WO2025227054A1 - Monoclonal antibodies binding to leptin and uses thereof - Google Patents

Abstract

The present disclosure is directed to antibodies binding to leptin and methods for use thereof.

Description

MONOCLONAL ANTIBODIES BINDING TO LEPTIN AND USES THEREOF

PRIORITY CLAIM

This application claims benefit of priority to U.S. Provisional Application Serial No. 63/638,688, filed April 25, 2024, the entire contents of which are hereby incorporated by reference.

REFERENCE TO A SEQUENCE LISTING

The application contains a sequence listing which has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing created on April 24, 2025, is named UTFHP0403WO.xml and is 183,371 bytes in size.

BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of medicine, metabolic disease, and immunology. More particularly, the disclosure relates to antibodies binding to leptin and uses thereof.

2. Background

Leptin is a hormone produced by adipocytes and is elevated in obesity; however, the congenital lack of leptin results in obesity. Upon cloning of the leptin gene, the original hope in the field of metabolic disease was that leptin would act as a break for further food intake and a trigger to increase energy expenditure. The hypothesis was that the injection of recombinant leptin would act as an effective weight loss mechanism. Unfortunately, the hypothesis was quickly overturned since obese individuals have high leptin levels, but the individual is leptin resistant. Not even injection of very large amounts of leptin can overcome this resistance. Thus, there remains a need for ways to address the various pathologic states that involve elevated leptin levels and leptin resistance. SUMMARY

Thus, in accordance with the present disclosure, provided is a method of detecting leptin in a sample comprising: (a) contacting a sample with an antibody or antibody fragment comprising clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) detecting leptin in the sample by detecting binding of the antibody or antibody fragment to leptin in the sample. In some embodiments, the sample is a body fluid, such as blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces. In some embodiments, detection comprises ELISA, RIA, lateral flow assay or Western blot. In some embodiments, the method further comprise performing steps (a) and (b) a second time and determining a change in leptin levels as compared to the first assay. In some embodiments, the antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1. In some embodiments, the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired variable sequences as set forth in Table 1. In some embodiments, the antibody or antibody fragment comprises light and heavy chain variable sequences comprising or consisting of clone-paired sequences from Table 2. In some embodiments, the antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2. In some embodiments, the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

The present disclosure also provides a method of treating a subject, comprising delivering to the subject an antibody or antibody fragment comprising clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. In some embodiments, the antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1. In some embodiments, the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired variable sequences as set forth in Table 1. In some embodiments, the antibody or antibody fragment comprises light and heavy chain variable sequences comprising or consisting of clone-paired sequences from Table 2. 1 some embodiments, the antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2. In some embodiments, the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In some embodiments, the antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LAL A, LALA- PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. In some embodiments, the antibody is a chimeric antibody or a bispecific antibody. In some embodiments, delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or a vector encoding the antibody or antibody fragment. In some embodiments, the subject exhibits one or more of liver fibrosis, diabetes, and metabolic syndrome. In some embodiments, the subject exhibits one or more of increased sensitivity to insulin, reduction in fat mass and/or weight loss, substance weight loss over time, reduced weight gain over time, improvement of diabetic phenotype, and reduced liver fibrosis. The subject may have cancer, such as leukemia, meningioma, adenocarcinoma, multiple myeloma, uterine cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, liver cancer, breast cancer, thyroid cancer and gallbladder cancer. Delivering may result in one or more of reduced tumor burden, reduced tumor size, cancer cell death, cancer remission, delay of cancer progression, and/or increase in patient survival. The subject may be treated with a second therapy, such as insulin, GLP-1 receptor antagonists, SGLT2 inhibitors, biguanides, sulfonylureas, insulin secretagogues, chemotherapy, radiotherapy, immunotherapy, or surgery.

Also provided is a monoclonal antibody, wherein the antibody or antibody fragment comprises clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. In some embodiments, the antibody or antibody fragment is encoded by clone- paired variable sequences as set forth in Table 1. In some embodiments, the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired variable sequences as set forth in Table 1. In some embodiments, the antibody or antibody fragment comprises light and heavy chain variable sequences comprising or consisting of clone-paired sequences from Table 2. In some embodiments, the antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2. In some embodiments, the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In some embodiments, the antibody is a chimeric antibody or a bispecific antibody. In some embodiments, the antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. In some embodiments, the antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody.

Also provided is a hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment comprises clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. In some embodiments, the hybridoma or engineered cell comprises sequences encoding an antibody or antibody fragment having light and heavy chain variable sequences according to clone-paired sequences from Table 1, or having at least 70%, 80%, 90% or 95% identity to clone-paired variable sequences from Table 1. In some embodiments, the hybridoma or engineered cell expresses an antibody or antibody fragment comprising or consisting of light and heavy chain variable sequences according to clone-paired sequences from Table 2, or having at least 70%, 80%, 90% or 95% identity to clone-paired variable sequences from Table 2. In some embodiments, the hybridoma or engineered cell expresses an antibody fragment that is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, Hab'E fragment, or Fv fragment In some embodiments, the hybridoma expresses an antibody that is a chimeric antibody or a bispecific antibody. In some embodiments, the antibody is an IgG, or a recombinant IgG antibody or antibody fragment may comprise an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA- PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. In some embodiments, the antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody.

Also provided is a vaccine formulation comprising one or more antibodies or antibody fragments comprising clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. In some embodiments, the one or more antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1. In some embodiments, the one or more antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired variable sequences as set forth in Table 1. In some embodiments, the one or more antibody or antibody fragment comprises light and heavy chain variable sequences comprising or consisting of clone-paired sequences from Table 2. In some embodiments, the one or more antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2. In some embodiments, the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')i fragment, or Fv fragment. In some embodiments, the antibody fragment comprises at least one recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In some embodiments, the one or more antibody is a chimeric antibody or a bispecific antibody. In some embodiments, the at least one antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. In some embodiments, the at least one antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody.

Also provided is a vaccine formulation comprising one or more expression vector(s) encoding a first antibody or antibody fragment as defined herein. In some embodiments, the expression vector(s) is Sindbis virus or VEE vector(s). In some embodiments, the vaccine is formulated for delivery by needle injection, jet injection, or electroporation. In some embodiments, the vaccine further comprises one or more expression vector(s) encoding a second antibody or antibody fragment as defined herein.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-C. Determination of binding affinity of LEPTIN antibodies using ELISA. Human (h-Lept) or mouse (m-Lept) LEPTIN recombinant protein (Sino Biological) was coated on a 96-well high binding plate over night at 2 pg/ml concentration at 4 °C in PBS. Purified antibodies in a series of concentrations were added for binding to LEPTIN antigen coated on the plate. Bound antibody was detected using a secondary detection antibody against human IgG conjugated with HRP (Jackson ImmunoResearch) with TMB substrate for absorbance detection at A450nm. (FIG. 1 A) Leptin mAb binding to human leptin protein in ELISA. (FIG. IB) Leptin mAb binding to mouse leptin protein in ELISA. (FIG. 1C) EC 50 determined using concentration titration curves from ELISA.

FIG. 2. Determination of kinetic binding affinity using Octet 96-Red instrument. Leptin antibody (30 pg/mL) was loaded onto the protein A biosensors for 4 min. Following incubation in kinetics buffer to establish baseline. Antibody loaded biosensors were exposed to a series of concentrations (starting 1875 nM, 3-fold titration down to 1.6 nM) of leptin protein (HIS tag at C terminus). Background subtraction was used to correct sensor drifting.

FIG. 3. Luciferase assay for leptin antibody screening.

FIG. 4. Plasmid ratio for transfection. The condition labeled 5 (Luc2P:LepR=40:20 (ng/well)) showed relatively good response and high sensitivity to increased leptin concentration among the six conditions.

FIG. 5. Comparison of dose response with antibody concentrations of 5 and 10 pg/ml. FIG. 6. Evaluation of antibody binding efficiency. Lep-P12E4 showed increased leptin binding capability as the Ab concentration increased. Lep-P14A12 showed enhanced effects compared to Lep-P12E4.

FIG. 7. pSTAT3 detection for leptin + leptin mAb. Blue arrow shows three mAb concentrations used for detecting pSTAT3 by Western blot. Bottom panel uses HEK293T cells.

FIG. 8. Evaluation of purified leptin activity. IgG: isotype control. Purified leptin showed comparable activity as the commercial source. Leptin mAb recognized and neutralized purified leptin with comparable efficiency to commercial source.

FIG. 9. Luciferase assay for leptin mAb neutralization efficiency - N2a cells.

FIG. 10. Test of leptin antibody (A).

FIG. 11. Test of leptin antibody (B).

FIGS. 12A-G. Structural characterization of a leptin-neutralizing antibody. (FIG. 12A) Coomassie blue-stained reducing SDS-PAGE gel of leptin-neutralizing antibody, Fab, Fabdeptin complex, and leptin. (FIGS. 12B-C) The interaction of the Fab with leptin mimics that of the leptin receptor (LEPR) CRH2 domain with leptin. The LEPR CHR2 domain interacts predominantly with the helix A of leptin, while the Fab interacts with both helices A and C as well as the loop between helices C and D of leptin. (FIG. 12D) The Fab loops and residues that adopt different orientations comparing Fabdeptin complex and the unbound Fab. Shown is the superposition of the variable regions of the unbound Fab (colored orange) to the variable regions of the Fab in the Fabdeptin complex (Fab light chain colored cyan, Fab heavy chain colored light blue). These superimposed with an r.m.s.d. of 0.44 A for 220 Ca atoms. The largest difference occurs in the CDR H3 loop (heavy chain residues 100-107). In the unbound Fab, residues 102-107 are unmodeled due to a lack of interpretable electron density. (FIG. 12E) Residues from both heavy and light chains of the Fab interact with conserved residues of the leptin’s helix A. Residues that have non-H atoms within 5.0 A or less between the Fab (Fab light chain colored cyan, Fab heavy chain colored light blue) and leptin (colored yellow) were identified in the program Ncont in the CCP4 program suite (Agirre et al., 2023). Leptin residues are prefixed with the letter ‘L’, Fab heavy chain residues with the letters ‘HC’, and Fab light chain residues with the letters ‘LC’ . Hydrogen bonds are depicted as dashed black lines. (FIG. 12F) Residues from both heavy and light chains of the Fab interact with conserved residues of the leptin’s helix C. For clarity, some of the interaction shown in FIG. 12D are not shown here. Hydrogen bonds are depicted as dashed black lines. (FIG. 12G) Residues from the Fab heavy chain interact with conserved residues of the leptin’ s helix C and the loop between the helices C and D. The charged carboxylate of LGlulOO points away from the hydrophobic cluster of HCPhe55, LHis88, and LPhe92. For clarity, some of the interactions shown in FIG. 12C are not shown here. Helices A and C as well as residues from the loop between helices C and D of leptin contribute to all the interactions with the heavy and light chains of the leptin- neutralizing antibody.

FIGS. 13A-D. Local leptin signaling is elevated in liver and kidney fibrosis. For liver fibrosis induction, Mup-uPA mice and control littermates fed a high-fat diet (HFD) for 12 weeks and then sacrificed. For kidney fibrosis induction, wild-type mice were injected with folic acid (200 mg/kg) or vehicle and sacrificed at D7. (FIGS. 13A-B) RT-qPCR analysis of total and long isoform leptin receptor (Lepr and Leprb, respectively) mRNA expression in livers (FIG. 13A) (n=4-7 per group) and kidneys (FIG. 13B) (n=4-6 per group). (FIGS. 13C- D) Immunoblot analysis of p-STAT3, STAT3, SOCS-3 protein expression in livers (FIG. 13C) (n=3 per group) and kidneys (FIG. 13D) (n=4 per group). Data are presented as mean+SEM and were analyzed by a one-tailed Student’s t test. *, p< 0.05; **, p< 0.01 ; **, pcO.001.

