WO2019180272A1 - Anti-leptin affinity reagents for use in the treatment of obesity and other leptin-resistance associated diseases - Google Patents

Abstract

The present invention relates to anti-leptin affinity ligands (e.g. anti-leptin antibodies) that specifically bind leptin and reduce its serum levels for use in a method of treating a leptin- resistance associated disease, such as obesity and/or overweight, in a subject in need thereof. It further relates to compositions comprising the same, as well as methods of treatment comprising administration of a therapeutically effective dosage thereof.

Description

ANTI-LEPTIN AFFINITY REAGENTS FOR USE IN THE TREATMENT OF OBESITY AND OTHER LEPTIN-RESISTANCE ASSOCIATED DISEASES

FIELD OF THE INVENTION

The present invention is in the field of medicine. More particularly, it relates to anti-leptin affinity reagents (e.g. anti-leptin antibodies) that specifically bind leptin and reduce its serum levels for use in a method of treating a leptin-resistance associated disease, such as obesity and/or overweight, in a subject in need thereof. It further relates to compositions comprising the same, as well as methods of treatment comprising administration of a therapeutically effective dosage thereof.

BACKGROUND OF THE INVENTION

Overweight and obesity are major risk factors for a number of chronic diseases, including diabetes, cardiovascular diseases and cancer. Overweight and obesity has become a global problem with at least 2.8 million people dying each year as a result of being overweight or obese.

Leptin is an anorexigenic hormone produced by white adipose tissue whose main function is to inform the brain, specifically the appetite regulating center in the hypothalamus, that the fat levels in the adipose tissue are elevated so that the brain responds by reducing appetite and food intake. Therefore, the higher the amount of fat, the more leptin is produced and secreted. However, in obesity patients a phenomenon of leptin resistance has been reported which results in the maintaining of high levels of food intake with the consequent increase in body weight.

Diverse factors have been reported to cause leptin resistance including hyperleptinemia, inflammation, endoplasmic reticulum (ER) stress and defective autophagy (Zhou Y. and Rui L., Front Med. 2013, 7(2), 207-22). Also, various molecular mechanisms of leptin resistance have been proposed, including a reduction in leptin transportation through the blood-brain barrier (BBB); and the impairment of the transmembrane receptor of neurons in the appetite regulating centre (Zhou Y. and Rui L., Front Med. 2013, 7(2), 207-22; Coppari R, and Bjorbaek C., Nat Rev Drug Discov. 2012, 1 1 (9), 692-708).

More specifically, it has been proposed that hyperleptinemia may result in leptin resistance due to the continuous stimulation of leptin transporters at the level of the BBB (i.e., LEPRa and LEPRe isoforms) that cause their desensitization and downregulation which prevents the access of circulating leptin to the brain (Zhou Y. and Rui L, Front Med. 2013, 7(2), 207- 22, El-Haschimi K, et al., J Clin Invest. 2000, 105(12), 1827-1832; Caro JF, et al., Lancet. 1996, 348(9021 ), 159-161 ; Banks WA et al., Peptides. 1999; 20(11 ): 1341-1345).

To date, several strategies have been proposed to allow a more efficient transport of leptin through BBB in leptin resistance conditions by using agonist molecules or modifications in the leptin molecule, Banks WA et al., Physiol Behav. 2011 30; 105(1 ): 145-9, albeit with little success. Accordingly, despite the efforts made in recent years, there is still a need to find a treatment effective in overcoming leptin resistance and in the treatment of obesity and other leptin-resistance associated diseases.

SUMMARY OF THE INVENTION

The inventors propose a completely new approach to treat leptin resistance based on the hypothesis that if hyperleptinemia results in BBB leptin transporters desensitization and downregulation, then it would be desirable to reduce leptin circulating levels so that both the transporter and the intracellular signaling route is re-sensitized and functional again. This strategy is counter-intuitive considering that the treatment is aimed to reduce the circulating levels of leptin while an increased effect of the hormone’s biological activity is desired and opposite to the therapeutic strategies described so far.

Yet, despite the multifactorial etiology and complexity of leptin resistance and obesity, the inventors showed that by inducing a temporary decrease of the circulating leptin levels (e.g., by the use of anti-leptin antibodies) food intake and body weight were reduced in vivo in an obese rat animal model in a dose-dependent manner (Figs. 1 and 2).

Accordingly, in a first aspect, the invention relates to an anti-leptin affinity reagent for use in a method of treating a leptin-resistance associated disease in a subject in need thereof, wherein said affinity reagent is administered by systemic administration.

The present invention also relates to the use of an anti-leptin affinity reagent in the manufacturing of a medicament for the treatment a leptin-resistance associated disease in a subject in need thereof, wherein said affinity reagent is administered by systemic administration. In addition, the present invention provides a method of treating a leptin-resistance associated disease comprising administering to a subject in need of such treatment a therapeutically effective amount of an anti-leptin affinity reagent, wherein said affinity reagent is administered by systemic administration.

