Patent Publication Number: US-2021188970-A1

Title: Leptin Antibodies

Description:
REFERENCE TO A SEQUENCE LISTING 
     A Sequence Listing in text format is incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 17124481.txt. The text file is about 24 KB, was created on Mar. 12, 2021, and is being submitted electronically via EFS-Web. 
     INTRODUCTION 
     Leptin is a hormone produced by adipocytes and is elevated in obesity. The congenital lack of leptin results in obesity and the metabolism field widely accepts the concept. Upon cloning of the leptin gene, the original hope was that leptin would act as a break for further food intake and a trigger to increase energy expenditure. The hope was that the injection of recombinant leptin would act as an effective weight loss mechanism. However, these hopes were quickly disappointed, since obese individuals have high leptin levels, but the individual is leptin resistant. Not even the injection of very high leptin levels can overcome this resistance. 
     We generated a battery of monoclonal antibodies against human and mouse leptin. These antibodies showed strong binding affinities to human leptin and significant neutralizing activity in vivo. More importantly, treatment of high-fat diet (HFD)-fed mice with neutralizing antibodies reduces body-weight gain and confirmed findings in mice with genetic knock-down of leptin. Our antibodies provide effective for treatment of obesity with high level of leptin but resistant to the conventional leptin treatment. 
     SUMMARY OF THE INVENTION 
     In an aspect the invention provides an antibody antigen binding domain which specifically binds human leptin, and comprises V H  or V L  CDR1, CDR2 and CDR3 sequences of an hLept antibody: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                   
               
             
            
               
                 V H   
                   
                   
                   
               
               
                   
               
               
                 hLept-1V H   
                 GGSVSRGSHY 
                 IHTDGST 
                 AREPGGALNF 
               
               
                   
                 (SEQ ID NO: 1) 
                 (SEQ ID NO: 2) 
                 (SEQ ID NO: 3) 
               
               
                   
               
               
                 hLept-2V H   
                 GYTFTGYY 
                 INPNSGGT 
                 ASGKTYYDFWSGGRRGMDV 
               
               
                   
                 (SEQ ID NO: 4) 
                 (SEQ ID NO: 5) 
                 (SEQ ID NO: 6) 
               
               
                   
               
               
                 hLept-3V H   
                 GGTFSSYA 
                 IIPIFGTA 
                 ARSQVPSSYYYGMDV 
               
               
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 8) 
                 (SEQ ID NO: 9) 
               
               
                   
               
               
                 hLept-5V H   
                 GFTFSSYA 
                 ISYDGSNK 
                 ARGREYYYYMDV 
               
               
                   
                 (SEQ ID NO: 10) 
                 (SEQ ID NO: 11) 
                 (SEQ ID NO: 12) 
               
               
                   
               
               
                 hLept-6V H   
                 GYTFTSYY 
                 INPSGGST 
                 ARGFGYGGKALDY 
               
               
                   
                 (SEQ ID NO: 13) 
                 (SEQ ID NO: 14) 
                 (SEQ ID NO: 15) 
               
               
                   
               
               
                 V L   
               
               
                   
               
               
                 hLept-1V L   
                 SSNIGSNT 
                 SNN 
                 ASWDDSLNGVV 
               
               
                   
                 (SEQ ID NO: 16) 
                   
                 (SEQ ID NO: 17) 
               
               
                   
               
               
                 hLept-2V L   
                 QSVSRY 
                 TSS 
                 QQTYSTPWT 
               
               
                   
                 (SEQ ID NO: 18) 
                   
                 (SEQ ID NO: 19) 
               
               
                   
               
               
                 hLept-3V L   
                 NSNIGAGYH 
                 GDT 
                 QSYDRSRGGWF 
               
               
                   
                 (SEQ ID NO: 20) 
                   
                 (SEQ ID NO: 21) 
               
               
                   
               
               
                 hLept-5V L   
                 NIARKS 
                 NDN 
                 QVWDNSDYV 
               
               
                   
                 (SEQ ID NO: 22) 
                   
                 (SEQ ID NO: 23) 
               
               
                   
               
               
                 hLept-6V L   
                 QNINSR 
                 KAS 
                 QQFDKYSIT 
               
               
                   
                 (SEQ ID NO: 24) 
                   
                 (SEQ ID NO: 25) 
               
               
                   
               
            
           
         
       
     
     The V H  and V L  CDR1, CDR2 and CDR3 sequences of the hLept antibodies can be combined in alternative combinations which bind human leptin, i.e. the CDRs of hLept-1V H  can be paired with the CDRs of hLept-1-V L,  hLept-2V L, hLept-3V L,  hLept-5V L  hLept-6V L.    
     In embodiments the antigen binding domain comprises:
     V H  and V L  CDR1, CDR2 and CDR3 sequences of the hLept antibody;   V H  or V L  sequences of the hLept antibody; and/or   V H  and V L  sequences of the hLept antibody.   