FIGS. 14A-G. Leptin neutralization diminishes kidney fibrosis. (FIG. 14A) Experimental setup of folic acid-induced kidney fibrosis. (FIG. 14B) Serum blood urea nitrogen (BUN) levels. (FIG. 14C) RT-qPCR analysis of fibrotic gene mRNA expression in the kidney (n=3-6 per group). (FIG. 14D) H&E and Masson’s trichome staining of kidney. Representative microphotographs are shown (n=3 per group). Scale bar equals 250 pm. (FIGS. 14E-F) RT-qPCR analysis of fibrotic (FIG. 14E) and inflammatory (FIG. 14F) gene mRNA expression in the kidney (n=4-5 per group). (FIG. 14G) Immunofluorescence staining of fibronectin (FN) and collagen I (COL1) of kidney. Representative microphotographs are shown (n=4 per group). Scale bar equals 100 pm. (FIGS. 14B-C and FIGS. 14E-F) Data are presented as mean+SEM and were analyzed by a two-tailed Student’s t test. *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p<0.0001.

FIGS. 15A-F. Leptin neutralization attenuates liver fibrosis. (FIG. 15 A) Experimental setup of Mup-uPA and HFD-induced liver fibrosis. (FIG. 15B) H&E and Masson’s trichome staining of liver. Representative microphotographs are shown (n=5 per group). Scale bar equals 250 pm. (FIGS. 15C-D) RT-qPCR analysis of fibrotic (FIG. 15C) and inflammatory gene (FIG. 15D) mRNA expression in the liver (n=4-7 per group). (FIG. 15E) Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (n=4 per group). (FIG. 15F) Immunofluorescence staining of fibronectin (FN) and collagen I (COL1) of liver. Representative microphotographs are shown (n=3-4 per group). Scale bar equals 100 pm. (FIGS. 15C-E) Data are presented as mean+SEM and were analyzed by an unpaired two-tailed t test. *, p< 0.05; **, p< 0.01. FIGS. 16A-E. Leptin neutralization alleviates lung fibrosis. (FIG. 16A) Experimental setup of bleomycin-induced lung fibrosis. (FIG. 16B) H&E and Masson’s trichome staining of lungs. Representative microphotographs are shown (n = 4 per group). H&E Scale bar equals 250 pm. Trichrome Scale bar equals 500 pm. (FIG. 16C) RT-qPCR analysis of fibrotic gene mRNA expression in the lung (n=6-12 per group). (FIG. 16D) RT- qPCR analysis of inflammatory gene mRNA expression in the lung (n=4-7 per group). (FIG. 16E) Immunofluorescence staining of fibronectin (FN) and collagen hybridizing peptide (CHP) of the lung. Representative microphotographs are shown (n=4 per group). Scale bar equals 100 pm. (FIG. 16C) Data are presented as mean+SEM and were analyzed by a two-tailed Student’s t test. *, p< 0.05; **, p< 0.01.

FIGS. 17A-D. Further characterization of a leptin-neutralizing antibody. (FIG. 17A) Overview of the Fabileptin complex structure. Leptin (colored yellow and with helices A and C in pink) is shown docked with the Fab light chain (colored cyan) and heavy chain (colored light blue). (FIG. 17B) Overview of the leptin receptor (LEPR) CRH2:leptin complex. Leptin (colored yellow and with helices A and C in pink) is shown docked with the CRH2 domain (colored green; PDB: 8X80 (Xie et al., 2023)). (FIG. 17C) Overview of the interleukin- 4 (IL-4) receptor a chain:IL-4 complex. IL-4 (multicolored) is shown docked with the IL-4R a chain (colored green; PDB: 1IAR). This interaction closely resembles the extended interaction of the leptin-neutralizing Fab with leptin. Buried surface areas are 810 A2 for the IL-4R a chain: IL-4 complex (Hage et al. , 1999), 760 A for the LEPR CRH2: leptin complex, and 870 A2 for the leptin-neutralizing Fab:leptin complex. The latter two according to the Protein Interfaces, Surfaces and Assemblies (PISA) service at the European Bioinformatics Institute (world- wide - web at ebi.ac.uk/pdbe/prot_int/pistart.html) (Krissinel & Henrick, 2007). (FIG. 17D) pSTAT3 response in HEK293 cells co-transfected with LEPRb and a pSTAT3::luciferase reporter plasmids and stimulated for 12 hours with leptin (40 ng/ml) in presence or absence of either LepAb or control IgG (n=3-4 per group). Data are presented as mean±SEM and were analyzed by a two-tailed Student’s t test. *, p< 0.05.

FIGS. 18A-B. Further effects of leptin neutralization in kidney fibrosis. Refer to FIG. 3 for experimental setup. (FIG. 18A) Body weight (n=3-6 per group). (FIG. 18B) Gross anatomy of kidney (n= 1 -2 per group). (FIG. 18 A) Data are presented as mean ± SEM and were analyzed by a two-way ANOVA test. No significant differences were detected.

FIG. 19. Further effects of leptin neutralization in liver fibrosis. Refer to FIG. 4 for experimental setup. Body weight (n=7 per group). Data are presented as mean+SEM and were analyzed by a two-way ANOVA. No significant differences were detected. FIGS. 20A-K. Leptin neutralization mitigates aorta and kidney fibrosis, but not heart fibrosis. (FIG. 20A) Experimental setup of angiotensin (Ang II)-induced heart and kidney fibrosis. (FIGS. 20B-C) Tail-cuff measurements of systolic (FIG. 20B) and diastolic (FIG. 20C) blood pressure (n=4-5 per group). (FIG. 20D) Ratio of ventricular weight to body weight (n=3-6 per group). (FIG. 20E) H&E and Masson’s trichome staining of heart. Representative microphotographs are shown (n=4 per group). Scale bar equals 50 pm. (FIGS. 20F-G) RT-qPCR analysis of fibrotic gene mRNA expression in the heart (FIG. 20F) and kidney (FIG. 20G) (n=3-6 per group). (FIGS. 20H-I) H&E and Masson’s trichome staining of kidney. Representative microphotographs (FIG. 20H) and quantification (FIG. 201) are shown (n=3-5 mice per group; 3-4 sections per mouse for quantification). Scale bar equals 50 pm. (FIGS. 20J-K) H&E and picrosirius red staining of aorta. Representative microphotographs (FIG. 20J) and quantification (FIG. 20K) are shown (n=3-5 mice per group; 5-10 sections per mouse for quantification). Scale bar equals 50 pm. (FIGS. 20B-D, FIGS. 20F-G, FIG. 201, and FIG. 20K) Data are presented as mean+SEM and were analyzed by one-way ANOVA. *, p< 0.05; **, p< 0.01; ***, p< 0.001; ****, p< 0.0001.

FIG. 21. In vivo assessment of leptin and palcociclib on MCV-7 Y537S cells. MCF- 7 Y537S cells were implanted subcutaneously into ovariectomized athymic, nude mice without exogenous P-estradiol supplementation. When tumors reached 100-150 mm³, mice were randomized (n = 10 tumors/arm) and received IgG, palbociclib (100 mg daily by oral gavage,

6 days/week), leptin (anti-leptin mAb hLep3; 10 mg/kg intraperitoneal (IP) injection, twice weekly), or palbociclib + leptin (anti-leptin mAb) as indicated. Tumor volumes were monitored every 3-4 days. The results, line graph and bar graphs were plotted as percentage of tumor volume change for each group + SEM. Statistical significance were calculated using unpaired two-tailed Mann- Whitney t-test.

FIG. 22. In vivo assessment of leptin and palcociclib on MCV-7 Y537S cells MCF-

7 Y537S cells were implanted subcutaneously into ovariectomized athymic, nude mice without exogenous P-estradiol supplementation. When tumors reached 100-150 mm³, mice were randomized (n = 10 tumors/arm) and received IgG, palbociclib (100 mg daily by oral gavage, 6 days/week), leptin (anti-leptin mAb; 10 mg/kg intraperitoneal (IP) injection, twice weekly), or palbociclib + leptin (anti-leptin mAb hLep3) as indicated. Tumor volumes were monitored every 3-4 days. The results, line graph and bar graphs were plotted as percentage of tumor volume change for each group + SEM. Statistical significance were calculated using unpaired two-tailed Mann- Whitney t-test. FIG. 23. In vivo assessment of leptin and irradiation on MCV-7 Y537S cells MCF- 7 Y537S cells were implanted subcutaneously into ovariectomized athymic, nude mice without exogenous P-estradiol supplementation. When tumors reached 100-150 mm3, mice were randomized (n = 10 tumors/arm) for IgG, leptin, irradiation (IR) and IR + leptin (anti-leptin mAh) group. Mice were treated with IgG, two doses of leptin (anti-leptin mAh hLep3; 10 mg/kg intraperitoneal (IP) injection), IR (2Gy consecutively for 6 days) and IR + leptin as indicated. Tumor volumes were monitored every 3-4 days. The results were plotted as average tumor volume measured for each group + SEM.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, use of leptin in patients as a therapeutic intervention in obesity has not come to fruition due to issues such as high leptin levels in obese patent and the development of leptin resistance. The present inventors generated a battery of monoclonal antibodies (mAbs) against leptin. These mAbs showed strong binding affinities to human leptin and significant leptin neutralizing activity in vivo. Therefore, they propose these antibodies for the effective treatment of obesity with high levels of leptin where there is resistance to conventional leptin treatment.

These and other aspects of the disclosure are described in detail below.

I. Leptin

Leptin is a protein hormone predominantly made by adipocytes (cells of adipose tissue). Its primary role is likely to regulate long-term energy balance. As one of the major signals of energy status, leptin levels influence appetite, satiety, and motivated behaviors oriented towards the maintenance of energy reserves (e.g., feeding, foraging behaviors).

The amount of circulating leptin correlates with the amount of energy reserves, mainly triglycerides stored in adipose tissue. High leptin levels are interpreted by the brain that energy reserves are high, whereas low leptin levels indicate that energy reserves are low, in the process adapting the organism to starvation through a variety of metabolic, endocrine, neurobiochemical, and behavioral changes.

Leptin is coded for by the LEP gene. Leptin receptors are expressed by a variety of brain and peripheral cell types. These include cell receptors in the arcuate and ventromedial nuclei, as well as other parts of the hypothalamus and dopaminergic neurons of the ventral tegmental area, consequently mediating feeding.

Although regulation of fat stores is deemed to be the primary function of leptin, it also plays a role in other physiological processes, as evidenced by its many sites of synthesis other than fat cells, and the many cell types beyond hypothalamic cells that have leptin receptors. Many of these additional functions are yet to be fully defined. In obesity, a decreased sensitivity to leptin occurs (similar to insulin resistance in type 2 diabetes), resulting in an inability to detect satiety despite high energy stores and high levels of leptin.

Predominantly, the "energy expenditure hormone" leptin is made by adipose cells, and is thus labeled fat cell specific. In the context of its effects, the short describing words central, direct and primary are not used interchangeably. In regard to the hormone leptin, central vs. peripheral refers to the hypothalamic portion of the brain vs. non-hypothalamic location of action of leptin; direct vs. indirect refers to whether there is no intermediary, or there is an intermediary in the mode of action of leptin; and primary vs secondary is an arbitrary description of a particular function of leptin.