The present invention further provides a composition comprising an anti-leptin affinity reagent of the invention for use in a method of treating a leptin-resistance associated disease in a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effects in body weight of the treatment with different concentrations of the antibody. Changes in body weight between the initial weight and the end of the experiment are expressed as a percentage (%). Antibody concentrations: Cone A: 10 pg/kg; Cone B: 4 pg/kg, and Cone C: 1 pg/kg.

Figure 2. Effects in food intake of the treatment with different concentrations of the antibody. Changes in food intake between the initial weight and the end of the experiment are expressed as a percentage (%). Antibody concentrations: Cone A: 10 pg/kg; Cone B: 4 pg/kg, and Cone C: 1 pg/kg.

Figure 3. A: Change in the circulating levels of leptin (in ng/mL, Mean and SEM) after treatment with the antibody. B: Change in the circulating levels of leptin (in %) after treatment with the antibody. Antibody concentrations: Cone A: 10 pg/kg; Cone B: 4 pg/kg, and Cone C: 1 pg/kg.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The terms "subject", or "individual"' are used herein interchangeably to refer to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a male or female human being of any age or race.

The term "treatment" as used herein refers to the prophylactic and/or therapeutic treatment. The term“therapeutic treatment” as used herein refers to bringing a body from a pathological state, disease or disorder back to its normal, healthy state. Specifically, unless otherwise indicated, includes the amelioration, cure, and/or maintenance of a cure (i.e., the prevention or delay of relapse) of a disease or disorder. Treatment after a disorder has started aims to reduce, alleviate, ameliorate or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, to slow the rate of progression, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse). It is noted that, this term as used herein is not understood to include the term“prophylactic treatment” as defined herein.

The term “prophylactic treatment” or “preventive treatment” as used herein refers to preventing a pathological state, disease or disorder. It is noted that, this term as used herein is not understood to include the term“therapeutic treatment” as defined herein.

The term“effective amount” or“therapeutically effective amount” as used herein refers to an amount that is effective, upon single or multiple dose administration to a subject (such as a human patient) in the prophylactic or therapeutic treatment of a disease, disorder or pathological condition as defined herein.

The term "combination" or“combination therapy” as used herein is meant to comprise the administration of the referred therapeutic agents to a subject in need thereof, in the same or separate pharmaceutical formulations, and at the same time or at different times. If the therapeutic agents are administered at different times they should preferably be administered sufficiently close in time to provide for the combined effect (e.g. potentiating or synergistic response) to occur. The particular combination of therapies to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and/or the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same disorder and/or they may achieve different effects (e.g., control of any adverse effects).

As used herein, the term "composition" or "compositions" may refer to a formulation(s) that it is suitable for injection and/or administration into an individual in need thereof. A "composition" may also be referred to as a "pharmaceutical composition." In certain embodiments, the compositions provided herein are substantially sterile and do not contain any agents that are unduly toxic or infectious to the recipient. The term "pharmaceutically acceptable carrier and/or excipient" is intended to include formulation used to stabilize, solubilize and otherwise be mixed with active ingredients to be administered to living animals, including humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

The term “antibody” as used herein refers to an immunoglobulin or an antigen-binding fragment thereof. Unless otherwise specified, the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The antibody can include a constant region, or a portion thereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes. For example, heavy chain constant regions of the various isotypes can be used, including: IgGi, lgG₂, lgG₃, lgG₄, IgM, IgA^ lgA₂, IgD, and IgE. By way of example, the light chain constant region can be kappa or lambda. In certain embodiments, the term "antibody" may also refer to antibody derivatives, such as antibody- based fusion proteins (e.g. including a region equivalent to the Fc region of an immunoglobulin) or antibodies further modified to contain additional non-proteinaceous moieties, such as water soluble polymers, e.g. polyethylene glycol (PEG).

The term“immunoglobulin G” or“IgG” refers to an antibody which contains two identical heavy chains and two identical light chains. The antigen binding site is formed by the intertwining of the light chain variable domain (VL) and the heavy chain variable domain (VH). Each V domain contains three short stretches of peptide known as the complementarity determining regions (CDRs); the CDRs are the prominent determinants of antigen binding affinity and specificity. The light chain contains one constant domain: CL. The heavy chain contains three constant domains: CH-i, CH₂, and CH₃. The CH₂ and CH₃ domains allow interactions of the IgG molecule with various components of the immune system by either binding C1 q, which activates the complement cascade and elicits complement-dependent cytotoxicity, or by binding to Fey receptors on immune effector cells, which elicits antibody-dependent cellular cytotoxicity.