     In embodiments the antigen binding domain is part of a monoclonal IgG antibody and/or a humanized antibody. 
     In other aspects the invention provides an expression vector encoding the antibody antigen binding domain or a cultured cell expressing the antibody antigen binding domain. 
     In another aspect the invention provides a method of using the antibody antigen binding domain to treat obesity or diabetes, comprising the step of administering the domain to a person in need thereof. 
     The invention includes all combinations of the recited particular embodiments. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A . Antibody therapy reduces body weight. 
         FIG. 1B . Antibody therapy reduces food intake. 
         FIG. 1C . Antibody therapy increases leptin transcription in brown and epididymal adipose tissue. 
         FIG. 1D . Antibody therapy reduces circulating leptin. 
         FIG. 1E . Antibody therapy reduces high fat diet effects on brown adipose tissue. 
     
    
    
     DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION 
     Unless the context indicates otherwise, the term “antibody” is used in the broadest sense and specifically covers antibodies (including full length monoclonal antibodies) and antibody fragments. An antibody molecule is usually monospecific, but may also be described as idiospecific, heterospecific, or polyspecific. Antibody molecules bind by means of specific binding sites to specific antigenic determinants or epitopes on antigens. “Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab&#39;, F(ab&#39;).sub.2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. 
     Natural and engineered antibody structures are well known in the art, e.g. Strohl et al.,  Therapeutic antibody engineering: Current and future advances driving the strongest growth area in the pharmaceutical industry,  Woodhead Publishing Series in Biomedicine No. 11, October 2012; Holliger et al. Nature Biotechnol 23, 1126 - 1136 (2005); Chames et al. Br J Pharmacol. 2009 May; 157(2): 220-233. 
     Monoclonal antibodies (MAbs) may be obtained by methods known to those skilled in the art. See, for example Kohler et al (1975); U.S. Pat. No. 4,376,110; Ausubel et al (1987-1999); Harlow et al (1988); and Colligan et al (1993). The mAbs of the invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art. 
     An “isolated polynucleotide” refers to a polynucleotide segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequence. 
     A “construct” means any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecule has been linked in a functionally operative manner, i.e. operably linked. A recombinant construct will typically comprise the polynucleotides of the invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the invention. 
     A “vector” refers any recombinant polynucleotide construct that may be used for the purpose of transformation, i.e. the introduction of heterologous DNA into a host cell. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. 
     An “expression vector” as used herein refers to a nucleic acid molecule capable of replication and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vectors comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desired, provide amplification within the host. The expression vector further comprises a promoter to drive the expression of the polypeptide within the cells. Suitable expression vectors may be plasmids derived, for example, from pBR322 or various pUC plasmids, which are commercially available. Other expression vectors may be derived from bacteriophage, phagemid, or cosmid expression vectors. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. 
     EXAMPLES 
     Panning and Selection of Anti-Leptin Monoclonal Antibodies using In-House Prepared scFv Phage Display Human Antibody Library 
     Leptin protein (NM-000230, Entrez 3952: Sino Biologics) was used to panning the phage display scFv library (diversity of 1×10 11 ). Binders were selected by phage ELISA by coating LEPTIN protein on 96-well plates (max-sorb plates, Nunc) and were detected with an anti-M13 phage antibody conjugated with horseradish peroxidase (HRP) and TMB substrate (cell signaling). DNA sequences contained in phage-mid vector is isolated using a plasmid preparation kit and sequenced (Genewiz). Complete heavy chain variable region and light chain variable sequences were amplified and expressed into full IgG using an expression vector system in HEK293 cells. 
     Selected LEPTIN binding hits were expressed as human IgGs using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen). Antibodies were purified using a column with protein A resin by a fast protein liquid chromatography (FPLC) separation unit. Purified LEPTIN binding antibodies were characterized for their biological properties. 
     Binding Affinity of Anti-Leptin Monoclonal Antibodies 
     Binding of LEPTIN by monoclonal antibodies was first screened by ELISA using supernatants collected from the B cell cultures. Human or mouse LEPTIN protein (Sino Biologicals) was coated on a 96-well high binding plate over night at 4° C. in PBS. B cell culture supernatants (5 μl medium and 95 μl of PBS) were added at for binding to LEPTIN antigen coated on the plate. Bound antibody was detected using a secondary antibody against rabbit IgG conjugated with HRP and TMB substrate. 
     Binding to human leptin in ELISA (1:20 diluted culture supernatants; signals: absorbance at 450 nM): 
     
