of embodiments 1-9, a] ₂ shows that 1 or 2 of the oxyntomodulin therapeutic peptides are covalently conjugated to the mAb.

Embodiment 22 is a method for preparing an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 15-21, comprising reacting an electrophile, preferably bromoacetamide or maleimide, introduced into the side chain of a therapeutic peptide with the sulfhydryl group of a cysteine residue of SEQ ID NO: 18 a monoclonal antibody or antigen-binding fragment thereof, thereby forming a covalent bond between the therapeutic peptide and the monoclonal antibody or antigen-binding fragment.

- 25 041709

Embodiment 23 is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 14-21 and a pharmaceutically acceptable carrier.

Embodiment 24 is a method for preparing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 14-21, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. .

Embodiment 25 is a kit containing the monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1-9 and 14-21.

Embodiment 26 is a method for increasing the half-life of a therapeutic peptide in a subject, the method comprising the steps of conjugating the therapeutic peptide to a monoclonal antibody or antigen-binding fragment thereof containing the complementarity-determining region 1 of the heavy chain (HCDR1), HCDR2, HCDR3 and determining complementarity light chain region 1 (LCDR1), LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 16, 17, 18, 19, 20 and 21, respectively, wherein the therapeutic peptide is conjugated to a monoclonal antibody or antigen-binding fragment thereof at the cysteine residue of SEQ ID NO : 18.

Embodiment 27 is the method of Embodiment 26 wherein the therapeutic peptide is selected from the group consisting of oxyntomodulin, glucagon-like peptide 1 (GLP1), tyrosine-tyrosine peptide (PYY), exendin (exenatide), amylin (pramlintide), alphamelanocyte-stimulating hormone (MSH), cocaine and amphetamine-regulated transcript (CART), neuropeptide Y Y1 receptor antagonists (NPY1), neuropeptide Y Y5 receptor antagonists (NPY5), neurotensin S, neuropeptide B, neuropeptide W, ghrelin, bombesin-like receptor 3 (BRS3) , galanin, cholecystokinin (CCK), orexin, melanin-concentrating hormone (MCH), oxytocin, and stresscopin.

Embodiment 28 is the method of Embodiment 27 wherein the therapeutic peptide is oxyntomodulin.

Embodiment 29 is the method of claim 28, wherein the oxyntomodulin has the polypeptide sequence of SEQ ID NO: 24.

Embodiment 30 is the method of any one of Embodiments 25-29, wherein the monoclonal antibody comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 12.

Embodiment 31 is a method according to any one of claims 25-30, wherein the monoclonal antibody comprises a heavy chain (HC) having the polypeptide sequence of SEQ ID NO: 13.

Embodiment 32 is the method of any one of Embodiments 25-31, wherein the monoclonal antibody comprises a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 14.

Embodiment 33 is the method of any one of Embodiments 25-32, wherein the monoclonal antibody comprises a light chain (LC) having the polypeptide sequence of SEQ ID NO: 15.

Embodiment 34 is an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising heavy chain complementarity determining region 3 comprising SEQ ID NO: 18, wherein the isolated monoclonal antibody, or antigen-binding fragment thereof, is capable of binding to a therapeutic peptide.

Examples

Example 1 Authentication and Preparation of mAb MSCB97 Selection of PH9L3 VL and PH9H5 VH as initial variable regions for construction.

Light chain variable region (VL) of PH9L3 antibody (SEQ ID NO: 3) (Teplyakov et al., Structural diversity in a human antibody germline library, mAbs Aug-Sep, 8(6):1045-63 (2016)) and variable heavy chain (VH) region of PH9H5 (SEQ ID NO: 4) (Teplyakov et al., Structural diversity in a human antibody germline library, mAbs Aug-Sep, 8(6):1045-63 (2016)) were chosen as initial variable regions for constructing mAbs suitable for conjugation with the peptide. PH9L3 consists entirely of human Ig variable region germline gene sequences and therefore does not contain any sequence mutations derived from in vivo affinity maturation, allowing for high affinity antigen-specific binding. CDR3 PH9H5 is the only segment that does not consist of human variable region germline gene sequences in this VH. CDR3 (SEQ ID NO: 5) PH9H5 is identical to CDR3 of an anti-human CCL2 antibody (CNTO 888, anti-CCL2 neutralizing antibody); see, for example, US 20100074886 A1, the corresponding CNTO 888 specification is incorporated herein by reference in its entirety. Received Fab containing pair PH9H5/PH9L3 VH/VL.

- 26 041709

The PH9L3, PH9H5 sequences and the human V-region and J-region germline sequences with which they are most similar were aligned to determine sequence identity or similarity to germline sequences. PH9H5 was aligned with a chain (SEQ ID NO: 6) of the linked human Ig Ig germline genes IGHV3-23*01 (PubMed ID: M99660) (SEQ ID NO: 7) and human IGHJ1*01 (PubMed ID: J00256) (SEQ ID NO : 8), with the only difference between the amino acid sequence of PH9H5 and the fused human sequence IGHV3-23*01-IGHJ1*01 being in the VH CDR3 region, which in the case of PH9H5 was SEQ ID NO: 5.

PH9L3 was aligned with a chain (SEQ ID NO: 9) of the linked human Ig germline genes IGKV3-11*01 (PubMed ID: X01668) (SEQ ID NO: 10) and IGKJ1*01 (PubMed ID: J00242) (SEQ ID NO: 11), with the only difference being one deviation in the junction of the V gene with the J gene.

Construction and production of Cys-substituted variants of PH9H5 and PH9L3.

PH9H5 VH variants that contain one cysteine substitution at selected CDR residues in all three V region CDRs were designed, generated and cloned as full human IgG1 constant region heavy chains into a mammalian host expression vector. The PH9H5/PH9L3 Fab structure was used to facilitate the selection of replacement CDRs that are more accessible for conjugation, and in some embodiments, additional glycine (Gly) residues were inserted on one side of the introduced cysteine residue to potentially increase the availability of cysteine for conjugation. Similar VL PH9L3 variants were constructed and made, except they were cloned as complete light chains with the human kappa constant region into an expression vector. A total of 24 single cysteine PH9H5 variant expression constructs and 22 single cysteine PH9L3 variant expression constructs were generated. The residues selected for replacement within PH9H5 VH (SEQ ID NO: 4) and within PH9L3 VL (SEQ ID NO: 3) are shown in FIG. 2.

The resulting expression constructs were used to express Cys variants by transiently co-transfecting each PH9H5-based Cys variant HC construct with a wild-type PH9L3 LC construct, or co-transfecting each PH9L3-based Cys variant LC construct with a wild-type HC PH9H5 construct. For initial trial transfections, HEK-derived Expi293 cells were used as the expression host cell in a volume of 20 ml. Most Cys variants of HC and LC showed a good level of expression when determined based on quantitative analysis in the culture of the supernatant.

The five original HC Cys variants (MSCB33-MSCB37) were expressed in Expi293 cells in a volume of 750 ml and the protein variants were purified. The yield after purification and the qualitative properties of the purified variants were almost identical and sufficient for the use of purified proteins in the initial peptide conjugation reactions.

Evaluation of peptide conjugation with HC Cys variants based on PH9H5.

Analytical determination of the mass of the MSCB33 protein and other protein variants showed the presence of cysteine adducts in the cysteine residues intended for conjugation (two per mAb), as well as the absence of a lysine residue at the C-terminus of the HC, which is usually observed in monoclonal antibodies obtained by recombination. To obtain the mAb conjugation variant, adducts were removed using a reduction process designed to preserve the original disulfide bonds in the mAb (see example 3). Initial trial conjugations with human peptide oxyntomodulin (OXM) analog (GCG Aib2, Gly16.24, Arg20, Leu27, Lys30 (PEG ₁₂ )-NH ₂ ) were performed by the maleimide method for all five Cys variants of the HC mAb. The efficiency of conjugation differed between mAb variants, which was qualitatively assessed by the products of the conjugation reaction and the relative percentage of each of them. Compared to other I102C variants containing flanking glycine residues, the highest efficiency, as determined by the highest percentage of homodimer, was observed with MSCB33, while when using the Y103C variants of MSCB35 or MSCB37, conjugation was weak or absent.

Several other Cys variants of HC and LC with cysteine substitutions in various CDRs (substitution of T28C, S30C and S54C in PH9H5_VH (SEQ ID NO: 129) and substitution of S30C and S92C in PHpL3_VL (SEQ ID NO: 128)) were expressed in a large volume in Expi293. Removal of cysteine adducts from these purified proteins by reduction has been difficult and these variants have not been further explored. Due to the problems encountered with most Cys variants and the initial high conjugation efficiency observed with the I102C variant of the PH9H5 mAb (MSCB33), development and further engineering efforts have focused on this particular variant.

Construction of the MSCB33 Fc region.

To reduce Fc function in vivo, MSCB33 was redesigned to include the inactive Fc region of human IgG4_PAA. Human IgG4_PAA has S228P/F234A/L235A mutations on the human IgG4 nG4m(a) allele (based on the IGHG4*01 allele as defined in the IMGT). An expression construct was created with MSCB33 VH fused to the IgG4_PAA Fc region and used along with the same LC expression construct that was used

- 27 041709 for the expression of MSCB33, obtaining IgG4_PAA-eapuaHTa MSCB33, designated as MSCB97. The amino acid sequences of VH, HC, VL and LC of MSCB97 are shown in SEQ ID NOs: 12, 13, 14 and 15, respectively.

An expression test of MSCB97 was performed transiently in a 20 ml volume in Expi293 cells and this mAb variant had a good level of expression. MSCB97 was purified from a large volume of Expi293 cells that were expressed. The yield of MSCB97 after purification was 264.53 mg/L and the quality was 85% monomeric molecules. Yields and quality from subsequent high volume expression steps were the same or better and indicated the stability with which this could be obtained.

Evaluation of the conjugation of peptides in the basis of MSCB97 and the possibility of carrying out the conjugation reaction in various volumes.

The LC-HC disulfide bonds differ between the IgG1 and IgG4 isotypes, so transferring the reduction and maleimide conjugation from the IgG1 mAb MSCB33 to the IgG4 mAb PAA MSCB97 via TCEP reduction and conjugation of the OXM-maleimide probe peptide described above was explored. It is known that the bond formed by maleimide conjugation is potentially reversible, so the bromoacetamide conjugation method has been adapted and successfully used, which leads to the creation of a more stable bond in a volume of 10 mg based on the original MSCB97.