The terms“antigen-binding domain” and“antigen-binding fragment” refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. For certain antigens, the antigen-binding domain or antigen-binding fragment may only bind to a part of the antigen. The part of the antigen that is specifically recognized and bound by the antibody is referred to as the “epitope” or “antigenic determinant.” Antigen-binding domains and antigen-binding fragments include Fab; a F(ab')₂ fragment (a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region); a Fv fragment; a single chain Fv fragment (scFv); a Fd fragment (having the two V_H and C_H1 domains); single domain antibodies (sdAbs; consisting of a single V_H domain), and other antibody fragments that retain antigen-binding function. The Fab fragment has V_H-C_H1 and V_L-C_L domains covalently linked by a disulfide bond between the constant regions. The F_v fragment is smaller and has V_H and V_L domains non-covalently linked. The scF_v contains a flexible polypeptide that links (1 ) the C-terminus of V_H to the N-terminus of V_L, or (2) the C-terminus of V_L to the N-terminus of V_H. The sdAbs include heavy chain antibodies naturally devoid of light chains and single-domain antibodies derived from conventional four chain antibodies. These antigen-binding domains and fragments are obtained using conventional techniques known to those with skill in the art, and are evaluated for function in the same manner as are intact immunoglobulins.

The term“specificity” as used herein may refer to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule can bind. The specificity of an antigen- binding molecule can be determined based on affinity and/or avidity.

The term“affinity” as used herein may refer to the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), and is considered a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen - binding protein: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the association constant (KA), which is 1/KD). It will be clear to the skilled person that the dissociation constant may be the actual or apparent dissociation constant. Typically, antigen-binding proteins will bind to their antigen with a dissociation constant (KD) of 10 ⁵ to 10 ¹² moles/liter or less, and preferably 10 ⁷ to 10 ¹² moles/liter or less and more preferably 10 ⁸ to 10 ¹² moles/liter (i.e., with an association constant (KA) of 10⁵ to 10¹² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles. Specific binding of an antigen-binding molecule to an antigen or antigenic determinant can be determined in any suitable manner known in the art, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein. The KD can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as koff, to the rate of its association, denoted kon (so that KD =koff/kon and KA = korAoff). The off-rate koff has units s ¹ (where s is the SI unit notation of second). The on-rate kon has units M V¹. The on-rate may vary between 10² M V¹ to about 10⁷ M V¹, approaching the diffusion-limited association rate constant for bimolecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation t_1/2=ln(2)/koff. The off -rate may vary between 10 ⁶ s ¹ (near irreversible complex with a t_1/2 of multiple days) to 1 s ¹ (t_1/2=0.69 s). The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well the known surface plasmon resonance (SPR) biosensor technique (see for example Ober et ah, Intern. Immunology, 13, 1551 -1559, 2001 ) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding kon, koff measurements and hence KD (or KA) values. This can for example be performed using the well-known BIACORE instruments.

The term “avidity” as used herein may refer to the measure of the strength of binding between an antigen-binding molecule and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule.

The term "recombinant antibody" as used herein refers to an antibody produced or expressed using a recombinant expression vector, where the expression vector comprises a nucleic acid encoding the recombinant antibody, such that introduction of the expression vector into an appropriate host cell results in the production or expression of the recombinant antibody.

The term“antibody mimetic” as used herein refers to protein-based scaffolds which have been engineered to bind therapeutic targets with affinity and specificity that match that of natural antibodies. Antibody mimetics have been developed utilizing an immunoglobulin-like fold, for example, fibronectin type III, NCAM and CTLA-4. Other mimetics scaffolds bearing no similarity to immunoglobulin folds have also been obtained. Non-limiting examples of said scaffolds are DARPins, anticalins, affibodies, adnectins, fynomers, etc. (see for instance, Weidle et al., 2013, Cancer Genomics & Proteomics, 10, 1 -18; Lofblom, J. et al., 201 1 , Curr. Opin. Biotechnol., 22, 843-848; Banta, S. et al., 2010, Annu. Rev. Biomed. Eng., 15, 93- 1 13). DETAILED DESCRIPTION

In a first aspect, the invention relates to an anti-leptin affinity reagent for use in a method of treating a leptin-resistance associated disease in a subject in need thereof, wherein said affinity reagent is administered by systemic administration.

The present invention also relates to the use of an anti-leptin affinity reagent in the manufacturing of a medicament for the treatment a leptin-resistance associated disease in a subject in need thereof, wherein said affinity reagent is administered by systemic administration.

In addition, the present invention provides a method of treating a leptin-resistance associated disease comprising administering to a subject in need of such treatment a therapeutically effective amount of an anti-leptin affinity reagent, wherein said affinity reagent is administered by systemic administration.

A leptin-resistance associated disease may refer to obesity, over-weight, type 2 diabetes, dyslipidemia, lipodystrophies, hepatic steatosis, non-alcoholic and alcoholic fatty liver diseases, severe insulin resistance, infertility, Leprechaunism/Donohue syndrome, Rabson- Mendenhall syndrome, and related complications. In a particular embodiment, said disease is an excess food intake associated disease. In a preferred embodiment, said disease is obesity and/or overweight.

Overweight and obesity may refer to abnormal or excessive fat accumulation that presents a risk to health. A measure of obesity is the body mass index (BMI), a subject’s weight (in kilograms) divided by the square of his or her height (in metres). Preferably, said subject is a human subject. A person with a BMI of 30 or more is generally considered obese. A person with a BMI equal to or more than 25 is generally considered overweight.