       
         
           
               
               
               
               
               
             
               
                   
               
             
            
               
                 hLept-1/ 
                 hLept-2/ 
                 hLept-3/ 
                 hLept-5/ 
                 hLept-6/ 
               
               
                 0.071 
                 2.5739 
                 0.1902 
                 0.7385 
                 0.663 
               
               
                   
               
            
           
         
       
     
     ELISA titration was used to determine the binding affinity of a panel of monoclonal antibodies to LEPTIN antigen. Binding constants (K D  and/or EC 50) of a panel of monoclonal antibodies were estimated using the 4 parameter curve fitting with Prism GraphPad program. For Biacore analysis, all experiments were performed at 25° C. at a flow rate of 45 μl/min An anti-human IgG Fc antibody (from ThermoFisher, at 50 μg/m1 each in acetate buffer, pH 5.0) was immobilized onto a carboxymethyl dextran sensorchip (CM5) using amine coupling procedures based on instruction from the manufacturer. Purified rabbit/human chimeric antibody to be tested was diluted at a concentration of 5 μg/m1 in 0.5% P20, HBS-EP buffer and injected on FC2 to reach 500 to 1000 RU. FC1 was used as the reference cell. Specific signals correspond to the difference of signals obtained on FC2 versus FC1. The analyte (recombinant human LEPTIN, apparent molecular weight 16 kDa on SDS-PAGE gel) was injected during 90 sec at series of concentration dilutions (100, 50, 25, 12.5, 6.25, and 3.13, 1.56 nM) in 0.5% P20, HBS-EP buffer. These concentrations were prepared from stock solution in 0.5% P20, HBS-EP. The dissociation phase of the analyte was monitored over a 30 minutes period. Running buffer was also injected under the same conditions as a double reference. After each running cycle, both flow cells were regenerated by injecting 20 to 45 μl of Glycine-HC1 buffer pH 1.5. Binding K D  on LEPTIN was calculated by k off /k on  kinetic rate for each LEPTIN monoclonal antibodies (Table 3). 
     Neutralizing Leptin Antibodies Confer Reduced Weight Gain. 
     We used a cohort of mice that had previously been exposed to 10 weeks of high fat diet exposure. We treated these mice twice a week either with PBS, low (“0.5 microgram/g BW), medium (5 microgram/g BW) or high (50 microgram /g BW) leptin antibody injections for up to 20 days; hLept-1, hLept-2, hLetp-3, hLept-5 and hLept-6 antibody injections provide consistent dose dependent results. As seen in  FIG. 1A , there is a dose-dependent effect of these injections in terms of impaired weight gain. While all animals initially lost some weight, control mice and lower doses of antibody had some effect, whereas with the highest dose of antibody, overall reduced weight reduction persisted. There was a dose-dependent reduction in food intake ( FIG. 1B ), as well as a dose-dependent transcriptional increase for leptin in brown and epididymal adipose tissue, with more limited compensatory effects in subcutaneous adipose tissue ( FIG. 1C ). Antibody treatment leads to an effective 40% reduction of circulating leptin levels, sufficient to achieve a significant degree of leptin sensitization. Leptin-neutralization not only effectively reduces the weight, but with the weight reduction we also see the expected improvements in glucose homeostasis ( FIG. 1D ). Furthermore, high fat diet generally affects brown adipose tissue negatively and lead to an increased “whitening” of BAT, and this process is significantly prevented and/or reversed by our antibody treatment ( FIG. 1E ). Concomitantly, we also observed a reduction in hepatic steatosis. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 CDR* sequences of heavy chain 
               
               
                 variable sequences of leptin antibodies 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                   
               