MSCB97 OXM Analog GCG Aib2, Glu16,24, Arg20, Leu27, Lys30-ε-(PEG ₁₂ )-NH ₂ Conjugate (Compound 2: Compound 2 and Conjugate 2 can be used interchangeably herein) (MSCB97 conjugated to H-Aib -QGTFTSDYSKYLDERRARDFVEWLLNTK-(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) ₁₂ NHCOCH ₂ Br)-NH ₂ (SEQ ID NO: 24) to form compound 2 (FIG. 11; SEQ ID NO: 27)), prepared with bromoacetamide method, analyzed to determine the activities of GLP-1R and GCGR in vitro. The activities compared to the reference peptides and reference unstructured peptide conjugates were acceptable and similar to those of the conjugate prepared with the same peptide as MSCB33 (IgG1). This showed that the only difference between MSCB97 (IgG4_PAA) and MSCB33 (IgG1), i.e. difference in isotype does not affect the activity of conjugates containing the same peptide. In addition, these data showed that the desired in vitro activities can be maintained in a peptide-mAb conjugate prepared by the bromoacetamide method, which provides a stable in vivo bond. Other OXM analogs were also conjugated to MSCB97 and the in vitro activities of these conjugates were evaluated. These conjugates had GLP-1R and GCGR activities similar to Compound 2, indicating the ability of MSCB97 to conjugate to various peptides while maintaining peptide activity.

Assessing the binding of the peptide-MSCB97 conjugate to human CCL2.

Although MSCB97 was chosen and designed to lack specific antigen binding, the most likely antigen that the mAb can bind to is human CCL2, based on the origin of the VH CDR3. Whether MSCB97 exhibits any specific binding to CCL2 was assessed using two peptide-MSCB97 conjugates with an OXM peptide analog (compound 2) or a PYY peptide analog (compound 1: compound 1 and conjugate 1 can be used interchangeably herein).

Potential binding to CCL2 was directly measured by surface plasmon resonance (SPR) in which the conjugates were immobilized on the surface using the anti-Fc capture method. Commercially available mouse anti-CCL2 mAbs were used as a positive control, and two non-specific human antibodies, CNTO 9412 and HH3B33, were used as negative controls. All controls were similarly immobilized on the surface, and recombinant human CCL2 was run over immobilized conjugates and controls at concentrations up to 400 nM. Based on predetermined assay criteria, accumulation of CCL2, indicative of specific antigen binding, was observed in the positive control, but not in the negative control and not in the peptide-MSCB97 conjugate (compound 1 or 2). This confirms that MSCB97 in the corresponding therapeutic form of the peptide-mAb conjugate does not bind to human CCL2.

Method for measuring binding using SPR.

Surface plasmon resonance (SPR) binding measurements were performed using a ProteOn XPR36 system (BioRad). The biosensor surface was prepared by binding a mixture of anti-human IgG Fc antibodies (Jackson, cat. no. 109-005-098) and anti-mouse IgG Fc antibodies (Jackson, cat. no. 315-005-046) to the surface of a GLC chip (BioRad, cat. 176-5011) coated with a modified alginate polymer layer according to the manufacturer's instructions for the amine coupling reaction. Approximately 5700 mAb response units (RU) were immobilized. Binding experiments were performed at 25°C in running buffer solution (DPBS; 0.01% P20; 100 μg/ml bovine serum albumin (BSA)). For a kinetic binding experiment, samples (Compound 2, Compound 1 and positive and negative control mAbs) were captured followed by administrations of recombinant human CCL2 (Thermo, cat. no. RMCP120) at concentrations of 5 (in 4-fold serial dilution). The association phase was monitored for

- 28 041709 min at 50 μl/min followed by a 5-minute flow of buffer solution (dissociation phase). The chip surface was regenerated with two 18 s pulses of 100 mM H ₃ PO ₄ (Sigma cat. no. 7961) at 100 μl/min.

The obtained data were processed using the ProteOn Manager software. First, the data was corrected against the background using intermediate plots. Subsequently, a double subtraction of the data standard was performed by injecting buffer solution instead of injecting analytes. Preset assay criteria for determining specific detectable binding of Compound 2, Compound 1, and a positive control to CCL2 required a dose-proportional response and >10 RU signal at the highest concentration and negative control signals <10 RU. Based on the analysis criteria, the results for each sample were reported as Yes or No for dose-dependent binding to human CCL2.

Example 2 mAb expression and purification.

Fully human monoclonal antibodies (mAbs) can be recombinantly expressed in a mammalian expression host cell and purified from cell culture supernatant using standard methods known in the art. For example, a cDNA sequence encoding a light (LC) and a heavy chain (HC) mAb, each including an appropriate signal peptide for secretion, can be cloned into separate mammalian expression vectors or into a single expression vector using standard molecular biology techniques. The expression vectors used can be any of the commercially available vectors such as pEE12.4, pcDNA™3.1(+) or pIRESpuro3 or any non-standard expression vector with similar functionality. In such vectors, transcription of each of the mAb heavy and light chains is induced by any known effective promoter such as the hCMV-MIE promoter. Plasmid DNA for transfection is prepared for separate LC and HC expression constructs or a single construct expressing both LC and HC using standard methods such as the QIAGEN Plasmid Midi Kit.

Purified plasmid DNA is prepared for transfection with a lipid-based transfection reagent such as Freestyle™ Max transfection reagent following the manufacturer's instructions and subsequently transfected into a standard mammalian host cell expression line such as CHO-S or HEK 293-F. If the LC and HC mAb are encoded by separate expression constructs, the two constructs are transfected simultaneously. Before and after transfection, mammalian cells are cultured to maintain or to express the mAb according to standard cell culture methods, with cell densities in the maintenance ranges, and the culture medium to be used and other cell culture conditions determined by the particular mammalian host cell line used. . These parameters are usually specified by the supplier who provided the cell line, or in the scientific literature. For example, CHO-S cells were maintained in CHO Freestyle™ medium in suspension with agitation at 125 rpm in a humidified incubator at 37°C and 8% CO ₂ and separated when the cell concentration ranged from 1.5 to 2.0x106 cells in ml.

Cell culture supernatants from transiently transfected mAb-expressing mammalian cells were harvested a few days after transfection, purified by centrifugation, and filtered. The duration of expression for CHO-S cells is typically four days, but can be adjusted and may differ for different mammalian host cell lines. Large volume transfections (>10 liters) are concentrated 10-fold using a concentrator such as Centramate. The MAb is purified from the clarified supernatant using a Protein A affinity chromatography column, such as HiTrap MabSelect Sure, using standard methods of coupling the mAb to a Protein A resin, washing the resin, and eluting the protein using a low pH buffer. Protein fractions were immediately neutralized by eluting into tubes containing pH 7 buffer and peak fractions were pooled, filtered and dialyzed against phosphate buffered saline (PBS), pH 7.2, overnight at 4°C. After dialysis, the mAb was again filtered (filter with a pore size of 0.2 μm) and the protein concentration was determined by absorption at a wavelength of 280 nm. The quality of the purified mAb protein was assessed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and analytical size exclusion HPLC, and endotoxin concentrations were measured by horseshoe crab amoebocyte lysate (LAL) assay. Purified mAbs were stored at 4°C.

Expression and purification of MSCB97 from transiently transfected CHO cells.

MSCB97 was expressed in ExpiCHO-S™ cells (ThermoFisher Scientific, Waltham, Massachusetts, USA; cat. no. A29127) by transiently transfecting the cells with purified MSCB97 expression construct plasmid DNA according to the manufacturer's recommendations. In other words, ExpiCHO-S™ cells were maintained in suspension in ExpiCHO™ expression medium (ThermoFisher Scientific, cat. no. A29100) in a shaking incubator at 37° C., 8% CO ₂ and 125 vol. /min Cells were subcultured so that dilutions of up to 6.0 x 106 cells per day could be achieved on the day of transfection.

- 29 041709 ml, maintaining cell viability at 98% or higher. Temporary transfections were performed using the ExpiFectamine™ CHO transfection kit (ThermoFisher Scientific, cat. no. A29131). For each ml of diluted cells to be transfected, one microgram of plasmid DNA was used, which was diluted in OptiPRO™ SFM complexing medium. ExpiFectamine™ CHO reagent was used at a 1:3 ratio (v/v, DNA:reagent) and also diluted in OptiPRO™. The diluted DNA and transfection reagent were combined for 1 min, allowing the DNA/lipid complex to form, and then added to the cells. After overnight incubation, ExpiCHO™ growth medium and ExpiFectamine™ CHO enhancer were added to the cells. Cells were cultured with shaking at 32° C. for 5 days before collecting culture supernatants.

Culture supernatants from transiently transfected ExpiCHO S™ cells were harvested by clarification by centrifugation (30 min, 6000 rpm) followed by filtration (0.2 μm PES membrane, Corning). Large volume transfections (5 to 20 L) were first concentrated 10-fold using a Pall Cenhamate tangential flow filtration system. Supernatant was added to Dulbecco's 10x phosphate buffered saline (DPBS, pH 7.2) to a final concentration of 1x before loading the resulting solution onto an equilibrated (DPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare; Little Chalfont, UK) at a relative concentration of ~20 mg protein per ml resin using an AKTA FPLC chromatography system. After loading, the column was washed with 10 column volumes of DPBS, pH 7.2. The protein was eluted with 10 column volumes of 0.1 M Na acetate, pH 3.5. The protein fractions were immediately neutralized by eluting into tubes containing 2.0 M tris(hydroxymethyl)aminomethane (Tris), pH 7, at an elution fraction volume of 20%. The peak fractions were pooled and the pH was adjusted to ~5.5 if necessary with an additional volume of Tris. The purified protein was filtered (0.2 μm) and concentration was determined by absorbance at 280 nm using a BioTek SynergyHT™ spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE and analytical size exclusion HPLC (Dionex HPLC system). Endotoxin concentrations were measured using a turbidimetric LAL test (Pyrotell®-T, Associates of Cape Cod).

Example 3 Conjugation of mAb and PYY cyclic peptides.

Method A. Partial mAb reduction with TCEP.