An anti-leptin affinity reagent as used herein refers to an affinity reagent or ligand that binds to leptin.

The term "affinity reagent" refers to a substance (or functional group) that selectively captures (binds to) a target molecule in a mixture of molecules due to a specific affinity between molecules, typically, antigen-antibody binding affinity. The leptin-specific affinity reagent may be a peptide, polypeptide, or protein it can also be a nucleic acid or peptide aptamer. For example, the leptin-specific affinity reagent may be an antibody or antibody- mimetic.

The term“nucleic acid aptamer” as used herein may refer to an isolated or purified nucleic acid that binds with high specificity and affinity to a target through interactions other than Watson-Crick base pairing. An aptamer has a three dimensional structure that provides chemical contacts to specifically bind to a target. Unlike traditional nucleic acid binding, aptamer binding is not dependent upon a conserved linear base sequence, but rather a particular secondary or tertiary structure. That is, the nucleic acid sequences of aptamers are non-coding sequences. Any coding potential that an aptamer may possess is entirely fortuitous and plays no role whatsoever in the binding of an aptamer to a target. A typical minimized aptamer is 5-15 kDa in size (15-45 nucleotides), binds to a target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind to other proteins from the same gene or functional family).

The term“antibody mimetic” as used herein may refer to protein-based scaffolds which have been engineered to bind therapeutic targets with affinity and specificity that match that of natural antibodies. Antibody mimetics have been developed utilizing an immunoglobulin-like fold, for example, fibronectin type III, NCAM and CTLA-4. Other mimetics scaffolds bearing no similarity to immunoglobulin folds have also been obtained. Non-limiting examples of said scaffolds are DARPins, anticalins, affibodies, adnectins, fynomers, etc. (see for instance, Weidle et al., 2013, Cancer Genomics & Proteomics, 10, 1-18; Lofblom, J. et al., 2011 , Curr. Opin. Biotechnol., 22, 843-848; Banta, S. et al., 2010, Annu. Rev. Biomed. Eng., 15, 93- 113).

The obese (ob) gene product, leptin, is an important circulating signal for the regulation of body weight (Zhang Y et al., Nature 1994, 372, 425-432). The term“leptin” as used herein refers to human leptin of amino acid sequence SEQ ID NO:1 (Leptin, Homo sapiens ; sequence (Ref.: http://www.uniprot.org/uniprot/P41 159 (v1 , February 1 , 1995)), rat leptin of amino acid sequence SEQ ID NO:2 (Leptin, Rattus norvegicus, sequence (Ref.: http://www.uniprot.org/uniprot/P50596 (v1 , October 1 , 1996)) and variants thereto comprising one or more amino acid substitutions, additions, or deletions that share at least 80%, preferably at least 90%, more preferably at least 95%, 97% or 99% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:2. . The term“leptin” also includes forms of the polypeptide with post-translational modifications, such as N-linked or O-linked glycosylated forms. SEQ ID NO:1

10 20 30 40 50

MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RIN DISHTQS

60 70 80 90 100

VSSKQKVTGL DFI PGLHPI L TLSKMDQTLA VYQQILTSMP SRNVIQISN D

110 120 130 140 150

LEN LRDLLHV LAFSKSCH LP WASGLETLDS LGGVLEASGY STEVVALSRL

160

QGSLQDMLWQ LDLSPGC

Signal peptide: Positions 1 -21 (in bold); Chain: Positions 22 -167.

SEQ ID N0:2

10 20 30 40 50

MCWRPLCRFL WLWSYLSYVQ AVPIHKVQDD TKTLIKTIVT RI NDISHTQS

60 70 80 90 100

VSARQRVTGL DFI PGLHPI L SLSKM DQTLA VYQQI LTSLP SQNVLQIAH D

110 120 130 140 150

LEN LRDLLH L LAFSKSCSLP QTRGLQKPES LDGVLEASLY STEVVALSRL

160

QGSLQDI LQQ LDLSPEC

Signal peptide: Positions 1 -21 (in bold); Chain: Positions 22 -167.

In a particular embodiment, said antibody is a monoclonal antibody, a recombinant antibody, an antigen-binding fragment or an antibody-derivative (e.g., a leptin-receptor immunoglobulin Fc fragment fusion protein). Preferably, said antibody is an Fc-containing antibody. The Fc region may be composed by two identical protein fragments derived from the CH₂ and CH₃ domains of the two heavy chains of the antibody, such as in In IgG, IgA and IgD antibody isotypes. It also may be composed by three heavy chain constant domains (domains CH₂, CH₃ and CH₄) in each polypeptide chain, like in IgM and IgE isotypes. Preferably, said antibody is a natural or recombinant intact immunoglobulin, e.g. an intact IgG. In a particular embodiment, said antibody is a high affinity antibody characterized by binding leptin with a dissociation constant (KD) of 10 ⁷ to 10 ¹² moles/liter or less, and more preferably 10 ⁸ to 10 ¹² moles/liter or less. This antibody may be genetically modified. For instance, the invention also encompasses engineered antibodies with altered affinity to human neonatal Fc receptor (FcRn) or to Fey receptors. In a particular embodiment, the Fc region is mutated so that it is increased the binding affinity of the antibody for FcRn (e.g. amino acid positions 428 and/or 250). This results in an increased serum half-live of the antibody (Tabrizi et al., Drug Discovery Today 2006,11 (1-2), 81-8).