               
                 hLept-1 
                 Ggtggctccgtcagc 
                 Atccacaccgatggg 
                 Gcgagagagcccggg 
               
               
                   
                 agaggtagtcactac 
                 agcacc 
                 ggcgccctgaatttc 
               
               
                   
                 (SEQ ID NO: 26) 
                 (SEQ ID NO: 27) 
                 (SEQ ID NO: 28) 
               
               
                   
                 GGSVSRGSHY 
                 IHTDGST 
                 AREPGGALNF 
               
               
                   
                 (SEQ ID NO: 1) 
                 (SEQ ID NO: 2) 
                 (SEQ ID NO: 3) 
               
               
                   
               
               
                 hLept-2 
                 Ggatacaccttcacc 
                 Atcaaccctaacagt 
                 Gcgagtgggaaaacg 
               
               
                   
                 ggctactat 
                 ggtggcaca 
                 tattacgatttttgg 
               
               
                   
                 (SEQ ID NO: 29) 
                 (SEQ ID NO: 30) 
                 agtggtgggagacgc 
               
               
                   
                   
                   
                 ggtatggacgtc 
               
               
                   
                   
                   
                 (SEQ ID NO: 31) 
               
               
                   
                 GYTFTGYY 
                 INPNSGGT 
                 ASGKTYYDFWSGGRR 
               
               
                   
                 (SEQ ID NO: 4) 
                 (SEQ ID NO: 5) 
                 GMDV 
               
               
                   
                   
                   
                 (SEQ ID NO: 6) 
               
               
                   
               
               
                 hLept-3 
                 Ggaggcaccttcagc 
                 Atcatccctatcttt 
                 Gcgagaagccaggta 
               
               
                   
                 agctatgct 
                 ggtacagca 
                 ccatcctcctactac 
               
               
                   
                 (SEQ ID NO: 32) 
                 (SEQ ID NO: 33) 
                 tacggtatggacgtc 
               
               
                   
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
                 GGTFSSYA 
                 IIPIFGTA 
                 ARSQVPSSYYYGMDV 
               
               
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 8) 
                 (SEQ ID NO: 9) 
               
               
                   
               
               
                 hLept-5 
                 Ggattcaccttcagt 
                 Atatcatatgatgga 
                 Gcgagaggtcgtgaa 
               
               
                   
                 agctatgct 
                 agcaataaa 
                 tactactactacatg 
               
               
                   
                 (SEQ ID NO: 35) 
                 (SEQ ID NO: 36) 
                 gacgtc 
               
               
                   
                   
                   
                 (SEQ ID NO: 37) 
               
               
                   
                 GFTFSSYA 
                 ISYDGSNK 
                 ARGREYYYYMDV 
               
               
                   
                 (SEQ ID NO: 10) 
                 (SEQ ID NO: 11) 
                 (SEQ ID NO: 12) 
               
               
                   
               
               
                 hLept-6 
                 Ggatacaccttcacc 
                 Atcaaccctagtggt 
                 Gcgagaggattcggc 
               
               
                   
                 agctactat 
                 ggtagcaca 
                 tacggtggtaaggcc 
               
               
                   
                 (SEQ ID NO: 38) 
                 (SEQ ID NO: 39) 
                 cttgactac 
               
               
                   
                   
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 GYTFTSYY 
                 INPSGGST 
                 ARGFGYGGKALDY 
               
               
                   
                 (SEQ ID NO: 13) 
                 (SEQ ID NO: 14) 
                 (SEQ ID NO: 15) 
               
               
                   
               
               
                 *CDRs are defined based on online antibody sequence analytical tool (IMGT). 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 List of CDR sequences for 
               
               
                 light chain of each antibodies 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                   
               
               
                 hLept-1 
                 Agctccaacatcgga 
                 agtaa 
                 Gcatcatgggatgac 
               
               
                   
                 agtaatact 
                 taat 
                 agcctgaatggtgtg 
               
               
                   
                 (SEQ ID NO: 41) 
                   
                 gta 
               
               
                   
                   
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 SSNIGSNT 
                 SNN 
                 ASWDDSLNGVV 
               
               
                   
                 (SEQ ID NO: 16) 
                   
                 (SEQ ID NO: 17) 
               
               
                   
               
               
                 hLept-2 
                 Cagagcgttagcagg 
                 acttc 
                 Caacagacttacagt 
               