A solution of mAb at a concentration of 10 mg/ml in Tris-acetate buffer solution (20 ml, 1 mm in EDTA) was treated with 3 equivalents of TCEP. The pH of the solution was adjusted to 6 and after 1 hour at room temperature (RT) high performance liquid chromatography with mass spectrometry (HLCMS) showed that the disulfide adducts at position C102 were completely reduced. The reduced mAb was purified by adsorption to protein A and eluting (4 column volumes with 100 mM acetic acid) to give 180 mg of the reduced mAb.

Conjugation of reduced mAbs and PYY cyclic peptides.

The lyophilized peptide (5 eq. to mAb) was added to the reconstituted mAbs described above. EDTA was added to give a final concentration of 1 mM and the pH was adjusted to 7. The concentration was adjusted to 8 mg/mL and the reaction mixture was kept under gentle stirring for 16 hours at room temperature. TCEP (0.5 eq. to mAb) was added and the reaction mixture was further aged for 4 hours at rt. with gentle stirring, after which the amount of compounds with high molecular weight (MW) was reduced to less than 3%.

The reaction mixture was adjusted to pH 5.5 and purified by ion exchange chromatography on CaptoSP resin using a gradient from 100% A (100 mM Tris acetate, pH 5.5) to 100% B (100 mM Tris acetate, pH 5, 5; 0.5 M NaCl) per 20 column volumes. Fractions containing the desired conjugate were pooled and 140 mg of conjugate was recovered, co-eluting a small amount of unreacted peptide. Final purification was performed by protein A adsorption and elution (4 column volumes, 100 mM acetic acid). The pH of the product was adjusted to 6 to give 120 mg of the conjugate (yield 60%) with >90% purity and <3% macromolecular content.

Method B

Purification of the mAb by hydrophobic chromatography (HIC).

A solution of mAb 20 mg/ml in Tris-acetate buffer solution was loaded onto a hydrophobic chromatography column (TOSOH TSKgel phenyl 7.5 x 21 cm) and eluted with a linear gradient (0-70% B/A, solvent A: 5% iPrOH, 1 M (NH ₄ ) ₂ SO ₄ , 100 mM phosphate buffer, pH 6.0, solvent B: 20% iPrOH, 100 mM phosphate buffer). mAb monomer peaks were pooled, concentrated (5-10 mg/mL) and dialyzed against 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (100 mM, pH 5.5).

Partial recovery with TCEP and conjugation of the reduced mAb to a peptide analog.

To purified mAbs (27 ml, 9.28 mg/ml) was added 4 eq. TCEP followed by the addition of EDTA (1 mM). After 2 hours at room temperature, LCMS showed that the disulfide adducts at the C102 position were completely reduced. Reconstituted mAbs were processed on a desalting column

- 30 041709 Zebra (7x10 ml, 7K MWCO, pre-equilibrate with 100 mM MOPS, pH 5.5) to remove freed cysteines/glutathione. To pooled fractions of reconstituted mAbs (28 ml) was added a solution of PYY cyclic peptide in Milli Q water (6.5 eq. to mAb, 15-20 mg/ml) followed by EDTA (1 mM). The pH of the reaction mixture was adjusted to 7.2-7.4 by adding dropwise 1N. NaOH. The reaction mixture was kept for 18 hours at room temperature with gentle stirring. The reaction mixture was kept for another 12 h after adding an additional 0.5 eq. TCEP to reduce the mAb-mAb dimer formed during the reaction and ensure conversion to the desired mAb homodimer. The pH of the reaction mixture was lowered to pH 5.5 by adding 2 M acetic acid and the crude conjugate was purified by hydrophobic chromatography and eluted with a linear gradient (0-100% B/A, solvent A: 5% iPrOH, 1M (NH ₄ ) ₂ SO ₄ , 100 mM phosphate buffer, pH 6.0, solvent B: 20% iPrOH, 100 mM phosphate buffer). Final purification was carried out by protein A adsorption (PBS) and elution (NaOAc, pH 3.5). The pH of the product was adjusted to 6 and dialyzed against PBS to obtain the final sample (56%).

In an alternative embodiment, the mAb was reconstituted with glutathione and/or cysteine. After removal of the reducing agent by tangential flow filtration (TFF), excess peptide was added to the recovered mAbs, optionally in the presence of 0.2-0.5 equivalents of TCEP.

Example 4 In Vitro Studies

The ability of compound 1 (SEQ ID NO: 2), which is a monoclonal antibody that binds to the PYY cyclic peptide (Fig. 3), to activate NPY receptors in vitro in clonal cells (HEK or CHO) expressing human, rat, mouse Y2 receptors was evaluated. and rhesus monkey and human Y1, Y4 and Y5 receptors. In these assays, PYY3-36, NPY and PP were included as study controls.

Cell lines.

Stable transfected clonal cell lines expressing NPY receptors have been developed for use in cAMP assays. In other words, HEK293 cell lines were transfected using the Lipofectamine 2000 kit (Invitrogen) according to the attached protocol, expression plasmids carrying sequences encoding human Y2 receptor (accession number: NM_000910.2), human Y5 receptor (accession number: NM_006174.2 ), the mouse Y2 receptor (accession number: NM_008731) and the Y2 receptor of the rhesus monkey (accession number: NM_001032832). 48 h after transfection, cells were replated with selective medium (DMEM high glucose and 10% fetal bovine serum (FBS), 50 IU penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 600 μg/ml G418). Cells were kept in selective medium for 2 weeks and then single clones were selected using the limiting dilution method. Subsequently, the transfected cells were cultured in high glucose DMEM (Cellgro) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin, and 600 μg/ ml G418.

In addition, CHO-K1 cell lines expressing human Y1 receptor (p/n 93-0397C2) and human Y4 receptor (p/n 95-0087C2) were purchased from DiscoverX Corporation. DiscoverX cells were cultured in F12 medium (Gibco) enriched with 10% FBS and G418 selection conditions (800 μg/ml). The rat Y2 receptor was expressed in the Glo-Sensor CHO-K1 line obtained from Promega Corporation. These cells were transfected with cAMP plasmid pGloSensor™-23F Camp for fluorescent cAMP assay, but were tested and approved for use in the Perkin-Elmer LANCE cAMP assay. Rat Y2 cells were grown in F12 medium (Gibco) supplemented with 10% FBS and 800 μg/ml G418.

All cell lines were placed in vials (4x106 cells/vial) and stored in liquid nitrogen until use. The day before analysis, the vials were thawed and their contents added to 15 ml of the appropriate medium. Cells were centrifuged at 450xg for 5 min, supernatants were aspirated and cells were resuspended in G418-free medium at a density of 0.2x106 cells/ml. Cells were plated (25 μl/well) in white 384-well collagen-coated Biocoat plates until a final density of 5000 cells/well was reached. Cell plates were incubated overnight in a humidified tissue culture incubator at 37° C. in a 5% CO ₂ /90% O ₂ atmosphere.

Experiment protocol.

The same cAMP analysis was performed for different receptors. The LANCE cAMP kit (Perkin Elmer Corporation; Waltham, MA, USA) was used in all experiments to quantify intracellular cAMP concentrations. On the day of analysis, the cell medium was decanted from the cells and 6 μl of peptides (2X concentration) were added to the wells. The peptides were prepared as 11 dilutions (starting at 100 nM or 10 μM with serial 1:3 dilutions) in stimulation buffer to assess dose response. Stimulation buffer solution consists of

- 31 041709 mm HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 500 μm IBMX and 0.1% bovine serum albumin (BSA) in HBSS (Hank's balanced salt solution). A further 6 μl of stimulation buffer containing forskolin (2X, 5 μM final concentration) and anti-cAMP LANCE antibody (1:100) was added to the cells. After incubation for 25 min at r.t. 12 μl of analytical detection mixture was added to each well. The detection mixture was obtained by diluting the biotin-cAMP (1:750) and Europium-W8044 (1:2250) complexes in the detection buffer solution included in the LANCE cAMP kit. The plate was incubated for 2 hours at RT and then read as a TR-FRET (Time-Resolved Fluorescence Resonant Energy Transfer) analysis on an Envision plate spectrophotometer (320 nm excitation, 615 and 665 nm emission). The fluorescence data of channel 1 (relative fluorescence units at 615 nm) and channel 2 (relative fluorescence units at 665 nm) and their ratio values were exported to an Excel file.

Data analysis.

Data obtained using an Envision plate spectrophotometer was expressed as relative fluorescence units (RFU) calculated as (615/665 nm) x 10,000. All samples were analyzed in triplicate. The data was analyzed using internal Crucible data analysis software developed by Eudean Shaw. Unknown cAMP concentrations in each well were interpolated from standard samples of known cAMP concentrations included in each plate. Parameters such as EC ₅₀ , Log(EC ₅₀ ), Hill curve slope (nH), top and bottom were determined by plotting cAMP concentration values against the logarithm of compound concentration, fitting with a 4-P model using a non-linear application of weighted least squares in R environment (open source http://cran.us.rproject.org/) implemented by Janssen R&D Nonclinical Statistics and Computing.

Table 1

In vitro data

Human isoform activity (EC50 nM) Cross-species activity (EC ₅₀ nM) Compound hY2 hYl hY4 hY5 mouse Y2 Rat Y2 Y2 rhesus monkey Compound 1 0.006 > 2910 > 2910 345.7 0.004 0.04 0.004 PYY3-36 0.08 66.4 136.7 12.3 0.03 0.50 0.06

Example 5. Pharmacokinetics (PK).

FC in DIO mice.

Male DIO C57BL/6N mice (20 weeks old, 14 weeks on a high fat diet) were purchased from Taconic Laboratory. Mice were housed singly per cage with AlphaDri bedding in a temperature controlled room with a 12 hour light/dark cycle. Mice were given ad libitum access to water and maintained on a high fat diet (D12492, Research Diet).