The anti-leptin antibody is preferably of the IgG isotype. In a particular embodiment, said antibody is a chimeric, humanized or human antibody, preferably of the IgG 1 , lgG2 or lgG4 isotypes, more preferably of the lgG2 or lgG4 isotype.

Numerous methods known to those skilled in the art are available for obtaining antibodies. Antibodies can be produced using recombinant DNA methods (See, e.g., U.S. 4,816,567, and Current Trends in Monoclonal Antibody Development and manufacturing (Steven Shire et al., Eds. Springer, 2010)), the disclosures of which are incorporated herein by reference in their entirety. Known antibody specific nucleic acid sequences and the V_H and V_L (or CDR) amino acid sequences encoded thereby, it is possible, using recombinant DNA techniques, to insert a nucleic acid of interest into an expression vector or otherwise express the nucleic acid of interest in a host cell to produce the desired antibody. In addition, as disclosed elsewhere in this application, modified versions of the antibodies described herein can be produced using known techniques, including, for example, random mutagenesis, error-prone PCR, and direct mutagenesis.

Monoclonal antibodies may also be produced by preparing immortalized cell lines capable of producing antibodies having desired specificity, for example against an antigen and/or epitope, such as leptin as disclosed in this application. Such immortalized cell lines may be produced in a variety of ways. In some embodiments, immortalized cell lines are produced from circulating B lymphocytes from human donors. In other embodiments, a small non- human animal, such as a mouse, is hyperimmunized with the desired immunogen. The vertebrate is then sacrificed, usually several days after the final immunization, the spleen cells removed, and the spleen cells immortalized. The most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1975) Nature 256:495-497. Other techniques, including EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Specific techniques for preparing monoclonal antibodies are described in Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, 1988 , the full disclosure of which is incorporated herein by reference. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer transgenic mouse strains that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected ( See, e.g., Green et al. (1994) Nature Genetics 7:13-21 , US 2003-0070185, U.S. 5,225,539, WO 96/34096, and WO 96/33735, the disclosures of which are incorporated herein by reference in their entirety mmortalized cell lines can be screened using standard methods, such as enzyme- linked immunosorbent assay (ELISA) or surface plasmon resonance analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen and/or epitope. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.

Another exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display technology mimics the mammalian immune system by cloning large libraries of antibody genes and selecting for binding to a desired target, such as leptin as disclosed in this application. Phage display is described, for example, in US 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991 ) Nature, 352: 624-628; Marks et al. (1991 ) J. Mol. Biol., 222: 581-597WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809, the disclosures of which are incorporated herein by reference in their entirety. It is also possible to produce antibodies that bind a specific antigen, such as leptin as disclosed in this application, by using a variable heavy domain and screening a library of complimentary variable domains to identify antibodies that retain the desired binding specificity. See Portolano et al., The Journal of Immunology (1993) 150:880-887 and Clarkson et al., Nature (1991 ) 352:624-628, the disclosures of which are incorporated herein by reference in their entirety.

Without willing to be bound by theory, as a methodology for reducing circulating leptin levels, a polyclonal antibody is expected to ensure a higher probability of success given that a greater proportion of the leptin molecule is being recognized by the antibodies. In a particular embodiment, said anti-leptin antibody are polyclonal antibodies (also referred herein as a polyclonal antibody serum). The polyclonal antibodies comprise at least two different antibodies targeting leptin, preferably targeting different epitopes of leptin. The polyclonal antibodies of the invention comprise preferably more than 10, 20, 50 or even 100 distinct antibodies, targeting leptin.

In a preferred embodiment, the polyclonal antibodies are immunoglobulin G (IgG) antibodies. Whereas other types of immunoglobulins may be present, polyclonal antibodies according to the invention are mainly immunoglobulins G, for example more than 80% of the polyclonal antibodies according to the invention are IgG, preferably more than 85%, 90% or 95%, even preferably more than 98% or 99% of the polyclonal antibodies are IgG.

The polyclonal antibodies of the invention can be obtained, for example, by immunization of an animal, preferably a non-human animal. Suitable animals according to this embodiment are chickens or mammals, especially rabbit, mouse, goat, guinea pigs, hamster, horse, rat, alpaca or sheep. In a particular embodiment, the antibodies are rabbit, horse or alpaca polyclonal antibodies, preferably rabbit polyclonal antibodies. The polyclonal antibodies may be isolated from one or more than one animal; for example the polyclonal antibodies may be a mixture of polyclonal antibodies isolated from 2, 3 or more rabbits, or 2, 3 or more mice. It is also envisaged in the context of the present invention that the polyclonal antibodies be isolated from at least 2 different types of animals, for example isolated from an immunized alpaca and an immunized rabbit.