In vertebrates, the nervous system consists of two main parts, the central nervous system (CNS) and the peripheral nervous system (PNS). The primary effect of leptins is in the hypothalamus, a part of the central nervous system. Leptin receptors are expressed not only in the hypothalamus but also in other brain regions, particularly in the hippocampus. Thus, some leptin receptors in the brain are classified as central (hypothalamic) and some as peripheral (non-hypothalamic).

Although leptin reduces appetite as a circulating signal, obese individuals generally exhibit a higher circulating concentration of leptin than normal weight individuals due to their higher percentage body fat. These people show resistance to leptin, similar to resistance of insulin in type 2 diabetes, with the elevated levels failing to control hunger and modulate their weight. A number of explanations have been proposed to explain this. An important contributor to leptin resistance is changes to leptin receptor signaling, particularly in the arcuate nucleus, however, deficiency of, or major changes to, the leptin receptor itself are not thought to be a major cause. Triglycerides crossing the blood brain barrier (BBB) can induce leptin and insulin resistance in the hypothalamus. Triglycerides can also impair leptin transport across the BBB.

Studies on leptin cerebrospinal fluid (CSF) levels provide evidence for the reduction in leptin crossing the BBB and reaching obesity-relevant targets, such as the hypothalamus, in obese people. In humans, it has been observed that the ratio of leptin in the CSF compared to the blood is lower in obese people than in people of a normal weight. The reason for this may be high levels of triglycerides affecting the transport of leptin across the BBB or due to the leptin transporter becoming saturated. Although deficits in the transfer of leptin from the plasma to the CSF is seen in obese people, they are still found to have 30% more leptin in their CSF than lean individuals. These higher CSF levels fail to prevent their obesity. Since the amount and quality of leptin receptors in the hypothalamus appears to be normal in the majority of obese humans (as judged from leptin-mRNA studies), it is likely that the leptin resistance in these individuals is due to a post leptin-receptor deficit, similar to the post-insulin receptor defect seen in type 2 diabetes.

When leptin binds with the leptin receptor, it activates a number of pathways. Leptin resistance may be caused by defects in one or more parts of this process, particularly the JAK7STAT pathway. Mice with a mutation in the leptin receptor gene that prevents the activation of STAT3 are obese and exhibit hyperphagia. The PI3K pathway may also be involved in leptin resistance, as has been demonstrated in mice by artificial blocking of PI3K signaling. The PI3K pathway also is activated by the insulin receptor and is therefore an important area where leptin and insulin act together as part of energy homeostasis. The insulin- PI3K pathway can cause POMC neurons to become insensitive to leptin through hyperpolarization.

Leptin is known to interact with amylin, a hormone involved in gastric emptying and creating a feeling of fullness. When both leptin and amylin were given to obese, leptin-resistant rats, sustained weight loss was seen. Due to its apparent ability to reverse leptin resistance, amylin has been suggested as possible therapy for obesity.

It has been suggested that the main role of leptin is to act as a starvation signal when levels are low, to help maintain fat stores for survival during times of starvation, rather than a satiety signal to prevent overeating. Leptin levels signal when an animal has enough stored energy to spend it in pursuits besides acquiring food. This would mean that leptin resistance in obese people is a normal part of mammalian physiology and, possibly, could confer a survival advantage. Leptin resistance (in combination with insulin resistance and weight gain) is seen in rats after they are given unlimited access to palatable, energy-dense foods. This effect is reversed when the animals are put back on a low-energy diet. This also may have an evolutionary advantage: allowing energy to be stored efficiently when food is plentiful would be advantageous in populations where food frequently may be scarce.

IL Monoclonal Antibodies and Production Thereof

An "isolated antibody" is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and includes enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most particularly more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody is prepared by at least one purification step.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable region (VH) followed by three constant domains (CH) for each of the alpha and gamma chains and four CH domains for mu and isotypes. Each L chain has at the N-terminus, a variable region (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. They gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

The term "variable" refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. , around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31- 35 (Hl), 50-65 (H2) and 95-102 (H3) in the Vn when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a "hypervariable loop" (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (Hl), 52-56 (H2) and 95-101 (H3) in the Vn when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a "hypervariable loop'VCDR (e.g., residues 27-38 (LI), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (Hl), 56-65 (H2) and 105-120 (H3) in the V_H when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (LI), 63, 74- 75 (L2) and 123 (L3) in the VL, and 28, 36 (Hl), 63, 74-75 (H2) and 123 (H3) in the V_subH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)).

By "germline nucleic acid residue" is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. "Germline gene" is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A "germline mutation" refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or can be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent 4,816,567) after single cell sorting of an antigen specific B cell, an antigen specific plasmablast responding to an infection or immunization, or capture of linked heavy and light chains from single cells in a bulk sorted antigen specific collection. The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al. , Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

A. General Methods

Methods for preparing and characterizing antibodies are well known in the art (see, e.g. , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265). The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection or vaccination with a licensed or experimental vaccine. As is well known in the art, a given composition for immunization can vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bisbiazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants in animals include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants and aluminum hydroxide adjuvant and in humans include alum, CpG, MFP59 and combinations of immunostimulatory molecules (“Adjuvant Systems”, such as AS01 or AS03). Additional experimental forms of inoculation to induce leptin-specific B cells is possible, including nanoparticle vaccines, or gene-encoded antigens delivered as DNA or RNA genes in a physical delivery system (such as lipid nanoparticle or on a gold biolistic bead), and delivered with needle, gene gun, transcutaneous electroporation device. The antigen gene also can be carried as encoded by a replication competent or defective viral vector such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively a virus like particle.

In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects that have been exposed to the pathogens, such as those who have been diagnosed as having contracted the disease, or those who have been vaccinated to generate protective immunity against the pathogen or to test the safety or efficacy of an experimental vaccine. Circulating anti-pathogen antibodies can be detected, and an antibody encoding or producing B cells from the antibody-positive subject can then be obtained.

The amount of immunogen composition used in the production of polyclonal antibodies varies based on the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also can be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells can be obtained from biopsied spleens, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs like lung or GI tract, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal or immune human are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells can be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion can vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. In some cases, transformation of human B cells with Epstein Barr virus (EBV) as an initial step increases the size of the B cells, enhancing fusion with the relatively large-sized myeloma cells. Transformation efficiency by EBV is enhanced by using CpG and a Chk2 inhibitor drug in the transforming medium. Alternatively, human B cells can be activated by co-culture with transfected cell lines expressing CD40 Ligand (CD 154) in medium containing additional soluble factors, such as IL-21 and human B cell Activating Factor (BAFF), a Type II member of the TNF superfamily. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71 -74, 1986) and there are processes for better efficiency (Yu et al. , 2008). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10‘⁶ to 1 x 10"⁸, but with optimized procedures one can achieve fusion efficiencies close to 1 in 200 (Yu et al., 2008). However, relatively low efficiency of fusion does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the medium is supplemented with hypoxanthine. Ouabain is added if the B cell source is an EBV- transformed human B cell line, in order to eliminate EBV-transformed lines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain can also be used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines can be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum- free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.

MAbs produced by either means can be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach can be used to generate monoclonal antibodies. Single B cells identified as responding to infection or vaccination because of plasmablast or activated B cell markers, or memory B cells labelled with the antigen of interest, can be sorted physically using paramagnetic bead selection or flow cytometric sorting, then RNA can be isolated from the single cells and antibody genes amplified by RT- PCR. Various single-cell RNA-seq methods are available to obtain antibody variable genes from single cells. Alternatively, antigen-specific bulk sorted populations of cells can be segregated into microvesicles and the matched heavy and light chain variable genes recovered from single cells using physical linkage of heavy and light chain amplicons, or common barcoding of heavy and light chain genes from a vesicle. Matched heavy and light chain genes from single cells also can be obtained from populations of antigen specific B cells by treating cells with cell-penetrating nanoparticles bearing RT-PCR primers and barcodes for marking transcripts with one barcode per cell. The antibody variable genes also can be isolated by RNA extraction of a hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates.

B. Antibodies of the Present Disclosure

Antibodies according to the present disclosure can be defined, in the first instance, by their binding specificity for leptin. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. For example, the epitope to which a given antibody binds can consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20) amino acids located within the antigen molecule (e.g., a linear epitope in a domain). Alternatively, the epitope can consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope).

Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody ‘‘interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Cross-blocking can be measured in various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke, Methods Mel. Biol. 248: 443-63, 2004), peptide cleavage analysis, high-resolution electron microscopy techniques using single particle reconstruction, cryoEM. or tomography, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer Prot. Sci. 9: 487-496, 2000). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuteri um-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuteri um-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface can retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e. t;., Ehring, Analytical Biochemistry 267: 252-259 (1999); Engen and Smith, Anal. Chem. 73: 256A-265A (2001).

'The term “epitope” refers to a site on an antigen to which B and/or T cells respond. 13- cell epitopes can be formed -from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary foldi ng are typically lost on treatment with denaturing solvents. An epitope typically i ncludes at least 3, and more usually, at least. 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling tMAPj, also known as Antigen Structure-based Antibody Profiling (ASAP), is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see U.S. Patent Publication 2004/0101920, herein specifically incorporated by reference in its entirely). Each category can reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapidfiltering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP can be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes.

The present disclosure includes antibodies that bind to the same epitope, or a portion of the epitope. Likewise, the present disclosure also includes antibodies that compete for binding to a target or a fragment thereof with any of the specific exemplary antibodies described herein. One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference, the reference antibody is allowed to bind to target under saturating conditions. Next, the ability of a test antibody to bind to the target molecule is assessed. If the test antibody is able to bind to the target molecule following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to die target molecule following saturation binding with the reference antibody, then the test antibody binds to the same epitope as the epitope bound by the reference antibody. To determine if an antibody competes for binding with a reference anti-leptin antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to leptin tinder saturating conditions followed by assessment of binding of the test antibody to leptin. In a second orientation, the test antibody is allowed to bind to leptin under saturating conditions followed by assessment of binding of the reference antibody io leptin. If, in both orientations, only the first (saturating) antibody is capable of binding to leptin, then it is concluded that tire test antibody and the reference antibody compete for binding to leptin As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a l-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans el al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody- binding assay available in the art. Structural studies with EM or crystallography also can demonstrate whether or not two antibodies that compete for binding recognize the same epitope.

The present disclosure provides monoclonal antibodies having clone paired CDRs from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such antibodies can be produced by the clones discussed below in the Examples section using methods described herein.

In another aspect, the antibodies can be defined by their variable sequence, which include additional “framework” regions. Furthermore, the antibody sequences can vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences can vary from those set out above in that (a) the variable regions can be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids can vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids can vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids can vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C, (e) the amino acids can vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids can vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing applies to the nucleic acid sequences and the amino acid sequences.

When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison can be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins- Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5: 151-153; Myers, E. W. and Muller W. (1988) CABIOS 4: 11- 17; Robinson, E. D. (1971) Comb. Theor 11 :105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy— the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730. Alternatively, optimal alignment of sequences for comparison can be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

One particular example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of an antibody sequence and the variable length of each gene requires multiple rounds of BLAST searches for a single antibody sequence. Also, manual assembly of different genes is difficult and error prone. The sequence analysis tool IgBLAST (world-wide-web at ncbi.nlm.nih.gov/igblast/) identifies matches to the germline V, D and J genes, details at rearrangement junctions, the delineation of Ig V domain framework regions and complementarity determining regions. IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches and allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene.