Mice were injected subcutaneously (sc) with 1 mg/kg of compound 1, 3 animals were sacrificed at each time point and blood was collected at t = 4, 8, 24, 48, 72, 120 and 168 hours. In addition, blood was collected from 3 intact animals. Approximately 300 μl of blood from each animal was collected via the jugular vein after decapitation under gas anesthesia induced with a mixture of 70% CO ₂ and 30% O ₂ . Blood samples (approximately 300 µl) were collected in K3E (EDTA) coated Sarstedt Microvette® tubes containing 12 µl (4% ratio) of complete protease inhibitor solution and 3 µl (1% ratio) of DPP-IV inhibitor. Blood samples were placed on liquid ice and then centrifuged at 10,000 rpm for ~4 min under chilled conditions (~5°C) to remove cells within 30 min of collection at each time point and all available plasma was transferred to 96 well plate. The well plate was stored on dry ice and then placed in a freezer at -80°C. The data obtained are presented in table. 2 and in FIG. 4.

Pharmacokinetics in rats.

Compound 1 was administered subcutaneously and intravenously to male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts, USA) at a dose level of 1.0 mg/kg in PBS (pH 7.0-7.6). Approximately 500 μl of blood was collected from three animals at each time point via the saphenous vein (t=l, 4, 24, 48, 72, 96, 168 and 240 hours post-dose). At 336 hours post-dose, blood samples were collected via the jugular vein after decapitation under gas anesthesia induced with a mixture of 70% CO 2 and 30% O ₂ . Blood samples were collected in K3E (EDTA) coated Sarstedt Microvette® tubes containing 20 µl (4% ratio) of complete protease inhibitor solution and 5 µl (1% ratio) of DPPIV inhibitor. Blood samples were placed on liquid ice and then centrifuged at 10,000 rpm for ~4 min under chilled conditions (~5°C) to remove cells within 60 min of collection at each time point, and all available plasma was transferred to 96 well plate. Compound 1 concentrations were measured using the LCMS method described below. The received data is shown

- 32 041709 in the table. 3.

FC in the Javan macaque.

All animals were fasted for at least 8 hours before dosing and the first four hours after blood sampling. Three animals received a single 1 mg/kg IV dose of Compound 1 and three animals received a single 1 mg/kg SC dose of Compound 1. Blood was collected pre-dose and at 1, 6, 10, 24, 36, 48, 72, 120, 168, 240, 336, 432 and 504 hours post-dose. An additional sample was collected 0.5 h after dosing for the IV group. Approximately 1 ml of blood from each animal was collected in CZE (EDTA) coated Sarstedt Microvette® tubes containing 4% protease inhibitor complete solution and 1% DPPIV inhibitor. Blood samples were placed on wet ice and then centrifuged for 30 min after collection at each time point, and the resulting plasma was divided into three parts and transferred in triplicate into a 96-well plate. The well plate was stored on dry ice and then placed in a freezer at -80°C. The data obtained are presented in table. 4 and in FIG. 5.

Intact mass spectrometric analysis for determination of plasma concentrations.

Plasma samples were processed by immunoaffinity capture using anti-human Fc antibody followed by reverse phase LC with high resolution mass spectrometric analysis and full scan on a TripleTOF time-of-flight mass spectrometer. Raw MS spectra were subjected to deconvolution to determine the molecular weights of the components in the injected samples. For quantitative determination, the molecular ion peak of the intact conjugate was used. Standard curve samples and quality control samples were prepared by adding the reference standard to plasma and processing using the same procedure and at the same time as the test samples. PK data for DIO mice, rats, and cynomolgus monkeys are shown in Table 1. 2-4 respectively. PK data for DIO mice and cynomolgus monkeys are also shown in FIG. 4 and 5, respectively.

Table 2 ______________ FC in RJ mice ________________

Analysis Dose (mg/kg) T1/2 h T _Max H S _MAC with NG/ML ACS _after h * ng / ml Intact 1.0 81.05 48 8750 995 460

Table 3

FC in rats

Route of administration Dose (mg/kg) T1/2 h Tmax H ^max ng/ml ACS _after h * ng / ml i/v 1.0 93.0 1 19.5 809.2 PC 1.0 88.7 48 4.2 602.0

Table 4

FC in the cynomolgus monkey

Route of administration Dose (mg/kg) T1/2 h Tmax H Smake ng/ml ATsposl Ch*ng/ml i/v 1.0 178.39 0.67 30 290 1207.39 PC 1.0 104.32 10 5590 712.19

Example 6 In vivo efficacy studies.

Weight loss in dietary obese (DIO) mice.

Acute dosing.

The ability of compound 1 to reduce food intake and body weight in C57B1/6 male DIO mice was evaluated after a single dose. Male DIO C57BL/6N mice (20 weeks old, 14 weeks on a high fat diet) were purchased from Taconic Laboratory. Mice were housed singly per cage with AlphaDri bedding in a temperature controlled room with a 12 hour light/dark cycle. Mice were given ad libitum access to water and maintained on a high fat diet (D12492, Research Diet). Animals were acclimated to the room at least 1 week before the start of the experiment.

The day before dosing, mice were grouped into cohorts of eight animals based on individual body weight. The next day at 15:00-16:00, animals were weighed and treated with vehicle (dPBS, pH 7.2), compound 1 at a dose of 0.1, 0.3, 1.0, 3.0, or 7.5 nmol /kg or dulaglutide at a dose of 0.3 nmol/kg by subcutaneous (s / c) administration. Body weight and food intake were measured at 24, 48 and 72 hours after dosing and percentage weight loss and food intake reduction were calculated. Statistical analysis was performed using two-way repeated measures analysis of variance with Tukey's post hoc test in the Prism software. All data are presented as mean±SEM (standard error of the mean) (FIGS. 6 and 7).

-33 041709

Weight loss in mice with alimentary obesity.

chronic dosage.

The ability of Compound 1 to reduce food intake and body weight, as well as to improve glucose homeostasis was evaluated at multiple dosing in male DIO G57B1/6 mice for 8 days. Male DIO C57BL/6N mice (20 weeks old, 14 weeks on a high fat diet) were purchased from Taconic Laboratory. Mice were housed singly per cage with AlphaDri bedding in a temperature controlled room with a 12 hour light/dark cycle. Mice were given ad libitum access to water and maintained on a high fat diet (D12492, Research Diet). Animals were acclimated to the room at least one week prior to the start of the experiment.

The day before dosing, mice were grouped based on individual body weight. At 15:00-16:00 each of the following 8 days, the animals and food consumed were weighed. Animals were treated with vehicle (dPBS, pH 7.2) or with dulaglutide at a dose of 0.3 nmol/kg via daily subcutaneous injection or with compound 1 at doses of 0.1, 0.3, 1.0, 3.0 nmol/kg via subcutaneous injection once every three days. After 8 days, the mice were fasted for 5 hours and then given an oral bolus of 2 g/kg glucose at t=0. After t=0, 30, 60, 90 and 120 min after glucose administration, the concentration of glucose in the blood was measured, and after t=0, 30 and 90 min blood was taken to measure insulin in plasma. Statistical analysis was performed using one-way ANOVA or two-way repeated measures ANOVA with Tukey's posterior test in the Prism software. All data are presented as mean±SEM (FIGS. 8 and 9 and Tables 5-8).

Table 5

Effect of compound 1 on body weight (g) during 8 days of treatment

Treatment Carrier Wednesday Compound 1 (nmol/kg) Dulaglutide (nmol/kg) Day N/A 0.1 0.3 1.0 3.0 0.3 -1 46.3± 0.6 46.3 ± 0.6 46.7± 0.8 46.3±0.6 46.3 ± 0.6 46.3±0.6 0 46.1±0.7 46.0 ± 0.6 46.8±0.7 46.3±0.6 46.3 ± 0.6 45.9±0.6 1 45.8± 0.7 45.0 ± 0.6 45.2±0.7 43.9± 0.5 43.9± 0.6 44.1 ± 0.6 2 45.2±0.7 43.9± 0.6 43.6± 0.7 42.4 ± 0.7* 41.8 ± 0.7* 42.2± 0.6* 3 45.1±0.7 43.9± 0.6 42.8± 0.6 41.0 ± 0.6* 40.1 ± 0.6* 40.6± 0.6* 4 44.7±0.7 43.4± 0.6 42.0 ± 0.6 39.9±0.6* 38.5± 0.6* 39.3± 0.6* 5 44.5±0.7 43.0 ± 0.6 41.5 ± 0.5* 39.1 ± 0.6* 36.8 ± 0.7* 38.1 ± 0.7* 6 44.5±0.7 43.1± 0.6 41.4± 0.5* 38.6 ± 0.6* 35.5± 0.8* 37.1 ± 0.8* 7 44.3±0.7 43.1 ± 0.7 41.3± 0.5* 38.2 ± 0.6* 34.8± 0.9* 36.4 ± 1.0* 8 45.2± 0.6 46.3±0.7 41.9 ± 0.5* 38.8±0.6 34.7± 10* 36.5± 1.1*

Values represent mean±SEM from 8 animals at each time point and per group.

* p<0.05 compared to vehicle; two-way repeated measures ANOVA, Tukey's multiple comparison test.

- 34 041709

Table 6

Effect of Compound 1 on Blood Glucose Concentration (mg/dL) in the Oral Glucose Tolerance Test (OGTT) after 8 days of treatment

Treatment Carrier Wednesday Comm. 1 Dose (nmol/kg) N/A 0, 1 0.3 10 Time; 0 148± 6 151 ± 4 137 ± 6 117 ± 5 ί after thirty 227 ± 15 213± 9 191 ± eleven 146± 9* stimulus (min) 60 272 ± 12 249 ± 10 208± 10* 203 ± 10* Glu 90 206 ± 9 186± eleven 146± 8* 135± 8* GOAT 120 209 ± 13 194 ± 15 160 ± 8* 136 ± 5* Total AUC (mg/dl/120 min) 26492 ± 1232 24598 ± 849 20786 ± 728* 18285 ± 769* Delta AUC (mg/dL/1 20 min) 8702 ± 1082 6433 ± 677 4379 ± 364* 4319 ± 522* thirty 73 ± eleven* 129 ± 5* 149 ± 10* 93 ± 9* 101 ± 9* 13,703± 913* 5004 ± 585 Dulaglu teed 0.3 76 ± 9* 151 ± 14* 174 ± 17* 104 ± 10* 119 ± 9* 15,758± 1054* 6668 ± 1846

Values represent mean±SEM from 8 animals at each time point and per group.

* p<0.05 compared to vehicle; repeated measures two-way analysis of variance, Tukey's multiple comparison test for glucose values; one-way analysis of variance, Tukey's multiple comparison test for AUC.