The polyclonal antibodies may indeed be obtained by immunization of an animal, and preferably of a mammal, with the whole leptin polypeptide or an antigenic fragment thereof, followed by a purification of the serum isolated from said animal, preferably mammal, for example by affinity chromatography with leptin.

In a preferred embodiment, said leptin is human leptin and said anti-leptin antibody (e.g. polyclonal serum) is an anti-human leptin antibody. This antibody may be specific to human leptin or may cross-react with leptin of other mammal species, such as mouse or rat. In a more preferred embodiment said anti-leptin antibody is an anti-human leptin antibody serum, preferably a human anti-human leptin antibody serum.

In another embodiment, said polyclonal antibody serum is a rabbit anti-rat leptin antibody serum, such as the one used in the Examples.

The anti-leptin affinity reagent of the present invention can be used either alone or in combination with other compositions in a therapy. For instance, an anti-leptin affinity reagent of the invention may be administered in combination with other drugs typically used in the treatment of leptin-resistance associated diseases, such as type 2 diabetes, obesity and/or overweight. This includes but is not limited to one or more from the group consisting of liraglutide, orlistat, naltrexone hycrochloride/bupropion hydrochloride (e.g., Mysimba™, Contrave™), metformin, SGLT2 inhibitors, and PP4 inhibitors.

Pharmaceutical Formulations

Pharmaceutical compositions comprising an anti-leptin affinity reagent of the invention can be prepared for storage by mixing the anti-leptin affinity reagent having the desired degree of purity with optional physiologically acceptable carriers and/or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), generally in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, and/or excipients are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glutamic acid, proline, glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, marmitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation may also contain more than one active compound as necessary for the particular indication being treated (e.g. obesity and/or overweight), preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The anti-leptin affinity reagent of the invention or a composition comprising thereof is administered by any suitable means of systemic administration, preferably selected from the group consisting of oral, sublingual or parenteral administration. In a particular embodiment, said systemic administration is parenteral administration, which includes for instance intramuscular, intravascular (e.g., intravenous or intraarterial), intraperitoneal, or subcutaneous administration. Preferably, said administration is intravascular, more preferably intravenous. In addition, the affinity reagent is suitably administered by pulse infusion, particularly with declining doses. Preferably the dosing is given by injections, most preferably intravenous injections.

The composition of the invention will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context may include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The affinity reagent (e.g., anti-leptin antibody) may be used as single agent and may be optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of affinity reagent (e.g., anti-leptin antibody) of the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used before.

The anti-leptin affinity reagent (e.g., anti-leptin antibody) of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.

In some embodiments, the dosage of the anti-leptin affinity reagent (e.g., anti-leptin antibody) will be in the range from about 0.05 mg/kg to about 20 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 6.0 mg/kg, 8.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. In other embodiments, the dosage of the anti-leptin affinity reagent (e.g., anti-leptin antibody) will be in the range from about 0.05 pg/kg to about 20 pg/kg. Thus, one or more doses of about 0.5 pg/kg, 1.0 pg/kg, 2.0 p/kg, 4.0 pg/kg, 6.0 pg/kg, 8.0 pg/kg or 10 pg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered daily, also may be administered intermittently, e.g. every 2 or 3 days, every week or every two or three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six doses of the affinity reagent). An initial higher loading dose, followed by one or more lower doses may be administered. An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the affinity reagent. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. In a particular embodiment, said affinity reagent is an anti-rat leptin antibody serum, preferably a rabbit anti-rat leptin antibody serum, such as the one used in the Examples. The amounts of anti-leptin antibody serum administered to rats in the Examples were extrapolated to a human dose as described in Reagan-Shaw et al., "Dose translation from animal to human studies revisited", The FASEB Journal, 2007, 22, 659-661 , using the equation:

Dose in humans (mg/kg) = Dose in animal (mg/kg)^*(km animal/km human)

Using the Km values shown in the table below:

Species Weight (kg) BSA K_m factor

Human

Adult 60 1.6 37

Child 20 0.8 25

Baboon 12 0.6 20

Dog 10 0.5 20

Monkey 3 0.24 12

Rabbit 1 8 0.15 1

Guinea pig 0 4 0.05 8

Rat 0 15 0.025 6

Hamster 0 08 0.02 5

Mouse 0 02 0.007 3

Values based on data from FDA Draft Guidelines (7). To convert:

dose in mg/kg to dose in mg/ in², multiply by K_m value.

The 1 pg/kg in rat corresponding to 0.16 pg/kg of body weight in a human adult, the 4 pg/kg in rat corresponding to 0.65 pg/kg of body weight in a human adult and 10 pg/kg in rat corresponding to 1.62 pg/kg of body weight in a human adult.