In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

In one approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions (z.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Yet another way of defining an antibody is as a “derivative” of any of the below- described antibodies and their antigen-binding fragments. The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen, but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule. Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non- naturally occurring amino acid residues. The term “derivative” encompasses, for example, as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example, antibodies, etc. , having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics. The term “derivative” additionally encompasses non-amino acid modifications, for example, amino acids that can be glycosylated (e.g., have altered mannose, 2-N- acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5- glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment, the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R. L. et al. (2002) “Lack Of Fucose On Human IgG N-Linkecl Oligosaccharide Improves Binding To Human Fcgamma RIH And Antibody-Dependent Cellular Toxicity.'' J. Biol. Chem. 277(30): 26733-26740; Davies J. et al. (2001) “Expression Of GriTIH In A Recombinant Anti- CD2Q CHO Production Cell Line: Expression Of Antibodies With Altered Glycoforms Leads To An Increase In ADCC Through Higher Affinity For FC Gamma RIH,” Biotechnology & Bioengineering 74(4): 288-294). Methods of altering carbohydrate contents are known to those skilled in the art, see, e.g., Wallick, S. C. et al. (1988), J. Exp. Med. 168(3): 1099-1109; Tao, M. H. et al. (1989), J. Immunol. 143(8): 2595-2601; Routledge, E. G. et al. (1995), Transplantation 60(8):847-53; Elliott, S. et al. (2003), Nature Biotechnol. 21 :414-21 ; Shields, R. L. et al. (2002), J. Biol. Chem. 277(30): 26733-26740).

A derivative antibody or antibody fragment can be generated with an engineered sequence or glycosylation state to confer preferred levels of activity in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibodydependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions as measured by bead-based or cell-based assays or in vivo studies in animal models.

A derivative antibody or antibody fragment can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative will possess a similar or identical function as the parental antibody. In another embodiment, an antibody derivative will exhibit an altered activity relative to the parental antibody. For example, a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody.

C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. Modified antibodies can be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. The following is a general discussion of relevant goals techniques for antibody engineering.

Hybridomas can be cultured, then cells lysed, and total RNA extracted. Random hexamers can be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR products can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization can be performed using antibodies collected from hyhridoma supernatants and purified by FPLC, using Protein G columns.

Recombinant full-length IgG antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant. Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows.

Expression of nucleic acids encoding antibodies, both for the purpose of subsequent antibody purification, and for immunization of a host, is also contemplated. Antibody coding sequences can be RNA, such as native RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, Nl-methyl-pseudouridine (NlmT) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In addition to turning off the immune/eIF2a phosphorylation-dependent inhibition of translation, incorporated N l m'P nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment. Such modifications could be used to enhance antibody expression in vivo following inoculation with RNA. The RNA, whether native or modified, can be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle.

Alternatively, DNA encoding the antibody can be employed for the same purposes. The DNA is included in an expression cassette comprising a promoter active in the host cell for which it is designed. The expression cassette is advantageously included in a replicable vector, such as a conventional plasmid or minivector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno- associated viruses, and lentiviruses are contemplated. Replicons encoding antibody genes such as alphavirus replicons based on VEE virus or Sindbis virus are also contemplated. Delivery of such vectors can be performed by needle through intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation when in vivo expression is desired.

The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.

Antibody molecules will comprise fragments (such as F(ab'), F(ab')2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. F(ab') antibody derivatives are monovalent, while Fiab'L antibody derivatives are bivalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules can contain substituents capable of binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g. , an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 + 1), glutamate (+3.0 + 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 + 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those that are within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgGi can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

Alternatively or additionally, it can be useful to combine amino acid modifications with one or more further amino acid modifications that alter Clq binding and/or the complement dependent cytotoxicity (CDC) function of the Fc region of an IL-23pl9 binding molecule. The binding polypeptide of particular interest can be one that binds to C Iq and displays complement dependent cytotoxicity. Polypeptides with pre-existing Clq binding activity, optionally further having the ability to mediate CDC can be modified such that one or both of these activities are enhanced. Amino acid modifications that alter Clq and/or modify its complement dependent cytotoxicity function are described, for example, in WO/0042072, which is hereby incorporated by reference.

One can design an Fc region of an antibody with altered effector function, e.g., by modifying Clq binding and/or FcyR binding and thereby changing CDC activity and/or ADCC activity. “Effector functions” are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. , B cell receptor; BCR), etc. Such effector functions can require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

For example, one can generate a variant Fc region of an antibody with improved Clq binding and improved FcyRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or ablated, a variant Fc region can be engineered with reduced CDC activity and/or reduced ADCC activity. In other embodiments, only one of these activities can be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa).

FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to the FcRn have been described (Shields et al., (2001). High resolution mapping of the binding site on human IgGl for FcyRI, FcyRII, FcyRIII, and FcRn and design of IgGl variants with improved binding to the FcyR, (I. Biol. Chem. 276:6591-6604). A number of methods are known that can result in increased halflife (Kuo and Aveson, (2011)), including amino acid modifications can be generated through techniques including alanine scanning mutagenesis, random mutagenesis and screening to assess the binding to the neonatal Fc receptor (FcRn) and/or the in vivo behavior. Computational strategies followed by mutagenesis can also be used to select one of amino acid mutations to mutate.

The present disclosure therefore provides a variant of an antigen binding protein with optimized binding to FcRn. In a particular embodiment, the variant of an antigen binding protein comprises at least one amino acid modification in the Fc region of the antigen binding protein, wherein the modification is selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317,

320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356,

359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393,

394, 395, 396, 397, 398, 399, 400, 401 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420,

421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fc region as compared to the parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat. In a further aspect of the disclosure the modifications are M252Y/S254T/T256E.

Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified, see for example Kontermann (2009) either by introducing an FcRn-binding polypeptide into the molecules or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fc receptors have been greatly reduced or fusing with FcRn binding domains of antibodies.

Derivatized antibodies can be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, particularly a human. Such alterations can result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased halflives of the antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of the antibodies or antibody fragments in the mammal, and thus reduces the frequency of the administration of the antibodies or antibody fragments and/or reduces the concentration of the antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor.

Beltramello el al. (2010) previously reported the modification of neutralizing mAbs, due to their tendency to enhance dengue virus infection, by generating in which leucine residues at positions 1.3 and 1.2 of CH2 domain (according to the IM GT unique numbering for C-domain) were substituted with alanine residues. This modification, also known as a “LALA” mutation, abolishes antibody binding to FcyRI, FcyRII and FcyRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their capacity to neutralize and enhance infection by the four dengue virus serotypes. LALA variants retained the same neutralizing activity as unmodified mAb but were completely devoid of enhancing activity. LALA mutations of this nature are therefore contemplated in the context of the presently disclosed antibodies.

Altered Glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody, or antigen binding fragment thereof, containing a substantially homogeneous glycan without sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which can be attached to heavy chain or light chain constant regions respectively. The aforementioned substantially homogeneous glycan can be covalently attached to the heavy chain constant region.

Another embodiment of the present disclosure comprises a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody, or antigen binding fragment thereof, is present in a substantially homogenous composition represented by the GNGN or G1/G2 glycoform. Fc glycosylation plays a significant role for therapeutic mAbs. The disclosure is in line with a recent study that shows increased anti-lentivirus cell-mediated viral inhibition of a fucose free anti-HIV mAb in vitro. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs).

The isolated monoclonal antibody, or antigen binding fragment thereof, comprising a substantially homogenous composition represented by the GNGN or G1/G2 glycoform exhibits increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibody without the substantially homogeneous GNGN glycoform and with GO, GIF, G2F, GNF, GNGNF or GNGNFX containing glycoforms. In one embodiment of the present disclosure, the antibody dissociates from Fc gamma RI with a Kd of 1 x 10 ^s M or less and from Fc gamma RIII with a Kd of 1 x 10‘⁷ M or less.

Glycosylation of an Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O- linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine can also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site. The glycosylation pattern can be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation site(s) that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites.

In certain embodiments, the antibody is expressed in cells that express beta (1,4)-N- acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL-23pl9 antibody. Methods for producing antibodies in such a fashion are provided in WO/9954342, WO/03011878, patent publication 20030003097 Al, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be altered to enhance or reduce or eliminate certain post-translational modifications, such as glycosylation, using genome editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CR1SPR). For example, CRISPR technology can be used to eliminate genes encoding glycosylating enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies.

Elimination of monoclonal antibody protein sequence liabilities. It is possible to engineer the antibody variable gene sequences obtained from human B cells to enhance their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs associated with sites containing: Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding recombinant antibodies.

Protein engineering efforts in the field of development of therapeutic antibodies clearly reveal that certain sequences or residues are associated with solubility differences (Fernandez- Escamilla et al., Nature Biotech., 22 (10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106 (29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21 (2), 385-392, 2010) Evidence from solubility-altering mutations in the literature indicate that some hydrophilic residues such as aspartic acid, glutamic acid, and serine contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine.

Stability. Antibodies can be engineered for enhanced biophysical properties. One can use elevated temperature to unfold antibodies to determine relative stability, using average apparent melting temperatures. Differential Scanning Calorimetry (DSC) measures the heat capacity, C_P, of a molecule (the heat required to warm it, per degree) as a function of temperature. One can use DSC to study the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAh structure, producing up to three peaks in the thermogram (from unfolding of the Fab, CH2, and CH3 domains). Typically unfolding of the Fab domain produces the strongest peak. The DSC profiles and relative stability of the Fc portion show characteristic differences for the human IgGi, IgG2, IgG?, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). One also can determine average apparent melting temperature using circular dichroism (CD), performed with a CD spectrometer. Far-UV CD spectra will be measured for antibodies in the range of 200 to 260 nm at increments of 0.5 nm. The final spectra can be determined as averages of 20 accumulations. Residue ellipticity values can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg/mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C/min. One can use dynamic light scattering (DLS) to assess the propensity for aggregation. DLS is used to characterize the size of various particles including proteins. If the system is not dispersed in size, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, and particle concentration. Since DLS essentially measures fluctuations in scattered light intensity due to particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays the particle population at different diameters. Stability studies can be done conveniently using DLS. DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pl of the protein. Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pls). This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step. The technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 pg/mL.

Solubility. One can determine the intrinsic solubility score of antibody sequences. The intrinsic solubility scores can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequences for residues 95-102 (Kabat numbering) in HCDR3 of each antibody fragment such as a scFv can be evaluated via the online program to calculate the solubility scores. One also can determine solubility using laboratory techniques. Various techniques exist, including addition of lyophilized protein to a solution until the solution becomes saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most straightforward method is induction of amorphous precipitation, which measures protein solubility using a method involving protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation gives quick and accurate information on relative solubility values. Ammonium sulfate precipitation produces precipitated solutions with well-defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using induction of amorphous precipitation by ammonium sulfate also can be done easily at different pH values. Protein solubility is highly pH dependent, and pH is considered the most important extrinsic factor that affects solubility. Autoreactivity. Generally, it is thought that autoreactive clones should be eliminated during ontogeny by negative selection, however it has become clear that many human and naturally occurring antibodies with autoreactive properties persist in adult mature repertoires, and the autoreactivity can enhance the antiviral function of many antibodies to pathogens. It has been noted that HCDR3 loops in antibodies during early B cell development are often rich in positive charge and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374- 1377, 2003). One can test a given antibody for autoreactivity by assessing the level of binding to human origin cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometric cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity also can be surveyed using assessment of binding to tissues in tissue arrays.