Table 7

Effect of Compound 1 on Insulin Concentration (ng/mL) in the OGTT Test Conducted After 8 Days of Treatment

Treatment Carrier Wednesday Dose (nmol/kg g) N/A Time 0 5, 6 ± ate 14 e stimulation: (min) thirty 11.3±3.0 and glucose 90 4.0±0.6 Total AUC (mg/dL/120 min) 711.4 ± 164.1. Comm. 1 0, 1 thirty + 0.4 6.6 ± 0.8* 2.4 + 0.2 415.6 ± 44.2 0.3 2.3 + 0.2 4.0±0.6* 1.7 + 0.2 264.3 ± 33.0* 10 1.3 ± 0.2* 2.2 ± 0.1* eleven ± 0.2 151.6 ± 12.5* thirty 0.4 ± 0.1* 1.2 ± 0.1* 0.4 ± 0.1* 73.9 ± 9.0* Dulaglutide 0.3 0.6 ± 0.1* 1.7 ± 0.2* 0.7 ± 0.2 108.8 ± 12.3*

Values represent mean±SEM from 8 animals at each time point and per group.

* p<0.05 compared to vehicle; repeated measures two-way analysis of variance, Tukey's multiple comparison test for glucose values; one-way analysis of variance, Tukey's multiple comparison test for AUC.

- 35 041709

Table 8

Effect of compound 1 on blood glucose concentration (mg/dl) after

Values represent mean±SEM from 8 animals at each time point and per group.

*p<0.05 compared to vehicle; one-way analysis of variance, Tukey's multiple comparison test.

Example 7 Synthesis strategy for the production of an oxyntomodulin mAb compound (mAb-OXM).

Human oxyntomodulin (OXM) is a 37-amino acid endogenous peptide (SEQ ID NO: 23) that has been shown to have good pharmacological characteristics in patients with type 2 diabetes mellitus. Pharmacological characteristics are provided due to GLP1R and GCGR agonism. Studies of OXM analogs identified a peptide variant that provided good glucose control and weight loss in a rodent model. The in vivo half-life of OXM in humans is on the order of a few minutes. Thus, additional modifications were made to OXM to provide a half-life to allow dosing once a week. The increase in half-life was achieved by increasing the stability of the OXM peptide to proteolysis and increasing the circulating half-life of the peptide through covalent attachment of a monoclonal antibody (mAb). The susceptibility of the peptide to DPP4 proteolysis was reduced by replacing the serine at position 2 with aminoisobutyric acid (Aib). The helical configuration of the peptide was stabilized by introducing a bifurcated salt bridge from Q20R to S16E and Q24E, and the potential susceptibility to oxidation was reduced using the M27L variant. The parent mAb MSCB97 (described above) was chosen to have weak intrinsic antigen binding, with an isoleucine to cysteine point mutation in the HCDR3 region (SEQ ID NO: 18) of the heavy chain (I102C) acting as the point of attachment of the synthetic peptide. The effector function was inactivated using IgG4 isotype PAA. A short oligoethylene glycol spacer was inserted between the mAb and the peptide to ensure unhindered access of the peptide to GLP1R and GCGR. The spacer also provides good water solubility, which facilitates the conjugation process. Attachment of the peptide-spacer complex to the mAb was performed by introducing a reactive bromoacetamide group to the distal end of the glycol spacer. The proximal end of the spacer was attached to the OXM variant via a K30 side chain. The amino acid sequence of glucagon and the OXM peptide variant are shown in SEQ ID NOs: 22 and 24, respectively. Conjugation was performed by reaction with the thiol functional group of the cysteine point mutation in the mAb heavy chain. The amino acid sequences of the mAb heavy and light chains are shown in SEQ ID NOs: 13 and 15, respectively.

Chemical synthesis of the mAb-OXM compound.

The general synthesis scheme for preparing the mAb-OXM compound is shown in FIG. 10.

Preparation of the oxyntomodulin (OXM) peptide variant.

Resin-bound protected peptide was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids (except N-terminal histidine, Na-BocHis(Boc)-OH and Lys30, Na-Fmoc-Lys(ivDDE)-OH) on PAL-PEG resin -polystyrene. In all cases, standard strategies for amino acid activation and Fmoc deprotection were used. After sequence completion, the C-terminal lysine side chain was selectively deprotected by treatment with dilute anhydrous hydrazine in N,N-dimethylformamide (DMF). Fmoc-dPEG ₁₂ -CO ₂ H was coupled to the released amine by activation with diisopropylcarbodiimide (DIC)/ethyl(hydroxyimino)cyanoacetate. The Fmoc group was removed and the resulting amine was bromoacetylated with a solution of bromoacetic anhydride in DMF.

The peptide was removed from the resin while removing all protective groups by treatment with trifluoroacetic acid (TFA) containing phenol, water and triisopropylsilane as scavengers. The crude peptide was isolated by cold ether precipitation and purified by reverse phase high performance liquid chromatography (RP-HPLC) using acetonitrile/water with 0.1% v/v. TFA as eluent. Pure peptide was collected as a fluffy white solid after lyophilization and stored at -80°C.

- 36 041709

Recovery of monoclonal antibody MSCB97.

The monoclonal antibody was isolated using an inserted Cys102 residue linked by a disulfide bridge to additional cysteine or glutathione residues taken from either the cytosol or the growth medium. Thus, prior to conjugation of the OXM peptide variant, it is necessary to pre-reduce these disulfides, followed by removal of unwanted cysteine. Reduction was performed using mAb immobilized on protein A resin beads. Reducing agent (tricarboxyethylphosphine, TCEP) was run through the column at pH 5 until reduction was complete (1 h). The advantage of TEP as a reducing agent under these conditions is that there is little disulfide bond conversion at lower pH, although reduction is efficient. After washing away the by-products, the recovered adsorbed protein was eluted from the column with an acidic buffer solution (sodium acetate at pH 3.5). The reconstituted mAbs were dialyzed twice against 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 5.5.

Smaller test lots were prepared by reconstituting 2.5 equivalents of TCEP with respect to mAb in solution at pH 5. Reactions were allowed to proceed for 2 hours at room temperature. Low molecular weight by-products were removed by gel filtration (PD10 column) and the recovered MSCB97 were eluted with 10 mM MOPS, pH 5.5.

Preparation of the mAb-OXM compound. A solution of reconstituted MSCB97 was added to a 7.6-fold excess of lyophilized OXM peptide variant. A 1 mM EDTA solution was added to protect the reactive thiols from metal-catalyzed oxidation and the pH of the reaction solution was raised to 7.3 by adding MOPS buffer (1 M, pH 8.1). The reaction mixture was kept for 18 hours at room temperature with gentle stirring. The reaction was stopped by adjusting the pH to 5.5 with 2M acetic acid and the crude conjugate was purified by adsorption to Protein A with washing to remove excess peptide, unfolded product and by-products. After elution of the product (sodium acetate, pH 3.6), dialysis was performed into an acetate buffer pH 5.

Three independent syntheses were performed starting at 250, 500 and 500 mg MSCB97 and the products were pooled into one batch.

Analysis of the mAb-OXM compound.

The properties of the resulting mAb-OXM compound were analyzed by (i) analytical hydrophobic chromatography (HIC);

(ii) measuring the mass of the starting material using liquid chromatography with electrospray ionization and mass spectrometry (LC-ESIMS);

(iii) analytical size exclusion chromatography (SEC); and (iv) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Stability in human and monkey plasma.

Plasma peptidases rapidly metabolize OXM. To determine the stability of compound mAbOXM (compound 2) (FIG. 11) to enzymatic degradation in human and monkey plasma, the stability of compound 2 was evaluated ex vivo. Compound 2 was incubated at 20 nM with fresh (not frozen) heparinized plasma at 37° C. for 168 hours. The bioassay was a functional cell bioassay that measured cAMP production in HEK cells transfected with the human GLP-1 receptor. The amount of cAMP produced was directly proportional to the concentration of active OXM analog, and the levels of active OXM analogs in the stability samples were determined in this assay by interpolation from reference standards of known concentrations. This assay was used to evaluate stability relative to a previously determined more stable control (control 1) and a previously determined less stable control (control 2). The data was used to determine relative stability (shown in FIGS. 12 and 13).

Example 8 In Vitro Studies In vitro activity of compound 2 on human, mouse, rat and cynomolgus monkey GLP1 and glucagon receptors.

The potency and species specificity of compounds of interest, including Compound 2, were evaluated in assays using HEK293 cells transfected to provide stable expression of human, cynomolgus, rat, or mouse GLP1 or GCG receptors. For each receptor assay, clonal cells were plated in 384-well plates at appropriate densities in the range of 2000-5000 cells/well. Compounds were diluted in HBSS enriched with 5 mM HEPES, 0.1% BSA and 0.5 mM IBMX and added to cells. Cell plates were incubated at room temperature for 5 or 10 min depending on the cell clone and subsequently lysed for cAMP measurement. cAMP concentrations were quantified using LANCE cAMP kits and an Envision plate spectrophotometer. Standard curves were included in each assay plate to back-calculate cAMP concentrations. Dose response curves were analyzed and EC ₅₀ values were calculated using Graphpad Prism or Crucible software. Data on EC ₅₀ are given in table. 9, 10.

- 37 041709

Table 9

Summary of EC ₅₀ (nM) values from human GLP1 receptor and glucagon cAMP assays

Human GCGR GLP1R Glucagon 0.05±0.01 0.96±0.21 Ser-8 GLP1 > 100 0.07±0.01 OHM 3.01±0.35 2.40±0.59 Compound 2 3.31 ± 0.22 0.43 ± 0.02 Dulaglutide > 100 0.17 ± 0.04

Values are mean±SEM for Experiments 3-7.

Table 10

Summary of EC ₅₀ (nM) values from mouse, rat, and cynomolgus receptor GLP1 and _______glucagon cAMP assays_______

Mouse Rat Javanese macaque GCGR GLP1R GCGR GLP1R GCGR GLP1R 0.09+ 2.13+ 1.57+ 4.73+ 0.03+ 1.96+ Glucagon 0.01 0.42 0.33 0.20 0.002 0.38 0.06± 0.14± 0.08± Ser-8 GLP1 > 100 0.005 > 100 0.03 > 100 0.002 6.31± 1.90+ 12.74+ 0.98+ 0.49+ 0.80+ OHM 2.58 0.31 3.54 0.16 0.08 0.07 Compound 0.82+ 0.11+ 5, 90+ 0.97+ 2.03+ 0.40+ 2 0.38 0.02 1.46 0.13 0.14 0.03 0.035± 0.13± 0.18± Dulaglutide > 100 0.002 > 100 0.03 > 100 0.01

Values are mean±SEM for Experiments 3-8.