In other embodiments, the anti-leptin affinity reagent, preferably a polyclonal antibody serum, such as the anti-rat leptin antibody serum used in the examples, is administered at doses of more than 0.16 pg/kg of body weight, such as at least 0.2 pg/kg, at least 0.3 pg/kg, 0.4 pg/kg, at least 0.5 pg/kg, at least 0.6 pg/kg, preferably equal to or of more than 0.65 pg/kg of body weight, such as at least 0.7 pg/kg, at least 0.8 pg/kg, 0.9 pg/kg, at least 1 pg/kg, at least 1.1 pg/kg, at least 1.2 pg/kg, at least 1.3 pg/kg, 1.4 pg/kg, at least 1.5 pg/kg, at least 1.6 pg/kg, more preferably at doses equal to or of more than 1.62 pg/kg of body weight. In other embodiments, the anti-leptin affinity reagent preferably a polyclonal antibody serum, such as the anti-rat leptin antibody serum used in the examples, is administered at doses from 0.2 pg/kg of body weight to 5 pg/kg of body weight, preferably from 0.65 pg/kg of body weight to 3 pg/kg of body weight, more preferably from 1 pg/kg of body weight to 2 pg/kg of body weight.

Administration schedule may be as described herein above. In a particular embodiment, anti-leptin affinity reagent administration is conducted every two days for 7 to 21 days, preferably from 10 to 15 days, more preferably for 12 days.

The anti-leptin affinity reagent or composition comprising thereof of the present invention is administered in a therapeutically effective amount to induce weight loss, to reduce body fat, to reduce food intake, to improve glucose homeostasis, or combinations thereof in a subject in need thereof, such as an obese subject or a subject with overweight.

In a particular embodiment, said anti-leptin affinity reagent or composition comprising thereof, is administered to a subject in need thereof in a therapeutically effective amount to induce weight loss, to decrease body mass and/or body fat, preferably by at least 2%, preferably by at least 4%, 5%, 6%, 7%, 8%, 9% or 10%, more preferably by at least 15%, most preferably by at least 20%, or higher.

In another particular embodiment, optionally in combination with the embodiment above, said anti-leptin affinity reagent or composition comprising thereof, is administered to a subject in need thereof in a therapeutically effective amount to reduce food intake or appetite, preferably in a therapeutically effective amount to reduce average daily food intake (e.g., in terms of calories) by at least 15%, 17%, 20%, 22%, 25%, 28%, 30%, 32%, 35%, or higher.

In a further particular embodiment, optionally in combination with one or more of the embodiments or preferred features described herein, the administered dose of said anti- leptin affinity reagent results in a reduction of leptin circulating levels in a treated individual of at least 5%, preferably of at least 10%, of at least 15%, of at least 20% or preferably of at least 25% or more. In a particular embodiment, the percentage of reduction is with respect to the beginning of the treatment. In another particular embodiment, the percentage of reduction is with respect to a control group or individual (in the absence of treatment). Typically a control group or individual, also referred as placebo group, has been submitted to the same conditions as the treatment group, receiving an administration of a placebo (vehicle in the absence of active ingredient). In another aspect, the invention relates to the use of an anti-leptin affinity reagent as described herein or of a non-medical composition comprising thereof as described herein in a subject suffering from over-weight.

It is contemplated that any features described herein can optionally be combined with any of the embodiments of any medical use, pharmaceutical composition, method of treatment, method of manufacturing a medicament and combination therapies of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term“another” may also refer to one or more. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprises” also encompasses and expressly discloses the terms“consists of” and“consists essentially of”. As used herein, the phrase "consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of” excludes any element, step, or ingredient not specified except for, e.g., impurities ordinarily associated with the element or limitation.

The term "or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation, "about", "around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.

In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15%. Accordingly, the term“about” may mean the indicated value ± 5% of its value, preferably the indicated value ± 2% of its value, most preferably the term“about” means exactly the indicated value (± 0%).

The following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.

EXAMPLES

Example 1.- Material and Methods

Anti-leptin antibody treatment of an obesity animal model

Twenty male normal weight rats (Sprague Dawley), from the central animal facilities of the Universidad de Santiago de Compostela, were maintained in controlled conditions of temperature, humidity, and illumination (12-h controlled photoperiod). They were allowed to acclimatize for 1 week on arrival.

After the acclimatization period, animals were randomized into four weight-matched groups (n=5/group). One group had ad libitum access to a standard diet (SAFE- Panlab, Spain), with 5.5% lipid, 23% protein, and 70% carbohydrate content. The other four groups were fed with a high-fat diet (HFD) (Open Source Diets, Research Diets; Brogaarden, Denmark) with 60% lipid, 20% protein, and 20% carbohydrate during two months.