Preferred residues (“Human Likeness”). B cell repertoire deep sequencing of human B cells from blood donors is being performed on a wide scale in many recent studies. Sequence information about a significant portion of the human antibody repertoire facilitates statistical assessment of antibody sequence features common in healthy humans. With knowledge about the antibody sequence features in a human recombined antibody variable gene reference database, the position specific degree of “Human Likeness” (HL) of an antibody sequence can be estimated. HL has been shown to be useful for the development of antibodies in clinical use, like therapeutic antibodies or antibodies as vaccines. The goal is to increase the human likeness of antibodies to reduce potential adverse effects and anti-antibody immune responses that will lead to significantly decreased efficacy of the antibody drug or can induce serious health implications. One can assess antibody characteristics of the combined antibody repertoire of three healthy human blood donors of about 400 million sequences in total and created a novel “relative Human Likeness” (rHL) score that focuses on the hypervariable region of the antibody. The rHL score allows one to easily distinguish between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in human repertoires.

D. Single Chain Antibodies

A single chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for singlechain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 x 10⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1 ,054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

In some embodiments, the recombinant antibodies of the present disclosure further comprise sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In some embodiments, the chains are modified with agents such as biotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e. , the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a stepwise manner, heterobifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker can react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide bond-containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered can prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- l,3'-dithiopropionate. The N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non- selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers. U.S. Patent 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest can be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.

U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.

E. Multispecific Antibodies

In some embodiments, antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. In some embodiments, bispecific antibodies bind two different epitopes of a single antigen. In some embodiments, bispecific antibodies combine a first antigen binding site with a binding site for a second antigen. In some embodiments, an anti-pathogen arm is combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and Fc gamma RIII (CD 16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies can also be used to localize cytotoxic agents to infected cells. These antibodies possess a pathogen-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-in terferon- a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc gamma RIII antibody and U.S. Patent 5,837,234 discloses a bispecific anti- ErbB2/anti-Fc gamma RI antibody. A bispecific anti-ErbB2/Fc alpha antibody is shown in WO98/02463. U.S. Patent 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain- light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and Cin regions. It is preferred to have the first heavy-chain constant region (Cm) containing the site necessary for light chain bonding, present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, DNA encoding the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.

In a particular embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Patent 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. , alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Techniques exist that facilitate the direct recovery of Fab’-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol. 16, 677-681 (1998). doi:10.1038/nbl0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

In some embodiments, a bispecific or multi specific antibody is formed as a DOCK- AND-LOCK™ (DNL^TM) complex (see. e.g. , U.S. Patents 7,521 ,056; 7,527,787; 7,534,866; 7.550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie ei al., FEBS Letters. 2005; 579: 3264: Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). In some embodiments, the DDD and AD peptides are attached to any protein, peptide, or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that can be attached to DDD or AD sequences.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60, 1991 ; Xu et al. , Science, 358(6359):85-90, 2017). A multivalent antibody can be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions. In some embodiments, the polypeptide chain(s) comprise VDl-(Xl)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. In some embodiments, the polypeptide chain(s) comprise: VH-CH1 -flexible linker- VH-CHl -Fc region chain; or VH- CHl-VH-CHl-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. In some embodiments, the multivalent antibody comprises from about two to about eight light chain variable region polypeptides. In come embodiments, the light chain variable region polypeptides comprise a light chain variable region and, optionally, further comprises a CL domain.

Charge modifications are particularly useful in the context of a multi- specific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Iones-type side products), which can occur in the production of Fab-based bi-/multi- specific antigen binding molecules with a VH/VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015/150447, particularly the examples therein, incorporated herein by reference in its entirety).

Accordingly, in particular embodiments, an antibody comprised in the therapeutic agent comprises:

(a) a first Fab molecule specifically binding to a first antigen; and

(b) a second Fab molecule specifically binding to a second antigen, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, wherein the first antigen is an activating T cell antigen and the second antigen is a target cell antigen, or the first antigen is a target cell antigen and the second antigen is an activating T cell antigen; and wherein i) in the constant domain CL of the first Fab molecule the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH 1 of the first Fab molecule the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index); or ii) in the constant domain CL of the second Fab molecule the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI of the second Fab molecule the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index).

In some embodiments, the antibody does not comprise both modifications in i) and ii). In some embodiments, the constant domains CL and CHI of the second Fab molecule are not replaced by each other (i.e., remain unexchanged).

In some embodiments, in the constant domain CL of the first Fab molecule the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CHI of the first Fab molecule the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In some embodiments, in the constant domain CL of the first Fab molecule the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the first Fab molecule the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid

(D) (numbering according to Kabat EU index).

In some embodiments, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid

(E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

In an even more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

F. Chimeric Antigen Receptors

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors that graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell, with transfer of their coding sequence facilitated by retroviral vectors. In this way, many target-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach show efficacy.

The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (<?.g., neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19.

The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.

Type I proteins are in fact two protein domains linked by a transmembrane alpha helix in between. The cell membrane lipid bilayer, through which the transmembrane domain passes, acts to isolate the inside portion (endodomain) from the external portion (ectodomain). It is not so surprising that attaching an ectodomain from one protein to an endodomain of another protein results in a molecule that combines the recognition of the former to the signal of the latter.

Ectodomain. A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence usually works fine. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in a scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used

The antigen recognition domain is usually an scFv. There are, however, many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g., CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact, almost anything that binds a given target with high affinity can be used as an antigen recognition region.

A spacer region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgGl. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For most scFv based constructs, the IgGl hinge suffices. However, the best spacer often has to be determined empirically.

Transmembrane domain. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Generally, the transmembrane domain from the most membrane proximal component of the endodomain is used. Interestingly, using the CD3-zeta transmembrane domain can result in incorporation of the artificial TCR into the native TCR, a factor that is dependent on the presence of the native CD3-zeta transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly expressed, stable receptor. Endodomain. This is the "business-end" of the receptor. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is CD3-zeta which contains 3 IT AMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed.

"First-generation" CARs typically had the intracellular domain from the CD3 chain, which is the primary transmitter of signals from endogenous TCRs. "Second-generation" CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improve the activity of T cells. More recent, "third generation" CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further augment potency.

G. ADCs

Antibody Drug Conjugates or ADCs are a new class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with infectious disease. ADCs are complex molecules composed of an antibody (a whole mAb or an antibody fragment such as a single-chain variable fragment, or scFv) linked, via a stable chemical linker with labile bonds, to a biological active cytotoxic/anti-viral payload or drug. Antibody Drug Conjugates are examples of bioconjugates and immunoconjugates.

By combining the unique targeting capabilities of monoclonal antibodies with drugs, antibody-drug conjugates allow sensitive discrimination between healthy and diseased tissue. This means that, in contrast to traditional systemic approaches, antibody-drug conjugates target and attack the infected cell so that healthy cells are less severely affected.

In the development ADC-based therapies, a drug is coupled to an antibody that specifically targets a certain cell marker (e.g., a protein that, ideally, is only to be found in or on certain cells). Antibodies track these proteins down in the body and attach themselves to the surface of cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the cell, which then absorbs or internalizes the antibody together with the drug. After the ADC is internalized, the drug is released. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other agents.

A stable link between the antibody and agents is a crucial aspect of an ADC. Linkers are based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (noncleavable) and control the distribution and delivery of the cytotoxic agent to the target cell. Cleavable and noncleavable types of linkers have been proven to be safe in preclinical and clinical trials. Brentuximab vedotin includes an enzyme-sensitive cleavable linker that delivers the potent and highly toxic antimicrotubule agent Monomethyl auristatin E or MMAE, a synthetic antineoplastic agent, to human specific CD30-positive malignant cells. Because of its high toxicity MMAE, which inhibits cell division by blocking the polymerization of tubulin, cannot be used as a single-agent chemotherapeutic drug. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAClO, a cell membrane protein of the tumor necrosis factor or TNF receptor) proved to be stable in extracellular fluid, cleavable by cathepsin and safe for therapy. Trastuzumab emtansine, the other approved ADC, is a combination of the microtubule- formation inhibitor mertansine (DM- 1), a derivative of the May tansine, and antibody trastuzumab (Herceptin®/Genentech/Roche) attached by a stable, non-cleavable linker.

The availability of better and more stable linkers has changed the function of the chemical bond. The type of linker, cleavable or non-cleavable, lends specific properties to the drug. For example, a non-cleavable linker keeps the drug within the cell. As a result, the entire antibody, linker, and agent enter the targeted cell where the antibody is degraded to the level of an amino acid. The resulting complex - amino acid, linker and agent - now becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in the host cell where it releases the agent.

Another type of cleavable linker, currently in development, adds an extra molecule between the cytotoxic/anti-viral drug and the cleavage site. This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics. Researchers are also developing a new method of peptide cleavage based on Edman degradation, a method of sequencing amino acids in a peptide. Future direction in the development of ADCs also includes the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and a emitting immunoconjugates and antibody-conjugated nanoparticles.

H. BiTES

Bi-specific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies that are investigated for the use as drugs. They direct a host's immune system, more specifically the T cells’ cytotoxic activity, against infected cells. BiTE is a registered trademark of Micromet AG.

BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other to an infected cell via a specific molecule.

Eike other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTEs form a link between T cells and target cells. This causes T cells to exert cytotoxic/anti-viral activity on infected cells by producing proteins like perforin and granzymes, independently of the presence of MHC I or co-stimulatory molecules. These proteins enter infected cells and initiate the cell’s apoptosis. This action mimics physiological processes observed during T cell attacks against infected cells.

I. Intrabodies

In a particular embodiment, the antibody is a recombinant antibody that is suitable for action inside of a cell - such antibodies are known as “intrabodies.” These antibodies can interfere with target function by a variety of mechanisms, such as by altering intracellular protein trafficking, interfering with enzymatic function, and blocking protein-protein or protein-DNA interactions. In many ways, their structures mimic or parallel those of single chain and single domain antibodies, discussed above. Indeed, single-transcript/single-chain is an important feature that permits intracellular expression in a target cell, and also makes protein transit across cell membranes more feasible. However, additional features are required.

The two major issues impacting the implementation of intrabody therapeutic are delivery, including cell/tissue targeting, and stability. With respect to delivery, a variety of approaches have been employed, such as tissue-directed delivery, use of cell-type specific promoters, viral-based delivery and use of cell-permeability/membrane translocating peptides. With respect to the stability, the approach is generally to either screen by brute force, including methods that involve phage display and can include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement/modification.

An additional feature that intrabodies can require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to subcellular regions such as the cytoplasm, nucleus, mitochondria and ER have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997).

By virtue of their ability to enter cells, intrabodies have additional uses that other types of antibodies can not achieve. In the case of the present antibodies, the ability to interact with the MUC1 cytoplasmic domain in a living cell can interfere with functions associated with the MUC1 CD, such as signaling functions (binding to other molecules) or oligomer formation. In particular, it is contemplated that such antibodies can be used to inhibit MUC 1 dimer formation.

J. Purification

In some embodiments, the antibodies of the present disclosure are purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it naturally occurs. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

In purifying an antibody of the present disclosure, it can be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide can be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps can be changed, or that certain steps can be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (/'.<?. , protein A) that bind the Fc portion of the antibody. Alternatively, antigens can be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter, or bead. The antibodies are bound to a support, contaminants removed (e.g. , washed away), and the antibodies released by applying conditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products can vary.

III. Passive Immunization and Treatment/Prevention

In one aspect, the inventors propose the treating and prevention of diabetes, metabolic disorder, insulin resistance, leptin resistance, obesity, weight gain, and liver fibrosis with the antibodies and antibody fragments of the present disclosure. In some cases, the antibodies or antibody fragments can be used in combination with drugs and therapies designed to treat or prevent of diabetes, metabolic disorder, insulin resistance, leptin resistance, obesity, weight gain, and liver fibrosis. Such agents include GLRP1 agonists, GLP-1, insulin, biguanides, thiazolidinediones, LYN Kinase activators, sulfonylureas, meglitinides, alpha-glucosidase inhibitors, incretins, gastric inhibitory analogs, dipeptidyl peptidase-4 inhibitors, amylin analogs, and SGLR2 inhibitors. Treatment will generally involve passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e. , sterile and syringeable. Alternatively, genetic therapy with expression vectors that encode and express anti-leptin antibodies can be employed.