Example 9 Activity of other related GPCRs.

Compound 2 activity was evaluated in assays using cells expressing multiple class B GPCRs. Compound 2 was tested in a panel of six in vitro cAMP assays using cells expressing CALCR, PTHR1, PTHR2, CRHR1, CRHR2 and VIPR1. Assays were performed according to the manufacturer's standard procedures, with a positive control included for each receptor tested.

In addition, compound 2 was tested in a stable cell line expressing the human GIP (glucose dependent insulinotropic peptide) receptor. Cells were seeded in 384-well plates and 24 hours later treated with compounds for 5 minutes at room temperature. cAMP concentrations were measured using the CisBio HTRF cAMP kit and the results were analyzed using the GraphPad Prism software. The results are presented in table. 11, 12.

Table 11

Compound 2 activity summary in cAMP assays

Compound Target RC ₅₀ (µM) Hill coefficient Lower dot crooked Upper point of the curve Max. answer Calcitonin CALCR 0.0007764259 1.5254 -6.2187 105 101.04 RTH(1-34) PTHR1 0.001080165 1.8672 -5.9767 101 93.574 Sauvagin CRHR1 0.0003082264 1.8133 -3.2011 102.19 106.16 Sauvagin CRHR2 0.003395158 1.3095 -1.175 102.76 107.04 TIP-39 PTHR2 0.0003934505 1.1032 -3.7746 100.33 100.71 Vasoactive intestinal peptide (VIP) VIPR1 0.0003567715 1.9827 0.96244 105 104.01 Compound 2 CALCR >0.1 0.070838 Compound 2 CRHR1 > 0.1 1.5625

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Compound 2 CRHR2 > 0.1 0.928 Compound 2 PTHR1 >0.1 0.86197 Compound 2 PThR2 > 0.1 2.2789 Compound 2 VIPR1 > 0.1 0.60373

Table 12

Compound 2 activity in the human GIP receptor assay_____

Analysis 1 Analysis 2 EU ₅₀ (nM) R-square EU ₅₀ (nM) R-square gip 2.31 1.00 1.28 0.99 Compound 2 > 100 > 100 > 100 > 100 Oxyntomodulin > 100 > 100 > 100 > 100

Example 10 In vivo studies. Efficacy of a single dose in DIO mice.

Food intake, body weight, glucose tolerance and plasma FGF21 concentration were assessed in DIO mice after a single dose of Compound 2 or the GLP1R agonist dulaglutide. One day before dosing, animals were weighed and divided into groups based on body weight (BW). The mice were dosed as indicated by subcutaneous injection. The next morning, the food was removed and a 6-hour fast began. BW and feed mass (FW) were recorded. Fasting blood glucose was measured and blood was collected for insulin measurement at 12:00. After 1 hour, mice were intraperitoneally injected with glucose (1 g/kg, 20% glucose, 5 ml/kg). An additional 20 μl of L blood was collected by tail bleeding 10 min later for plasma insulin determination. Blood glucose levels were measured with a glucometer 10, 30, 60, and 90 minutes after glucose administration. Subsequently, mice were sacrificed by CO ₂ inhalation and post-mortem blood samples were taken via cardiac puncture. The results are presented in table. 13-19.

Table 13

Effect of compound 2 and dulaglutide on food intake (in grams) in DIO mice up to 18 hours post-treatment

Treatment 24 hours before dosing 18 hours after dosing carrier medium 3.1±0.1 2.9±0.1 Dulaglutide (0.3 nmol/kg) 3.1±0.1 1.5 ± 0.1* ^l Compound 2 (1.0 nmol/kg) 3.2±0.1 2.5 ± 0.1 ^L # Compound 2 (2.0 nmol/kg) 3.4±0.1 2.2 ± 0.1* ^l # Compound 2 (4.0 nmol/kg) 3.2±0.1 1.7 ± 0.1* ^l Compound 2 (8.0 nmol/kg) 3.4±0.1 1.4 ± 0.2* ^l

*p<0.01 compared to vehicle (18 hours post-dose);

l p<0.001 compared with the corresponding value 24 hours before dosing;

# p<0.0001 vs. dulaglutide (0.3 nmol/kg) 18 hours post-dose.

Two-way analysis of variance, Sidak's test for multiple comparisons.

Values are the mean±SEM of 8 animals at each time point and per group, excluding vehicle data given 24 hours prior to dosing (n=7).

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Table 14

Effect of compound 2 and dulaglutide on percent change in body weight in DIO mice 18 hours post-treatment

Treatment Mass change (%) carrier medium -0.7±0.3 Dulaglutide (0.3 nmol/kg) -4.1 ± 0.3* Compound 2 (1.0 nmol/kg) -1.9 ± 0.3* ^l Compound 2 (2.0 nmol/kg) -2.8 ± 0.4* ^l Compound 2 (4.0 nmol/kg) -4.4 ± 0.3* Compound 2 (8.0 nmol/kg) -5.2 ± 0.3* ^l

* p<0.01 compared to vehicle;

λ p<0.05 compared to dulaglutide (0.3 nmol/kg).

Two-way repeated measures ANOVA, Tukey's multiple comparison test. Data shown for 18 hour timestamp only

Percent change in weight was calculated relative to body weight on day 0 (before dosing).

Values are mean±SEM from 8 animals in each group.

Table 15

Effect of Compound 2 and Dulaglutide on Blood Glucose Concentration (mg/dL) in the Intraperitoneal Glucose Tolerance Test (IPGTT) in DIO Mice at 24 Hours Post-Treatment

Treatment Time (min) 0 10 thirty 60 90 carrier medium 215+9 353+20 436 ± 19 424+25 391+25 Dulaglutide (0.3 nmol/kg) 140±7 264±18 267 ± 22 228±12 193 ± 9 Compound 2 (1.0 nmol/kg) 169±6 349 ± 17 411 ± 30 411 ± 19 384±18 Compound 2 (2.0 nmol/kg) 138+11 288+19 349 ± 27 303 ± 29 229+20 Compound 2 (4.0 nmol/kg) 114+8 235+23 237 ± 34 239 ± 30 195+25 Compound 2 (8.0 nmol/kg) 96+7 206+13 168 ± 22 166+19 131+12

Values are mean±SEM from 8 animals at each time point and per group.

Table 16

Actual AUC of blood glucose concentration in IPGTT in DIO mice 24 h after treatment

Treatment Actual AUC of blood glucose concentration carrier medium 16539±1400 Dulaglutide (0.3 nmol/kg) 8471 ± 1204* Compound 2 (1.0 nmol/kg) 19 214 ± 1596 ^l Compound 2 (2.0 nmol/kg) 13805 ± 1840 Compound 2 (4.0 nmol/kg) 9827 ± 1046* Compound 2 (8.0 nmol/kg) 6035 ± 742*

* p<0.05 compared to vehicle;

λ p<0.05 compared to dulaglutide (0.3 nmol/kg) and JNJ-64151789 (4.0 and 8.0 nmol/kg).

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM from 8 animals in each group.

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Table 17

Effect of compound 2 and dulaglutide on glucose concentration after 6-hour fasting (mg/dL) in DIQ mice 24 hours after treatment

Treatment Glucose concentration (mg/dl) carrier medium 215±9 Dulaglutide (0.3 nmol/kg) 140±7* Compound 2 (1.0 nmol/kg) 169 ± 6* Compound 2 (2.0 nmol/kg) 138 ± 11* Compound 2 (4.0 nmol/kg) 114 ± 8* Compound 2 (8.0 nmol/kg) 96 ± 7* ^l

* p<0.05 compared to carrier medium;

^Λ p<0.05 compared to dulaglutide (0.3 nmol/kg).

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM from 8 animals in each group.

Table 18

Effect of Compound 2 and Dulaglutide on Plasma Insulin Concentrations (ng/mL) in DIO Mice Before Sugar Load (24 h post-treatment) and 10 min after sugar load during IPGTT

Treatment Plasma insulin concentration (ng/ml) Before the sugar load 10 minutes after the sugar load carrier medium 2.7±0.4 2.0±0.3 Dulaglutide (0.3 nmol/kg) 1.6 + 0.2 1.7 + 0.2 Compound 2 (1.0 nmol/kg) 2.8±0.4 1.8±0.2 Compound 2 (2.0 nmol/kg) 2.3±0.5 2.9±0.4 Compound 2 (4.0 nmol/kg) 2.1±0.2 2.5±0.5 Compound 2 (8.0 nmol/kg) 1.5±0.4 2.7±0.3

Values are mean±SEM from 8 animals in each group.

Table 19

Effect of Compound 2 and Dulaglutide on Plasma FGF21 Concentration (ng/mL) in DIO Mice 26 Hrs Post-Treatment

Treatment Plasma FGF21 concentration (ng/ml) carrier medium 0.4 ± 0.1 Dulaglutide (0.3 nmol/kg) 0.8 ± 0.2 Compound 2 (1.0 nmol/kg) 0.7 ± 0.1 Compound 2 (2.0 nmol/kg) 1.5±0.3 Compound 2 (4.0 nmol/kg) 2.1 ± 0.4 ^L Compound 2 (8.0 nmol/kg) 3.5±0.3*

* p<0.0001 compared to all groups;

^Λ p<0.05 compared to all groups except compound 2 (2.0 nmol/kg).

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM from 8 animals in each group.

Example 11 In vivo studies. Multiple dosing in DIO mice.

The effects of the dual GLP1R/GCGR agonist Compound 2 and the GLP1R agonist dulaglutide in DIO mice were monitored during multiple dosing for 9 days. To control dosing frequency, all animals were dosed daily. The frequency of administration was determined based on PK data in the DIO mouse for compound 2 and dulaglutide. Animals in the dulaglutide group received this drug daily. Animals treated with vehicle were dosed with vehicle daily.