Then, three HFD groups, were treated intravenously with a rabbit polyclonal anti- rat leptin antibody IgG serum (Reference: 1399990321 Immunostep, Spain) at three different concentrations (1 , 4 and 10pg/kg) every two days for a period of 12 days. Body weight and food intake were measured during the experimental period. During the treatment period, blood was collected through the tail vein to obtain serum and plasma. To perform cannulation of the tail vein with a catheter (I.V. catheter 24 GA), both for obtaining blood and for admitting the treatment, the animals underwent general anesthesia (inhalation of isoflurane) and to visualize the vein remained on a thermal blanket at 37°C constant temperature during the process. For the collection of blood serum, serum separator tubes (BD Vacutainer™ SST™ II Advance Tubes) of 5 ml. volume were used and kept on ice. The whole blood samples were rotated in a refrigerated centrifuge (4 ° C) at 3500g for 15 minutes. Finally, the eluted serum was stored at -80 ° C in sterile 1.5 mLmicrocentrifuge tubes. Finally, animals were euthanized and decapitated, and the blood were collected and kept at -80°C until analysis. All animal experiments and procedures involved in this study were approved by the Ethical Committee at the University of Santiago de Compostela, in accordance with the European Union Normative for the use of experimental animals.

Body weight

Body weight was analyzed for each animal individually, on a daily basis, using a scale.

Food intake

The weight of the food provided to the animals was determined daily individually for each animal.. Daily food consumption was calculated from a 3-day average.

Leptin analysis

Circulating leptin levels were determined using a commercial kit (Wako, USA) and according to the manufacturer^'s instructions. Example 2.-Results

Anti-leptin antibody treatment reduces body weight gain and food intake in rats fed with HFD.

The treatment group received a diet of 60% fat ad libitum and was treated daily with 1 , 4 or 10pg/kg of a polyclonal anti-leptin antibody while monitoring body weight gain. At the end of the experiment, the animals that received 10pg/kg and 4pg/kg doses of antibody had significantly less weight than the HFD control animals; the 10 pg/kg dose was the most effective with a with a 4% reduction in body weight followed by the 4 pg / kg-dose with a 2% reduction in body weight (Fig. 1 ). However, the 1 pg/kg dose did not appear to reduce the weight in comparison with the HFD control group (Fig. 1 ). In parallel with the observed decrease in body weight, food intake was also significantly lower in the anti-leptin antibody- treated animals than in the HFD control group (Fig. 2). Also, the average circulating levels of leptin throughout the experimental time were found to be decreased for 10pg/kg- and 4pg/kg-doses (Fig. 3).

Claims

1. An anti-leptin affinity reagent for use in a method of treating a leptin-resistance associated disease in a subject in need thereof, wherein said affinity reagent is administered systemically, preferably is administered parenterally.

2. The anti-leptin affinity reagent according to claim 1 , wherein said affinity reagent is an antibody, preferably an Fc-containing antibody.

3. The anti-leptin affinity reagent for use according to any of claims 1 or 2, wherein said affinity reagent is a chimeric, humanized or human antibody.

4. The anti-leptin affinity reagent for use according to any of claims 1 to 3, wherein said affinity reagent is a polyclonal antibody serum, preferably more than 80% of the polyclonal antibodies in said antibody serum are IgG antibodies.

5. The anti-leptin affinity reagent for use according to any of claims 1 to 4, wherein said leptin is human leptin.

6. The anti-leptin affinity reagent for use according to any of claims 4 or 5, wherein said polyclonal antibody serum is an anti-human leptin antibody serum.

7. The anti-leptin affinity reagent for use according to any of claims 1 to 4, wherein said leptin is rat leptin.

8. The anti-leptin affinity reagent for use according to any of claims 4 or 7, wherein said polyclonal antibody serum is a rabbit anti-rat leptin antibody serum.

9. The anti-leptin affinity reagent for use according to any of claims 1 to 8, wherein said affinity reagent is administered intravascularly, preferably intravenously.

10. The anti-leptin affinity reagent for use according to any of claims 1 to 9, wherein said polyclonal antibody serum is administered at doses equal to or of more than 0.65 pg/kg of body weight, preferably equal to or of more than 1.62 pg/kg of body weight.

1 1. The anti-leptin affinity reagent for use according to any of claims 1 to 10, wherein the administered dose of said affinity reagent results in a reduction of leptin circulating levels in a treated individual of at least 15%, of at least 20% or of at least 25% or more with respect to a control placebo group or individual.

12. The anti-leptin affinity reagent for use according to any of claims 1 to 11 , wherein said treatment comprises the administration of the anti-leptin affinity reagent more than once.

13. A composition comprising an anti-leptin affinity reagent as defined in any of claims 1 to

12 for use in a method of treating a leptin-resistance associated disease in a subject in need thereof.

14. The anti-leptin affinity reagent for use according to any of claims 1 to 12 or the composition for use according to claim 13, wherein said disease is overweight and/or obesity.

15. The anti-leptin affinity reagent for use according to any of claims 1 to 12 and 14, or the composition for use according to any of claims 13 or 14, wherein said subject is a human subject.