               
                   
                 tat 
                 atcc 
                 accccgtggacg 
               
               
                   
                 (SEQ ID NO: 43) 
                   
                 (SEQ ID NO: 44) 
               
               
                   
                 QSVSRY 
                 TSS 
                 QQTYSTPWT 
               
               
                   
                 (SEQ ID NO: 18) 
                   
                 (SEQ ID NO: 19) 
               
               
                   
               
               
                 hLept-3 
                 Aactccaacatcggg 
                 ggtga 
                 Cagtcctatgacaga 
               
               
                   
                 gcaggttatcat 
                 cact 
                 agccggggtggttgg 
               
               
                   
                 (SEQ ID NO: 45) 
                   
                 ttt 
               
               
                   
                   
                   
                 (SEQ ID NO: 46) 
               
               
                   
                 NSNIGAGYH 
                 GDT 
                 QSYDRSRGGWF 
               
               
                   
                 (SEQ ID NO: 20) 
                   
                 (SEQ ID NO: 21) 
               
               
                   
               
               
                 hLept-5 
                 Aacattgcaagaaaa 
                 aatga 
                 caggtgtgggataat 
               
               
                   
                 agt 
                 taac 
                 agtgattatgtc 
               
               
                   
                 (SEQ ID NO: 47) 
                   
                 (SEQ ID NO: 48) 
               
               
                   
                 NIARKS 
                 NDN 
                 QVWDNSDYV 
               
               
                   
                 (SEQ ID NO: 22) 
                   
                 (SEQ ID NO: 23) 
               
               
                   
               
               
                 hLept-6 
                 Cagaatattaatagt 
                 aaggc 
                 Caacagtttgataaa 
               
               
                   
                 agg 
                 gtct 
                 tattcgatcact 
               
               
                   
                 (SEQ ID NO: 49) 
                   
                 (SEQ ID NO: 50) 
               
               
                   
                 QNINSR 
                 KAS 
                 QQFDKYSIT 
               
               
                   
                 (SEQ ID NO: 24) 
                   
                 (SEQ ID NO: 25) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Binding affinities of anti-Leptin antibodies determined by ELISA 
               
            
           
           
               
               
               
            
               
                   
                 Antibody name 
                 EC50 
               
               
                   
                   
               
               
                   
                 hLept-1 
                 1-500 nM 
               
               
                   
                 hLept-2 
                 1-500 nM 
               
               
                   
                 hLept-3 
                 1-500 nM 
               
               
                   
                 hLept-5 
                 1-500 nM 
               
               
                   
                 hLept-6 
                 1-500 nM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 APPENDIX I 
               
               
                   
               
               
                 Variable DNA sequences of anti-leptin antibodies 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 &gt;hLept1_HCv 
               
               
                 (SEQ ID NO: 51) 
               
               
                 CAGGTACAGCTGCAGCAGTTGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCT 
               
               
                   
               
               
                 GCAATGTCTCTGGTGGCTCCGTCAGCAGAGGTAGTCACTACTGGACCTGGATCCGGCAGCCCCC 
               
               
                   
               
               
                 AGGAAAGGGACTGGAGTGGATTGGGTATATCCACACCGATGGGAGCACCAACTTCAATCCCTCC 
               
               
                   
               
               
                 CTCAAGAGTCGAGTCACCATGTCACTAGACAGGTCCAGGAACCAGTTCTCCCTGACGCTGAGCT 
               
               
                   
               
               
                 CTGTGACCGCTACGGACACGGCCGTTTATTATTGTGCGAGAGAGCCCGGGGGCGCCCTGAATTT 
               
               
                   
               
               
                 CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 
               
               
                   
               
               
                 &gt;hLept1_LCv 
               
               
                 (SEQ ID NO: 52) 
               
               
                 AATTTTATGCTGACTCAGCCACCCTCAACGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT 
               
               
                   
               
               
                 GTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAAC 
               
               
                   
               
               
                 GGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT 
               
               
                   
               
               
                 GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTG 
               
               
                   
               
               
                 ATTATTATTGTGCATCATGGGATGACAGCCTGAATGGTGTGGTATTCGGCGGAGGGACCACGGT 
               
               
                   
               
               
                 GACCGTCCTG  
               
               
                   
               
               
                 &gt;hLept2_HCv 
               
               
                 (SEQ ID NO: 53) 
               