As noted above, combination therapies can be employed to increase the effectiveness of a given agent including the antibodies of the present disclosure. As such, it can be desirable to combine these compositions with other agents effective in the treatment of the disease of interest, such as diabetes. A diabetes therapeutic is capable of improving one of more symptoms of diabetes (or other disease state described herein) in a subject. More specifically, these other compositions would be provided with the described antibodies in a combined amount to benefit the patient. This process can involve administering to the patient an antibody and the other agent(s) or factor(s) at the same time. This can be achieved by administering a single composition or pharmacological formulation that includes both agents, or by providing two distinct compositions or formulations, at the same time, wherein one composition includes the antibody the other includes the second agent(s).

The antibody treatment can precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and the antibody are applied separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the antibody would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one can provide both modalities within about 12-24 hours of each other and, more specifically, within about 6-12 hours of each other. In some situations, it can be desirable to extend the time period for treatment significantly where several days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations. A. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprising anti-leptin antibodies. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington’s Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical, or delivered by mechanical ventilation.

Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

2. ADCC

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or fragments thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. By “antibody having increased/reduced antibody dependent cell-mediated cytotoxicity (ADCC)”, it is meant an antibody having increased/reduced ADCC as determined by any suitable method known to those of ordinary skill in the art.

As used herein, the term “increased/reduced ADCC” is defined as either an increase/reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or a reduction/increase in the concentration of antibody, in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The increase/reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation, and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the increase in ADCC mediated by an antibody produced by host cells engineered to have an altered pattern of glycosylation (e.g., to express the glycosyltransferase, GnTIII, or other glycosyltransferases) by the methods described herein, is relative to the ADCC mediated by the same antibody produced by the same type of non -engineered host cells.

3. CDC

Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is the processes in the immune system that kill pathogens by damaging their membranes without the involvement of antibodies or cells of the immune system. There are three main processes. All three insert one or more membrane attack complexes (MACs) into the pathogen which cause lethal colloid-osmotic swelling, i.e. , CDC. It is one of the mechanisms by which antibodies or antibody fragments have an anti-viral effect.

IV. Antibody Conjugates

In some embodiments, antibodies of the present disclosure are linked to at least one agent to form an antibody conjugate. To increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety can be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g. , cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which can be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹ chromium, ³⁶chlorine, ⁵⁷ cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵ sulphur, technicium^99m and/or yttrium⁹⁰. ¹²⁵I is often preferred for use in certain embodiments, and technicium"¹⁷¹ and/or indium¹¹¹ are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure can be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure can be labeled with technetium⁹⁹¹¹¹ by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques can be used, e.g., by incubating pertechnate, a reducing agent such as SNCF, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTP A) or ethylene diaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BOD1PY-TMR, BOD1PY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

Additional types of antibodies contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, haptenbased affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

Molecules containing azido groups can also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binding agents.

Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4- hydroxyphenyljpropionate are disclosed.

In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O’Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.

V. Immunodetection Methods

In still further embodiments, the present disclosure provides immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting leptin. The methods can also be used to screen various antibodies for appropriate/desired reactivity profiles.

Other immunodetection methods include specific assays for determining the presence of leptin in a subject. A wide variety of assay formats are contemplated, but specifically those that would be used to detect leptin in a fluid obtained from a subject, such as saliva, blood, plasma, sputum, semen, or urine. The assays can be advantageously formatted for nonhealthcare (home) use, including lateral flow assays (see below) analogous to home pregnancy tests. In some embodiments, the assays are packaged in the form of a kit with appropriate reagents and instructions to permit use by the subject of a family member.

Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In general, the immunobinding methods include obtaining a sample suspected of containing leptin and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying leptin or related antigens from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the leptin or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving leptin immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.

The immunobinding methods also include methods for detecting and quantifying the amount of leptin or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing leptin and contact the sample with an antibody that binds leptin or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed can be any sample that is suspected of containing leptin, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.

Contacting the chosen biological sample with the antibody under effective conditions and for a period sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e.. to bind to leptin. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages using a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection can itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand can be linked to a detectable label. The second binding ligand is itself often an antibody, which can thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system can provide signal amplification if this is desired.

One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is like the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

A. ELISAs

Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like can also be used.

In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the leptin is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by the addition of another anti-leptin antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection can also be achieved by the addition of a second anti-leptin antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing leptin are immobilized onto the well surface and then contacted with the anti-leptin antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-leptin antibodies are detected. Where the initial anti-leptin antibodies are linked to a detectable label, the immune complexes can be detected directly. Again, the immune complexes can be detected using a second antibody that has binding affinity for the first anti-leptin antibody, with the second antibody being linked to a detectable label.

Irrespective of the format employed, ELIS As have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

In ELIS As, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25 °C to 27 °C or can be overnight at about 4 °C or so.

Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.

To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of leptin antibodies in a sample. In competition-based assays, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample.

Here, the inventor proposes the use of labeled leptin monoclonal antibodies to determine the amount of leptin antibodies in a sample. The basic format would include contacting a known amount of leptin monoclonal antibody (linked to a detectable label) with leptin. The leptin is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample.

B. Western Blot

The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in each sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Samples can be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells can also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus, or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers can be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

The proteins of the sample are separated using gel electrophoresis. Separation of proteins can be by isoelectric point (pl), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their nonspecific protein binding properties (z.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.

C. Lateral Flow Assays

Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized and costly equipment, though many laboratory-based applications exist that are supported by reading equipment. Typically, these tests are used as low resources medical diagnostics, either for home testing, point of care testing, or laboratory use. A widely spread and well-known application is the home pregnancy test.

The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt- sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically, there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the final porous material - the wick - that simply acts as a waste container. Lateral Flow Tests can operate as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Patent 6,485,982. D. Immunohistochemistry

In some embodiments, the present disclosure provides immunodetection kits for use with the immunodetection methods described above. As the antibodies can be used to detect leptin, the antibodies can be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to leptin, and optionally an immunodetection reagent.

In some embodiments, the leptin antibody is pre-bound to a solid support, such as a column matrix and/or well of a microtiter plate. In some embodiments, the immunodetection reagents of the kit take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, several exemplary labels are known in the art and all such labels can be employed in connection with the present disclosure.

In some embodiments, the kit further comprises a suitably aliquoted composition of leptin, whether labeled or unlabeled, to prepare a standard curve for a detection assay. In some embodiments, the kit comprises antibody -label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. In some embodiments, the components of the kit are packaged either in aqueous media or in lyophilized form.

The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody is placed, or preferably, suitably aliquoted. The kit of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers can include injection or blow-molded plastic containers into which the desired vials are retained.

E. Immunodetection Kits

In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect leptin, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to leptin, and optionally an immunodetection reagent.

In certain embodiments, the leptin antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtiter plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, several exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

The kits may further comprise a suitably aliquoted composition of leptin, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

VI. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 - 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 Biologies) was used for panning the phage display scFv library (diversity of 1 X 101 1). 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). Antibody variable DNA sequences (scFv) contained in phage-mid vector are isolated using a plasmid preparation kit (Qiagen) and sequenced. CDR sequences are analyzed using the online software and are shown in Tables 3 and 4 (amino acids) and Tables 5 and 6 (DNA) for heavy chain and light chain of each antibody, respectively. Complete heavy chain variable region and light chain variable sequences (Tables 1 and 2) were amplified and expressed into full IgG using an expression vector system in human embryonic kidney (HEK293) cells.

Selected Leptin binding antibodies were expressed as human IgGl using a mammalian expression vector system in HEK293 cells using a shaker flask CO2 incubator. Antibodies were purified using a column with protein A resin (GenScript) using a fast protein liquid chromatography (FPLC) separation unit. Purified Leptin binding antibodies were characterized for their biological properties.

Example 2 - Binding affinity of anti-leptin monoclonal antibodies

ELISA titration was used to determine the binding affinity of a panel of monoclonal antibodies to LEPTIN antigen (FIG. 1). Binding affinities (EC50, shown in the Figure 1C) of the leptin monoclonal antibodies were determined using 4-parameter curve fitting with GraphPad Prism program.

For kinetic binding affinity measurements, antibody (30 pg/mL) was loaded onto the protein A biosensors for 4 min. Following incubation in kinetics buffer to establish baseline. Antibody loaded biosensors were exposed to a series of concentrations (0.1-100 nM) of leptin protein (HIS tag at C terminus). Background subtraction was used to correct sensor drifting. All experiments were performed with shaking at 1,000 rpm accordingly to the manufacturer’s suggestion. Kinetic sensorgrams for antibodies are shown in FIG. 2. ForteBio’s data analysis software was used to determine association rate (kon) and dissociation rate (koff) and KD was calculated using the ratio of koff/kon. The binding affinities of anti-leptin mAbs are shown in Table 7. Example 3 - Neutralization of Leptin Alleviates Organ Fibrosis

Identification and characterization of a leptin-neutralizing antibody. The inventors previously described the identification of a human monoclonal IgG antibody (hLep3; in the following simply referred to as ‘LepAb’) capable of neutralizing human as well as mouse leptin (Zhao et al., 2019). However, the structural basis of how LepAb neutralizes leptin is unknown, which hampers its application in clinic. The inventors therefore determined the structure of LepAb-Leptin complex.

Utilizing papain digestion, the inventors prepared a Fab from the LepAb, which they subjected to crystallization either in complex with human leptin or alone. Reducing SDS-PAGE analysis confirmed the successful purification of the Fab and the Fab: leptin complex with distinct bands corresponding to each component (FIG. 12A). Using X-ray crystallography, the inventors determined the structure of the Fabdeptin complex as well as the isolated unbound Fab.

While the Fab: leptin interaction (FIG. 12B and FIG. 17A) overall mimics that of the LEPR CRH2 domain with leptin (FIG. 12C and FIG. 17B), the inventors observed several noteworthy differences. The leptin receptor CRH2 domain forms more hydrogen bonds with leptin than the Fab does (5 versus 3), yet the Fab epitope involves a broader region, including leptin’s helix C and residues from the C-D loop. This interaction hears greater similarity to the binding geometry of interleukin-4 to its receptor (Stroud & Wells, 2004; Hage et al., 1999), with comparable buried surface areas (Fabdeptin, 870 A² ; IL4R:IL4, 810 A²) (FIG. 17C). Of note, the inventors observe only minor conformational changes in the Fab upon complex formation (FIG. 12D). In the Fabdeptin complex, residues of leptin helix A interact with residues from both the Fab light chain (Asn-26, Ala-31, Gly-32, Tyr-33, His-34, Tyr-51, Arg- 95) and the Fab heavy chain (Gln-100, Val-101, Tyr-106, Tyr-107) (FIG. 12E). Key interactions include a hydrogen bond between the Fab heavy chain Tyr-107 and leptin Asp-9 and a salt bridge between the Fab light chain His-34 and leptin Asp-9. Similarly, leptin helix C engages with residues from the Fab heavy chain (Ser-31, Ile-52, Phe-55, Val-101, Pro-102, Ser-103, Ser-104, Tyr-106, Tyr-107) and Fab light chain (Tyr-33, Arg-95, Gly-98, Glu-100) (FIGS. 12F-G). Crucial interactions include hydrogen bonds between Fab light chain Tyr-33 and leptin Asp-79 and between Fab light chain Arg-95 and leptin Asn-78. Two residues from leptin’s C-D loop (Pro-99, Glu-100) further stabilize the Fab heavy chain interaction. The observed epitope overlaps between the Fab: leptin and LEPR CRH2: leptin complexes offer valuable insights into the antibody’s mechanism of action (FIG. 12D and FIG. 12G). Importantly, several conserved residues are critical for binding specificity, underscoring their significance for leptin’s interactions with the LEPR as well as LepAb (FIGS. 12A-G, Table SI).