-41 041709

Animals receiving Compound 2 at 1.0, 2.0, or 4.0 nmol/kg were treated with compound every third day, and on non-treated days they received vehicle. One day before dosing, the animals were weighed and divided into groups according to body weight. All mice were dosed as indicated by subcutaneous injection at 13:00-15:00. In these treatment groups, food intake and body weight were monitored daily at 13:00-15:00.

Starting 24 hours after the first dose, three groups of mice received the same diet (PF) as animals treated with compound 2 at a dose of 1.0, 2.0 or 4.0 nmol/kg. The amount of food in the container for mice receiving the same diet was adjusted to match the average amount of food consumed over the previous 24 hours by mice in the compound treated group to which they were matched. Animals from all PF groups were injected with vehicle daily. On day 9 (or day 10 for the same feeding group), the animals were fasted for 6 hours and their body weight and fasting glucose were measured. Blood was collected for measurements of insulin, FGF21 and plasma lipids. Data on food intake, body weight, body composition, fasting glucose concentrations, fasting insulin concentrations, fasting FGF21 concentrations, and fasting plasma lipid concentrations are summarized in Table 1. 20-26.

Table 20

Effect of compound 2 and dulaglutide on daily food intake (in grams) in DIO mice during 9 days post-treatment

Time (days) Treatment 0 1 2 3 4 5 6 7 8 9 Carrier Wednesday 2.7 ± 0.1 2.9±0.0 3, 1 ± 0.1 2.9 ± 0, 1 2.9 ± 0, 1 3.0 ± 0.1 2.9 ± 0.1 3.0 ± 0, 1 3.0 ± 0, 1 2.9 ± 0.1 Dulaglutide (0.3 nmol/kg) 3.1+ 0.2 1.5 + 0.2* 1.3 + 0.2* 1.7 + 0.3* 1.9 + 0.2* 2.1 + 0.2* 2.2± 0.2* 2.3 ± 0.2 2.9+ 0.4 2.5± 0.2 Compound 2 (1.0 3.1±0.1 +1 =#= CO 2.5± 0, 1*# 2.6± 0.2 2.4±0.1# 2.7±0.2# 2.6 ± 0, 1# 2.9±0.2# 2.8±0.1# 2.7 ± 0.1 nmol/kg) Compound 2 (2.0 nmol/kg) 3.1± 0.1 2.0 ± 0.1* 2.0 ± 0.1* ^l 2.7± ^0.1L 2.5 ± 0.2 ^L 2.3 ± 0.1 2.5± 0.2 2.7 ± 0, 1 2.8± 0.3 2.7 ± 0.1 Compound 2 (4.0 nmol/kg) 2.9±0.2 1.5± 0.1* 1.5±0.2* 2.1 ± 0.2* 1.7 ± 0.1* 1, 6 ± 0.1* 1.6± 0.1* 1.8 ± 0.2* 2.2 ± 0.3* ^l 2.1 ± 0.1*

* p<0.05 compared to vehicle;

^Λ p<0.05 compared to dulaglutide (0.3 nmol/kg);

# p<0.05 compared to compound 2 (4.0 nmol/kg).

Two-way repeated measures ANOVA, Tukey's multiple comparison test.

Animals from all PF groups received average amounts of food (data not shown) relative to their respective treatment groups.

Values are mean±SEM from 6-10 animals at each time point and per group.

- 42 041709

Table 21

Effect of compound 2 and dulaglutide on percent change in body weight in DIO mice during 9 days of treatment

Time (days) T 0 1 2 3 4 5 6 7 8 9 V 0.0 -0.1±0.2 -0.1±0.2* -0.2±0.2* -0.7±0.3* -0.5±0.3* 0.2 ± 0.5* 0.1 ± 0.6* 0.0 ± 0.5* -1.7±0.6* 1 0.0 -3.7± 0.3 -6.2±0.6 -7.8±0.8 -9.2±0.8 -10, 1 ±0.8 -10.2 ±0.8 -10.7 ±1.0 -11.1 ±0.9 -13.1 ±1.1 2 0.0 -1.9± 0.4 -2.9 ± 0.5 ^L -3.2 ± 0.4 ^L -4.4 ± 0.7 ^L & -4.7 ± 0.8 ^L & -4.5 ± 0.8 ^L & -5.3 ± 1.0 ^L & -5.2 ± 1.1 ^L & — 6, 6 ± 1.1 ^l & 3 0.0 -1.6± 0.3 -2.7 ± 0.4 ^L -3.5 ± 0.4 ^L -3.8 ± 0, Z ^l -3.8 ± 0.4 ^L -3.9± ^0.5L -3.5 ± 0.5 ^L -3.4 ± 0.5 ^L -4.5 ± 0.5 ^L 4 0.0 -2.8± 0.4 -4.7±0.5 -4.9 ± 0.6 ^L -7.1±0.7 -8.0±0.8 -8.0±0.8 -9.1±0.8# -10.0 ±0.9# -11.5 ±0.9# 5 0.0 -3.0 ± 0.4 -4.5±0.4 -5.1 ± 0.5 ^L -5.3 ± 0.5 ^L -5.8 ± 0.5 ^L -6, 1 ± 0.5 ^L -5.8 ± 0.5 ^L -6.2 ± 0.4 ^L -6.9 ± 0.4 ^L 6 0.0 -3.7± 0.3 -6, 8 ± 0.3 -8.8 ± 0.5$ -11.6 ± 0.6#$ -14.2± ^0.7L #$ -15.6 ± 0.9 ^L #$ -18.4 ± 1.2 ^L #$ -20.4 ± ^1.5L #$ -21.9 ± 1.6 ^L #$ 7 0.0 -3.6± -5.6± -6, 9 ± -7.9± -9.1± -10.5 -11.4 -12.3 -13.3 0, 1 0.1 0.1 0.2 0.2 ±0.3 ±0.3 ±0.3 ±0.3

T: treatment;

V: carrier medium;

1: dulaglutide (0.3 nmol/kg);

2-3: compound 2 (1.0 nmol/kg);

4-5: compound 2 (2.0 nmol/kg);

6-7: compound 2 (4.0 nmol/kg);

* p<0.01 compared to all other treatments;

λ p<0.05 compared to dulaglutide (0.3 nmol/kg);

# p<0.05 compared to the corresponding PF group;

$ p<0.05 compared to JNJ-64151789 (1.0 and 2.0 nmol/kg);

& p<0.05 compared to JNJ-64151789 (2.0 nmol/kg).

Two-way repeated measures ANOVA, Tukey's multiple comparison test.

The % weight change was calculated based on body weight on day 0 (before dosing).

Values are mean±SEM from 10 animals at each time point and per group.

- 43 041709

Table 22

Effect of compound 2 and dulaglutide on body composition (weight and percent _____ body weight) in DIO mice after multiple dosing _____

Time (days) Weight (g) % body weight Treatment Lean tissue Fats Lean tissue Fats carrier medium 24.0±0.2 10.6±0.5 57.5±0.8 25.4±0.8 Dulaglutide (0.3 nmol/kg) 22.1 ± 0.3* 7.6 ± 1.1 61.0±1.7 22.8±1.2 Compound 2 (1.0 nmol/kg) 23.1±0.5 9.5±1.3 57.4±2.5 23.1 ± 2.5 Compound 2 (1.0 nmol/kg) PF 23.9±0.3 10.6±0.3 57.7 ± 0.7 25.5±0.5 Compound 2 (2.0 nmol/kg) 22.2 ± 0.4* 8.2±0.7 59.7±1.1 22.0±1.6 Compound 2 (2.0 nmol/kg) PF 23.4±0.1 9.8±0.4 58.5±0.8 24.4±0.8 Compound 2 (4.0 20.9± 7.4±0.7 61.9±2.4 21.6 ± 1.1 nmol/kg) 0.3* Compound 2 (4.0 nmol/kg) PF 21.4±0.6 9.3±0.5 56.8 ± 1.4 24.7±1.0

* p<0.05 compared to vehicle.

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM from 5 animals in each group.

Table 23

Effect of compound 2 and dulaglutide on glucose concentration after 6-hour fasting (mg/dL) in DIO mice after multiple dosing

Treatment Glucose concentration (mg/dl) carrier medium 170±5 Dulaglutide (0.3 nmol/kg) 123±5* Compound 2 (1.0 nmol/kg) 164 ± 5 ^L Compound 2 (1.0 nmol/kg) PF 168 ± 7 ^L Compound 2 (2.0 nmol/kg) 140±7* Compound 2 (2.0 nmol/kg) PF 155 ± 6 ^L Compound 2 (4.0 nmol/kg) 98±4*# Compound 2 (4.0 nmol/kg) PF 133 ± 6*

* p<0.05 compared to vehicle;

^Λ p<0.05 compared to dulaglutide (0.3 nmol/kg);

# p<0.05 compared to JNJ-64151789 (4.0 nmol/kg) PF.

One-way analysis of variance, Tukey's multiple comparison test

Values are mean±SEM from 10 animals in each group.

- 44 041709

Table 24

Effect of Compound 2 and Dulaglutide on Plasma Insulin Concentration after 6 Hour Fasting (ng/mL) in DIO Mice after Multiple Dosing

Treatment Plasma insulin concentration (ng/ml) carrier medium 3.0±0.4 Dulaglutide (0.3 nmol/kg) 1.4 ± 0.2* Compound 2 (1.0 nmol/kg) 2.0±0.3 Compound 2 (1.0 nmol/kg) PF 2.0±0.1* Compound 2 (2.0 nmol/kg) 1.1 ± 0.1* ^l Compound 2 (2.0 nmol/kg) PF 2.3±0.3 Compound 2 (4.0 nmol/kg) 0.5±0.1* Compound 2 (4.0 nmol/kg) PF 1.2 ± 0.1*

* p<0.05 compared to carrier medium;

^Λ p<0.05 compared to compound 2 (2.0 nmol/kg) PF.

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM from 10 animals in each group.