               
                 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT 
               
               
                   
               
               
                 GCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACA 
               
               
                   
               
               
                 AGGGCTTGAGTGGATGGGACGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTT 
               
               
                   
               
               
                 CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGC 
               
               
                   
               
               
                 TGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTGGGAAAACGTATTACGATTTTTGGAG 
               
               
                   
               
               
                 TGGTGGGAGACGCGGTATGGACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA  
               
               
                   
               
               
                 &gt;hLept2_LCv 
               
               
                 (SEQ ID NO: 54) 
               
               
                 GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA 
               
               
                   
               
               
                 CTTGCCGGGCAGGTCAGAGCGTTAGCAGGTATTTAAATTGGTTTCAGCAGAAACCAGGGAAAGC 
               
               
                   
               
               
                 CCCTAAGCTCCTCATCTATACTTCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCC 
               
               
                   
               
               
                 AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCTACCT 
               
               
                   
               
               
                 ACTACTGTCAACAGACTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAA 
               
               
                   
               
               
                 A  
               
               
                   
               
               
                 &gt;hLept3_HCv 
               
               
                 (SEQ ID NO: 55) 
               
               
                 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCT 
               
               
                   
               
               
                 GCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACA 
               
               
                   
               
               
                 AGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTC 
               
               
                   
               
               
                 CAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC 
               
               
                   
               
               
                 TGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAGCCAGGTACCATCCTCCTACTACTA 
               
               
                   
               
               
                 CGGTATGGACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA  
               
               
                   
               
               
                 &gt;hLept3_LCv 
               
               
                 (SEQ ID NO: 56) 
               
               
                 CAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCT 
               
               
                   
               
               
                 GCACTGGGGGCAACTCCAACATCGGGGCAGGTTATCATGTACATTGGTACCAGCAACTTCCAGG 
               
               
                   
               
               
                 AGCAGCCCCCAAACTCCTCATCTATGGTGACACTAATCGGCCCTCAGGGGTCCCTGACCGATTC 
               
               
                   
               
               
                 TCTGGCTCTCAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGACGATGAGG 
               
               
                   
               
               
                 CTGATTATTACTGCCAGTCCTATGACAGAAGCCGGGGTGGTTGGTTTTTCGGCGGAGGGACCCA 
               
               
                   
               
               
                 GCTGACCGTCCTA  
               
               
                   
               
               
                 &gt;hLept5_HCv 
               
               
                 (SEQ ID NO: 57) 
               
               
                 CAGGTGAAGCTGGTGGAGTGGTCGCTGAGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT 
               
               
                   
               
               
                 GCGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA 
               
               
                   
               
               
                 GGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTG 
               
               
                   
               
               
                 AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC 
               
               
                   
               
               
                 TGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGTCGTGAATACTACTACTACATGGA 
               
               
                   
               
               
                 CGTCTGGGGCAAAGGGACCACGGTCAGCGTCTCCTCA  
               
               
                   
               
               
                 &gt;hLept5_LCv 
               
               
                 (SEQ ID NO: 58) 
               
               
                 CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACAT 
               
               
                   
               
               
                 GTGGGGGATACAACATTGCAAGAAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCC 
               
               
                   
               
               
                 TGTGTTGGTCATGTATAATGATAACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCC 
               
               
                   
               
               
                 AACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATT 
               
               
                   
               
               
                 ACTGTCAGGTGTGGGATAATAGTGATTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA 
               
               
                   
               
               
                 &gt;hLept6_HCv 
               
               
                 (SEQ ID NO: 59) 
               
               
                 CAGGTGCAGTTGATGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCT 
               
               
                   
               
               
                 GCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACA 
               
               
                   
               
               
                 AGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTC 
               
               
                   
               
               
                 CAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCC 
               
               
                   
               
               
                 TGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGATTCGGCTACGGTGGTAAGGCCCT 
               
               
                   
               
               
                 TGACTACTGGGGCCAGGGAACCACGGTCACCGTCTCTTCA  
               
               
                   
               
               
                 &gt;hLept6_LCv 
               
               
                 (SEQ ID NO: 60) 
               
               
                 GACATCCAGATGACCCAGTCTCCTCCCACCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCA 
               
               
                   
               
               
                 CTTGCCGGGCCAGTCAGAATATTAATAGTAGGTTGGCCTGGTATCAGCAGAAACCAGGGAGAGC 
               
               
                   