To evaluate the functional consequences LepAb binding to leptin, the inventors cotransfected HEK293 cells with LEPRb and a pSTAT3: luciferase reporter plasmids and treated them with leptin in presence of either LepAb or control IgG antibody. This assay revealed that LepAb effectively inhibited leptin-induced STAT3 activation compared to the IgG control (FIG. 17D). Taken together, these structural and functional analyses demonstrate that LepAb neutralizes leptin by preventing it from binding to and thus also activating its receptor.

Assessment of local leptin signaling in mouse models of liver and kidney fibrosis. The majority of leptin’s effects are mediated through LEPRb expressed in both the brain and peripheral tissues (Gorska et al., 2010; Zhao et al., 2021). To investigate leptin’s role in fibrogenesis, the inventors first assessed total and long isoform leptin receptor mRNA expression in two distinct fibrosis settings, namely a model of liver fibrosis (Mup-uPA transgenic mice fed a high-fat diet) and a model of kidney fibrosis (mice treated with a bolus of folic acid). Intriguingly, following fibrosis induction, total as well as long isoform leptin receptor mRNA expression was significantly upregulated in the liver and kidney of these mice (FIGS. 13A-B). Next, the inventors assessed active leptin signaling by analyzing the protein levels of phosphorylated STAT3 (p-STAT3), total STAT3, and SOCS3 in the fibrotic tissues. Immunoblotting revealed markedly elevated p-STAT3 and SOCS3 protein levels in the liver of high-fat diet (HFD)-fed Mup-uPA mice (FIG. 13C) and elevated p-STAT3 and SOCS3 protein levels the kidneys of folic acid- treated mice (FIG. 13D). These findings indicate that fibrogenesis is accompanied by elevated local leptin signaling, emphasizing leptin’ s potential as a therapeutic target for fibrosis treatment.

Leptin neutralization diminishes kidney fibrosis in folic acid-treated mice. Given that leptin signaling is elevated following folic acid-induced acute kidney injury, the inventors evaluated the therapeutic effects of increasing doses of LepAb (1-20 mg/kg) compared to IgG control antibody in this model (FIG. 14A). Body weight remained stable across all groups following folic acid application (FIG. 18 A). LepAb administration resulted in a dose-dependent reduction in blood urea nitrogen (BUN) levels, indicating improved renal function (FIG. 14B). Notably, LepAb treatment also resulted in a dose dependent decrease in the mRNA expression of several fibrotic markers (FIGS. 14B-C). Of note, no significant differences in BUN levels or fibrosis marker expression were observed between the highest and second highest antibody dose (20 mg/kg and 10 mg/kg). LepAb treatment also mitigated the folic acid-induced reduction in kidney size (FIG. 18B). Based on these findings, the 10 mg/kg dose was selected for subsequent experiments. To further assess the effects of leptin neutralization on kidney fibrosis, the inventors performed histological analysis of the kidneys from mice treated with the 10 mg/kg dose. These analyses revealed that LepAb treatment reduced ECM deposition and improved kidney architecture compared to IgG control treatment (FIG. 14D). RT-qPCR analysis demonstrated that LepAb treatment significantly decreased the mRNA expression of key fibrogenic genes such as Tgfbl, Collal, Colla2, and Col3al (FIG. I4E) as well as pro- inflammatory genes such as Ccl2 (Mcpl\ Illb, 116, and Ifng in the kidney (FIG. 14F). Immunofluorescence staining corroborated these findings, showing markedly reduced fibronectin and collagen I accumulation in LepAb-treated mice compared to IgG control- treated mice (FIG. 14G). These results demonstrate that leptin blockade effectively diminishes fibrosis development following acute kidney injury.

Leptin neutralization attenuates liver fibrosis in Mup-uPA mice fed a high-fat diet. Nonalcoholic steatohepatitis (NASH) significantly increases the risk of hepatocellular carcinoma (HCC), with up to 50% of new HCC cases arising independently of viral infections (Dhamjia et al., 2019). Obesity-related NASH can progress to liver fibrosis, cirrhosis, and eventually HCC (Nakagawa iet al., 2014; Febbraio et al., 2019; Basha et al., 2023). The inventors’ previous findings indicated that leptin signaling is increased under conditions of HFD-induced liver fibrosis in Mup-uPA transgenic mice(30). To evaluate the effects of leptin neutralization on NASH progression, Mup-uPA mice were fed HFD for 10 weeks and then treated with LepAb or IgG control for an additional 2 weeks (FIG. 15 A). Histological analysis showed that LepAb treatment effectively reduced hepatocyte ballooning and ECM deposition in the liver (FIG. 15B). Consistently, RT-qPCR analysis revealed significant reductions in the mRNA expression of key fibrotic and inflammatory markers (FIGS. 15C-D). Moreover, LepAb treatment improved liver function, as demonstrated by lower serum levels of aspartate aminotransferase (AST), although alanine aminotransferase (ALT) levels were unaffected (FIG. 15E). Immunofluorescence staining corroborated these results, showing decreased fibronectin and collagen I deposition in the liver of LepAb-treated mice (FIG. 15F). Notably, body weight was not affected by either LepAb or IgG treatment, indicating that the observed therapeutic effects were independent of body weight changes (FIG. 19). These findings underscore the importance of leptin signaling for NASH pathogenesis and suggest that neutralizing leptin effectively attenuates obesity-associated liver fibrosis.

Leptin neutralization alleviates lung fibrosis in bleomycin-treated mice. Pulmonary fibrosis is a serious and debilitating condition, and early intervention is key to managing its progression (Martinez et al., 2017). To evaluate the therapeutic potential of leptin neutralization in this setting, the inventors treated mice with bleomycin to induce lung fibrosis and administered LepAb or control IgG to them (FIG. 16A). Histological analyses revealed that LepAb treatment prevented the deterioration of alveolar architecture and reduced the deposition of ECM in the lungs (FIG. 16B). RT-qPCR analyses showed that LepAb administration markedly decreased the mRNA expression of key fibrogenic factors such as Tgfbl, Ccn2 (Ctgf), Collal, Colla2, and ColSal as well as inflammatory factors such as Ccl2 (Mcpl), lllb, and 116 (FIGS. 16C-D). These findings were further supported by immunofluorescence staining, which showed reduced fibronectin deposition and collagen hybridizing peptide (CHP) in the lungs of LepAb-treated mice compared to IgG-treated control mice (FIG. 16E). Collectively, these data indicate that leptin neutralization effectively alleviates fibrosis development following lung injury.

Leptin neutralization mitigates aorta and kidney fibrosis, but not heart fibrosis in angiotensin Il-treated mice. Leptin has been reported to contribute to the elevation of blood pressure in obesity, but its effects in non-obese populations, are less clear (Barash et al., 1996; Simonds et al., 2014; Kusminski & Scherer, 2015; Brown et al., 2015; von Schnurbein et al., 2019). To assess the impact of leptin neutralization on hypertension-driven organ fibrosis, the inventors infused lean mice with angiotensin II (Ang II) and treated them with either LepAb or control IgG for 14 days (FIG. 20 A). Tail-cuff blood pressure measurements showed no significant differences in systolic or diastolic blood pressure between LepAb- and IgG-treated mice, independent of whether they were infused with Ang II to induce hypertension or saline as a control (FIGS. 20B-C). Similarly, there were no significant differences in the ventricular weight to body weight ratio between the treatment groups following Ang II infusion (FIG. 20D). Histological analysis of left ventricular sections revealed no changes in cardiac morphology or fibrosis between groups (FIG. 20E). In line with these findings, the mRNA expression of fibrosis-related genes including Collal, Colla2, Col3al, and Ccn2 (Ctgf) in the heart was comparable between LepAb- and IgG-treated groups following Ang II infusion (FIG. 20F). In contrast, LepAb treatment effectively reduced kidney fibrosis and aortic media thickness in Ang Il-infused mice (FIGS. 20G-K). These findings suggest that while leptin neutralization does not impact hypertension-induced cardiac fibrosis, it significantly mitigates hypertension-induced kidney fibrosis, indicating that leptin signaling plays a central role in the fibrotic process in the kidney under these conditions.

Discussion. Leptin has wide-ranging effects on peripheral tissues, primarily through its impact on energy balance, metabolism, and tissue homeostasis (Muoio & Lynis Dhom, 2002). While leptin promotes glucose and lipid metabolism in adipose tissue (Pico el al., 2022), muscle (Ceddia, 2005), and liver (Polyzos el al., 20i5), it also contributes to tissue inflammation, remodeling, and fibrosis in organs such as the heart, kidney, and liver (Liu el al., 2022). Understanding leptin's peripheral effects is essential for developing therapeutic strategies to manage not only obesity-related complications but also other chronic diseases in which leptin signaling is impacted. Antibody-based therapies targeting signaling through the leptimLEPRb complex hold significant promise in this context.

Here, the inventors used X-ray crystallography to determine the structure of a potent leptin-neutralizing antibody, as an unbound Fab as well as in complex with leptin. The inventors’ structural analyses provide critical insights into the mechanistic basis of this antibody’s actions and further deepens the inventors’ understanding about how leptin engages its receptor. The resolution of the Fabdeptin complex revealed that electrostatic interactions, shape complementarity, and minor conformational changes contribute to antigen binding. Consistent with other antibody-antigen complexes, the Fab antibody undergoes limited induced fit upon leptin binding, enhancing shape complementarity at the interface (Davies & Cohen, 1996).

Using the neutralizing antibody, the inventors establish leptin as a significant contributor to peripheral tissue fibrosis, demonstrated by the marked reductions in fibrotic processes observed in the kidney, liver, and lung following antibody application. These results indicate that leptin suppression could serve as a novel therapeutic strategy for the treatment of fibrosis. These findings demonstrate that leptin signaling, mediated by JAK-STAT and PI3K- AKT pathways, is activated during fibrotic processes in peripheral tissues, promoting inflammation and ECM deposition. Neutralizing leptin effectively disrupts these pathways, reducing fibrotic and inflammatory responses across various tissues. These findings suggest that leptin acts as a pro-fibrotic hormone whose controlled neutralization can yield health benefits.

In this study, the inventors utilized a set of well-established mouse models of kidney, liver, and lung fibrosis, specifically folic acid-treated wild-type mice, HFD-fed Mup-uPA transgenic mice, and bleomycin-treated wild-type mice. In all these models, they demonstrate that antibody-mediated leptin neutralization effectively mitigates fibrotic processes without apparent adverse effects on body weight. While the results strongly support the therapeutic potential of leptin neutralization, certain limitations must be acknowledged. Optimal LepAb dosage can vary across fibrotic conditions, and the long-term safety of leptin-targeted therapies requires further exploration, though the inventors currently anticipate no negative effects. Additionally, the molecular mechanisms underlying leptin’s effects in different organs remain to be elucidated. Furthermore, expanding this research to include female mice and obese models will enhance the translational relevance of these findings.