Table 25

Effect of compound 2 and dulaglutide on plasma FGF21 concentration after 6-hour fasting (ng/ml) in DIO mice after multiple dosing

Treatment FGF21 (ng/ml) carrier medium 0.7 ± 0.1 Dulaglutide (0.3 nmol/kg) 0.5±0.1 Compound 2 (1.0 nmol/kg) 0.7±0.2 Compound 2 (1.0 nmol/kg) PF 0.4 ± 0.1 Compound 2 (2.0 nmol/kg) 0.6±0.1 Compound 2 (2.0 nmol/kg) PF 0.2+0.1 Compound 2 (4.0 nmol/kg) 1.5 ± 0.3* ^l Compound 2 (4.0 nmol/kg) PF 0.3 ± 0.1

* p<0.05 compared to all groups;

^Λ p<0.05 compared to compound 2 (4.0 nmol/kg) PF.

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM of data from 10 animals from each group except Compound 2 (1.0 nmol/kg) and Compound 2 (4.0 nmol/kg) PF where N=9.

-45 041709

Table 26

Effect of compound 2 and dulaglutide on plasma lipid concentrations in DIO mice after multiple dosing

Time (days) Treatment Free fatty acids (µmol/l) Free glycerin (mmol/l) Triglycerides (mg/dl) Total cholesterol (mg/dl) carrier medium 66.2 ± 3.9 0.6±0.0 30.1 ± 2.8 142.1 ± 5.2 Dulaglutide (0.3 nmol/kg) 73.2 ± 12.0 0.5±0.1 24.1 ± 2.3 146.1 ± 3.3 Compound 2 (1.0 nmol/kg) 71.8±5.4 0.5 ± 0.0* 32.5±4.5 149.1 ± 2.3 Compound 2 (1.0 nmol/kg) PF 58.8 ± 6.1 0.6±0.1 33.7 ± 4.1 161.4±2.0 Compound 2 (2.0 nmol/kg) 73.8 ± 4.6 0.5 ± 0.0* 30.4 ± 3.4 128.0 ± 6, 6# Compound 2 (2.0 nmol/kg) PF 84.8 ± 25.3 0.6 ± 0.0* 34.5 ± 3.3 149.9±4.4 Compound 2 (4.0 nmol/kg) 71.2 ± 6.4 0.4 ± 0.0* 16.1 ± 2.0$ 85.7 ± 4.9 ^L Compound 2 (4.0 nmol/kg) PF 102.6 ± 14.5 0.7±0.0 31.8 ± 1.1 137.8±5.7

* p<0.05 compared to compound 2 (4.0 nmol/kg) PF;

λ p<0.05 compared to all other groups;

# p<0.05 compared to compound 2 (1.0 nmol/kg) and compound 2 (2.0 nmol/kg) PF;

$ p<0.05 compared to all groups except the dulaglutide group (0.3 nmol/kg).

One-way analysis of variance, Tukey's multiple comparison test.

Values are mean±SEM from 10 animals/group for free fatty acids, 8-10 animals/group for free glycerol, 9-10 animals/group for triglycerides and total cholesterol.

Example 12 In vivo studies. Single dose efficacy in cynomolgus monkeys.

In cynomolgus monkeys not taking any biological agents, food intake was monitored daily for three weeks prior to treatment. At the start of treatment, animals were divided into four groups to obtain a similar mean body weight (n=8-9, mean body weight 7.4-7.9 kg for each group). Animals in each group received one subcutaneous dose corresponding to 1, 3, 5, or 7.5 nmol/kg Compound 2. Food intake was monitored daily for an additional 14 days. Body weight was measured on the day of dosing and 4, 7, 10, 14 and 21 days after dosing. The percentage change in food intake was determined daily by comparing food intake on that day with the average daily food intake during the seven days prior to dosing. The results are shown in FIG. 14 and 15.

Example 13 Pharmacokinetic (PK), Pharmacokinetic (PK)/Pharmacodynamic (PD) analysis.

FC in DIO mice.

Compound 2 time course was evaluated in DIO mice (Table 27). Animals (n=3 per time point) received s.c. injection at a dose of 10 nmol/kg.

-

Claims (20)

Table 27 Plasma time course (nM) of Compound 2 measured by bioassay and immunoassay. Time watch) Content in plasma (nM) Biological analysis Immunological analysis 0 N/A N/A 7 6.9±2.0 16.7 ± 6.7 24 32.6 ± 3.5 71.0±8.9 48 55.5±2.4 111.9 ± 16.4 72 45.3 ± 4.0 95.7 ± 9.7 96 42.3 ± 14.5 86.8 ± 6.8 120 26.5 ± 6.9 58.1 ± 14.2 Values are mean±SEM from 3 animals at each time point and per group. Pharmacokinetics in rats. Pharmacokinetic parameters of compound 2 were evaluated in Sprague-Dawley rats. Bioassay data from samples collected at 0, 24, 48, 72, 144, 216, 312, 408, and 504 hours post dosing were used to determine pharmacokinetic parameters (Table 28). Table 28 Pharmacokinetic parameters of compound 2 in Sprague-Dawley rats Parameter Subcutaneous administration Intravenous administration Smake (NM) 5.7±2.1 N/A Tmax (CHESY) 64.0 ± 13.9 N/A AUCo-οο (nM*hours) 456 ± 154 865 ± 185 ti/2 (hours) 38.5 ± 12.8 26.2 ± 6.2 Data are mean +/- standard deviation, content values are taken from biological analysis. FC of the Javan macaque. The pharmacokinetic parameters of compound 2 were evaluated in the cynomolgus monkey. Animals received an i.v. or s.c. injection at a dose of 6.45 nmol/kg. Bioassay data from samples collected at 0, 1, 6, 24, 48, 72, 120, 240, 336, 432, and 528 hours post dosing were used to determine pharmacokinetic parameters (Table 29). Table 29 Pharmacokinetic parameters of compound 2 in cynomolgus monkeys Parameter Subcutaneous administration Intravenous administration Smake (NM) 89.1 ± 8.7 N/A Tmax (CHESY) 56.0 ± 13.9 N/A AUCo-οο (nM*hours) 11700±1380 14 700 ± 5260 ti/2 (hours) 51.9±6.2 55.9 ± 41.7 Values are mean ± standard deviation. Those skilled in the art will appreciate that changes can be made to the embodiments described above without departing from the general inventive concept presented herein. Thus, it is to be understood that the present invention is not limited to the specific embodiments described, but is intended to cover modifications within the spirit and scope of the present invention as defined herein. All documents cited here are incorporated by reference. CLAIM 1. An isolated non-targeted monoclonal antibody or antigen-binding fragment thereof, containing the complementarity determining region 1 of the heavy chain (HCDR1), HCDR2, HCDR3 and the complementarity determining region 1 of the light chain (LCDR1), LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 16, 17 , 18, 19, 20, and 21, respectively. - 47 041709 2. An isolated non-targeted monoclonal antibody or an antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 12 and a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 14 . 3. An isolated untargeted monoclonal antibody according to claim 1 or 2, additionally containing an Fc region. 4. The isolated non-targeted monoclonal antibody of claim 3, comprising a heavy chain (HC) having the polypeptide sequence of SEQ ID NO: 13 and a light chain (LC) having the polypeptide sequence of SEQ ID NO: 15. 5. An isolated nucleic acid encoding a non-targeted monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4. 6. A vector containing an isolated nucleic acid according to claim 5, wherein the vector is for expressing the isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof. 7. A host cell containing the vector according to claim 6, wherein the host cell is designed to express the vector. 8. A method for obtaining an isolated non-targeted monoclonal antibody or its antigen-binding fragment, including culturing the host cell according to claim 7 under conditions that allow obtaining a monoclonal antibody or its antigen-binding fragment, and recovering the non-targeted monoclonal antibody or its antigen-binding fragment from the cell or culture. 9. A conjugate comprising a non-targeted monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 and at least one pharmacologically active group conjugated to it, wherein the conjugate has an increased half-life compared to the pharmacologically active group individually. 10. The conjugate of claim 9, wherein the pharmacologically active group is a therapeutic peptide. 11. The conjugate of claim 10, wherein the therapeutic peptide is conjugated to a monoclonal antibody or antigen-binding fragment thereof at a cysteine residue of SEQ ID NO: 18. 12. The conjugate of claim 10 or 11, wherein the therapeutic peptide is conjugated to the monoclonal antibody or antigen-binding fragment thereof via a linker. 13. The conjugate of claim 12, wherein the linker is 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 an inserted heterocycle. 14. The conjugate of any one of claims 10-13, wherein the therapeutic peptide is selected from the group consisting of oxyntomodulin, glucagon-like peptide 1 (GLP1), exendin (exenatide), amylin (pramlintide), alpha melanocyte stimulating hormone (MSH), cocaine - and amphetamine-regulated transcript (CART), neuropeptide Y Y1 receptor antagonists (NPY1), neuropeptide Y Y5 receptor antagonists (NPY5), 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 of claim 14, wherein the therapeutic peptide is an oxyntomodulin containing the polypeptide sequence of SEQ ID NO: 24. 16. A method for producing a conjugate according to any one of claims 10-15, comprising introducing an electrophile, preferably bromoacetamide or maleimide, introduced into the side chain of a therapeutic peptide, reacting with a sulfhydryl group of a cysteine residue of SEQ ID NO: 18 of a monoclonal antibody or its antigen-binding fragment to form , thus, a covalent bond between the therapeutic peptide and the monoclonal antibody or antigen-binding fragment thereof. 17. A pharmaceutical composition containing a therapeutically effective amount of a conjugate according to any one of claims 9 to 15 and a pharmaceutically acceptable carrier. 18. A method for producing a pharmaceutical composition containing a conjugate according to any one of claims 9 to 15, including the combination of a monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. 19. A method for increasing the half-life of a therapeutic peptide in a patient, comprising conjugating the therapeutic peptide to a monoclonal antibody or antigen-binding fragment thereof containing complementarity-determining region 1 of the heavy chain (HCDR1), HCDR2, HCDR3 and complementarity-determining region 1 of the light chain (LCDR1), LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NO: 16, 17, 18, 19, 20 and 21, respectively, and the therapeutic peptide is conjugated to a monoclonal antibody or its antigen-binding fragment in the cysteine residue of SEQ ID NO: 18. 20. The method of claim 19, wherein the therapeutic peptide is selected from the group consisting of oxyntomodulin, glucagon-like peptide 1 (GLP1), exendin (exenatide), amylin (pramlintide), alpha-melanocyte-stimulating hormone (MSH), cocaine- and amphetamine-regulated transcript (CART), -