               
               
                 CCCTAAACTCCTGATCTATAAGGCGTCTACTTTAGAGAGTGGGGTCCCATCGAGGTTCAGCGGC 
               
               
                   
               
               
                 AGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCAGATGACTTTGCAACTT 
               
               
                   
               
               
                 ATTACTGCCAACAGTTTGATAAATATTCGATCACTTTCGGCGGAGGGACCAAGATGGAGATCAA 
               
               
                   
               
               
                 A  
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 APPENDIX II 
               
               
                   
               
               
                 Variable amino acid sequences 
               
               
                 of anti-leptin antibodies 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 &gt;hLept1_HCv_AA 
               
               
                 (SEQ ID NO: 61) 
               
               
                 QVQLQQLGPGLVKPSETLSLTCNVSGGSVSRGSHYWTWIRQPPGKGLEWI 
               
               
                   
               
               
                 GYIHTDGSTNFNPSLKSRVTMSLDRSRNQFSLTLSSVTATDTAVYYCARE 
               
               
                   
               
               
                 PGGALNFWGQGTLVTVSS  
               
               
                   
               
               
                 &gt;hLept1_LCv_AA 
               
               
                 (SEQ ID NO: 62) 
               
               
                 DFMLTQPPSTSGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIY 
               
               
                   
               
               
                 SNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCASWDDSLNGVV 
               
               
                   
               
               
                 FGGGTTVTVL  
               
               
                   
               
               
                 &gt;hLept2_HCv_AA 
               
               
                 (SEQ ID NO: 63) 
               
               
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGR 
               
               
                   
               
               
                 INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGK 
               
               
                   
               
               
                 TYYDFWSGGRRGMDVWGQGTMVTVSS 
               
               
                   
               
               
                 &gt;hLept2_LCv_AA 
               
               
                 (SEQ ID NO: 64) 
               
               
                 DIQLTQSPSSLSASVGDRVTITCRAGQSVSRYLNWFQQKPGKAPKLLIYT 
               
               
                   
               
               
                 SSNLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQ 
               
               
                   
               
               
                 GTKLEIK  
               
               
                   
               
               
                 &gt;hLept3_HCv_AA 
               
               
                 (SEQ ID NO: 65) 
               
               
                 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 
               
               
                   
               
               
                 IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSQ 
               
               
                   
               
               
                 VPSSYYYGMDVWGQGTMVTVSS  
               
               
                   
               
               
                 &gt;hLept3_HCv_AA 
               
               
                 (SEQ ID NO: 66) 
               
               
                 QSVLTQPPSVSGAPGQRVTISCTGGNSNIGAGYHVHWYQQLPGAAPKLLI 
               
               
                   
               
               
                 YGDTNRPSGVPDRFSGSQSGTSASLAITGLQADDEADYYCQSYDRSRGGW 
               
               
                   
               
               
                 FFGGGTQLTVL  
               
               
                   
               
               
                 &gt;hLept5_HCv_AA 
               
               
                 (SEQ ID NO: 67) 
               
               
                 QVKLVEWSLSVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAV 
               
               
                   
               
               
                 ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGR 
               
               
                   
               
               
                 EYYYYMDVWGKGTTVSVSS  
               
               
                   
               
               
                 &gt;hLept5_LCv_AA 
               
               
                 (SEQ ID NO: 68) 
               
               
                 QSVLTQPPSVSVAPGKTARITCGGYNIARKSVHWYQQKPGQAPVLVMYND 
               
               
                   
               
               
                 NDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNSDYVFGTG 
               
               
                   
               
               
                 TKVTVL  
               
               
                   
               
               
                 &gt;hLept6_HCv_AA 
               
               
                 (SEQ ID NO: 69) 
               
               
                 QVQLMQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGI 
               
               
                   
               
               
                 INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGF 
               
               
                   
               
               
                 GYGGKALDYWGQGTTVTVSS  
               
               
                   
               
               
                 &gt;hLept6_LCv_AA 
               
               
                 (SEQ ID NO: 70) 
               
               
                 DIQMTQSPPTLSASVGDRVTITCRASQNINSRLAWYQQKPGRAPKLLIYK 
               
               
                   
               
               
                 ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQFDKYSITFGG 
               
               
                   
               
               
                 GTKMEIK