Leptin has been suggested to contribute to obesity-associated cardiac fibrosis (Xue et al. , 2016). Here, the inventors evaluated its impact on Ang II- induced cardiac and renal fibrosis in lean mice, providing complementary insights to diet-driven models. Notably, LepAb treatment had no effect on hypertension-induced cardiac fibrosis following Ang II infusion, potentially due to low baseline leptin levels in lean mice. However, LepAb substantially reduced hypertension-induced renal fibrosis, suggesting a tissue- and/or insult-dependent role for leptin signaling in fibrotic processes. Future studies should explore leptin’s contribution to cardiac fibrosis in obese models to better understand its broader therapeutic implications.

In conclusion, this study demonstrates that anti-leptin therapy is a promising strategy for treating fibrosis in multiple organs. These findings also highlight the intersection of metabolic, inflammatory, and fibrotic processes, emphasizing the importance of developing comprehensive treatment strategies for chronic diseases. Overall, this study lays the groundwork for future research into leptin-targeted therapies and their clinical applications.

Example 4 - Materials & Methods for Example 3

Animal models. The Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center at Dallas approved all animal studies. All mice were housed under standard laboratory conditions (12 h light/ 12 h dark cycle) and provided with ad libitum access to food and water.

Transgene and high-fat diet-induced liver fibrosis model. 12- week old male Mup- uPA transgenic mice (Nakagawa et al. , 2014) and their transgene-negative littermates were fed a lard-based high-fat diet (60% calories from fat; Bio-Serv #S 1850) for 10 weeks to promote obesity and then grouped to receive either LepAb or control IgG injections for another 2 weeks. These injections were performed intraperitoneally, twice per week at a dose of 10 mg/kg. The mice were sacrificed after 2 weeks of antibody injection and tissues were collected for analysis.

Folic acid-induced kidney fibrosis model. 10- 12- week-old male C57B1/6 mice were injected intraperitoneally with a single dose of folic acid (200 mg/kg in 0.3 M NaHCO₃, pH 7.4; Sigma Aldrich F7876) or vehicle (0.3 M NaHCO₃, pH 7.4) to induce acute kidney injury and subsequent fibrosis and then grouped to receive either LepAb or control IgG injections for 21 days, starting on day 1. These injections performed intraperitoneally, every other day at a dose of 1-20 mg/kg (as indicated). The mice were sacrificed at D28, and tissues were collected for analysis.

Bleomycin -induced lung fibrosis model. 12-week-old male C57B1/6 mice were administered intratracheally with a single dose of bleomycin (1.5 units/kg; Sigma- Aldrich #B2434) or vehicle (Saline) to induce acute lung injury and subsequent fibrosis and then grouped to receive either LepAb or control IgG injections for 14 days, starting on day 1. These injections performed intraperitoneally, every other day at a dose of 10 mg/kg. The mice were sacrificed at DI 4, and tissues were collected for analysis. Another group of mice was not treated with any antibody and sacrificed at DI 4.

BP measurement. Minipumps (1002D; Alzet, Cupertino, CA) were implanted subcutaneously in 11- to 12-week-old mice to deliver Angll (1000 ng/kg per minute) or vehicle (saline) (Zhang et al., 2012). For Tail-cuff BP measurements, mice were infused with Angll or vehicle for 2 weeks and BP was measured using CODA-HT8 Blood Pressure Analysis System (CODA® High Throughput system, Kent Scientific, CT) (Schiattarella et al., 2019).

Serum BUN measurement. The levels of serum BUN were measured using an Invitrogen Urea Nitrogen Colorimetric Detection kit (Invitrogen™, E1ABUN).

Serum AST and ALT measurement. The measurement of serum AST and ALT was performed by the Metabolic Phenotyping Core of UTSW Medical Center.

HEK293 cell culture, transfection and luciferase assay. HEK293 cells were cultured in DMEM (Gibco, 11965092) supplemented with 10% FBS, and penicillin-streptomycin in a humid incubator with 5% CO2 at 37°C. This cell line was seeded into a 96-well plate and reached 80% confluence. The cells were then co-transfected with LEPRb and a pSTAT3: luciferase reporter plasmids and treated them with leptin (40ng/ml) in presence of either LepAb or control IgG antibody. Before adding to the wells, leptin and LepAbs were mixed in a 1.5- ml Eppendorf tubes with gentle shaking for 1 hour. Then, the mixture was added into the well for 24 hours. After that, the wells were washed with cold PBS twice and then performed for luciferase analysis using ONE-Glo™ Luciferase Assay System (Sanchez et al., 2020).

Leptin-neutralizing antibody discovery and production. The generation and purification of the human leptin-neutralizing antibody (hLep3; herein ‘LepAb’) is described in detail elsewhere Zhao et al., 2019).

Human leptin production. A pSUMO-TCS(A)-LEP W100E plasmid for human leptin (W100E variant (Zhang et al., 1997)) expression in bacterial cells was cloned by Gibson assembly. This plasmid encodes a fusion protein featuring an N-terminal hexahistidine- affinity tag, SUMO, a TEV protease-cleavable linker, and mature human leptin (amino acids 22-167; with W121E substitution). The pSUMO-TCS(A)-LEP W100E plasmid was transformed into the expression host E. coli BL21 (DE3) and overexpression was induced overnight at 30°C. The cells were lysed and Ni-NTA purification was performed. This yielded a recombinant protein of -95% purity by SDS-PAGE. The hexahistidine-affinity-tag and SUMO were subsequently removed by TEV protease digestion. Ni-NTA extraction of the removed portions and utilized TEV protease resulted in a recombinant protein of -98% purity by SDS page (see Fig. 12A). The pSUMO-TCS(A)-LEP W100E plasmid sequence is available upon request.

Fab generation and Fab:leptin complex formation. LepAb (25 mg/ml) was digested with agarose-immobilized papain (Thermo Scientific #20341) to produce Fab and Fc antibody fragments. For complex formation, the purified Fab (12 mg/ml) was incubated with recombinant human leptin (15 mg/ml) overnight at 4°C. An HL Sephacryl S-200 HR column (Cytiva #171 19501) was utilized for size exclusion chromatography employing a 20 mM Tris- HC1 pH 7.5, 150 mM NaCl buffer. Peak fractions containing the desired protein or protein complexes were pooled and concentrated to 20 mg/ml using a Pierce 10K MWCO Protein Concentrator (Thermo Scientific #88528).

Crystallization, data collection, and structure determination. Fab: leptin complex and unbound Fab crystals were grown by the hanging drop vapor diffusion method at 20°C in 24-well VDX trays using a 1:1 ratio of protein/reservoir solution. The Fab:leptin complex was applied at 25 mg/ml in a buffer containing 20 mM HEPES, pH 7.4 and 75 mM NaCl against a reservoir solution containing 18% PEG-8000 and 20% glycerol. The unbound Fab was applied to VDX trays at 25 mg/ml in a buffer containing 20 mM HEPES, pH 7.4 and 75 mM NaCl against a reservoir solution containing IM LiCl, 0.1 M citrate, pH 4.0 and 20% (w/v) PEG-8000. The obtained crystals were cryo-protected with 18% (w/v) PEG 8,000 and 20% (v/v) glycerol, diffracted to a minimum Bragg spacing (dmin) of 3.10 A and exhibited the symmetry of space group Pl with cell dimensions of a = 51.9 A, b = 48.5 A, c = 125.0 A, alpha = 90.3°, beta = 85.2° and gamma = 70.4° and contained two complexes per asymmetric unit. Crystals of the unbound Fab diffracted to a minimum Bragg spacing (dmin) of 3.25 A and exhibited the symmetry of space group P2i2i2 with cell dimensions of a = 70.2 A, b = 207.9 A, c = 72.8 A and contained two molecules of Fab per asymmetric unit. All diffraction data were collected at beamline 19-ID (SBC-CAT) at the Advanced Photon Source (Argonne National Laboratory, Argonne, Illinois, USA) and processed in the program HKL-3000 (Minor et al. , 2006) with applied corrections for effects resulting from absorption in a crystal and for radiation damage (Borek et al., 2003; Otwinowski et al., 2003), the calculation of an optimal error model, and corrections to compensate the phasing signal for a radiation-induced increase of non-isomorphism within the crystal (Borek et al., 2013; 2010). The data for both crystals displayed significant levels of anisotropy.

Phases were obtained via a molecular replacement (MR) experiment in the program Phaser (McCoy et al. , 2007) using modified search models from the previously determined human leptin structure (PDB: 1AX8) and a model for the heavy and light chains of the Fab obtained from the AlphaFold2 CoLab server (Mirdita et al., 2022; Jumper et al., 2021). Completion of this model was performed by multiple cycles of manual rebuilding in the program Coot (Emsley et al., 2010) alternated with positional and isotropic atomic displacement parameter (ADP) refinement to a resolution of 3.10 A using the program Phenix (Adams et al., 2010) with a random 10% of all data set aside for an Rfree calculation. Due to the low resolution of the diffraction data for both crystals, torsion-angle non-crystallographic symmetry restraints and secondary structure restraints were used during model refinement. The model and electron density for chains A (Fab heavy chain), B (Fab light chain) and C (leptin) of the LeptimFab complex are the most complete. The more complete model of leptin in this complex includes an ordered helix E and the linker between helices D and E, primarily due to a close crystallographic lattice contact to this ordered region. Data collection and structure refinement statistics are summarized in Table S2.

RNA isolation and RT-qPCR. Tissue samples were lysed at 4 °C in TRIzol (Thermo Fisher Scientific #15596018) and RNA was isolated using the RNeasy Mini Kit (Qiagen #74106). RNA concentrations were determined on a NanoPhotometer (Implen) and cDNA synthesis was carried out using the PrimeScript 1st strand cDNA Synthesis Kit (Takara #6110A). RT-qPCR was using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific #A25778) on QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific). Gene expression was normalized to the housekeeping gene Rpll9 using the AACT method. PCR specificity was confirmed by melting curve analysis. Primer sequences listed in Table S3.

Protein extraction and immunoblot. Tissues were lysed at 4 °C in radioimmunoprecipitation assay (RIPA; Sigma- Aldrich R0278) buffer supplied with protease and phosphatase inhibitors (Roche #04906837001) and subsequently cleared by centrifugation. Protein concentrations were determined using the Pierce BCA Protein Assay (Thermo Fisher Scientific #23225). Proteins were separated into 2 gels (Bio-Rad #5671095) and transferred onto 2 membranes (Bio-Rad #1704271). The following primary antibodies were used at 1: 1,000 dilutions: phospho-STAT3 (Cell Signaling Technology #4139), and STAT3 (Sigma- Aldrich #A4700), SOCS3 (Cell Signaling Technology #52113).). Membranes were incubated with IRDye-conjugated secondary antibodies and (LI-COR) and scanned on an Odyssey DLx Imager (LI-COR). The obtained images were analyzed using Image Studio software (version 3.0; LI-COR).

Histological analysis. Tissues were fixed overnight at room temperature in a 10% neutral-buffered formalin and thereafter stored in 50% ethanol. Fixed tissues were dehydrated, embedded in paraffin, and cut into 4-7 pm sections. Hematoxylin and eosin (H&E), picrosirius red, and Masson’s trichrome staining of deparaffinated sections was carried out according to established protocols. For immunofluorescence staining, deparaffinated sections treated for 20 mins at 95°C in R-buffer A (Electron microscopy sciences, 62706-10) for antigen retrieval, blocked with 5% normal goat serum for 60 mins at 22°C, and, incubated with primary antibodies overnight at 4°C, incubated with fhiorochrome-conjugated secondary antibodies at 37°C for 1 hour, and then mounted with anti-fade mounting medium (Vectashield). The following primary antibodies were used: fibronectin (1:500 dilution; Chemicon #AB1943), collagen I (1:500 dilution; Thermo Fisher Scientific #PA5-95137). For the Collagen Hybridizing Peptide, the product R-CHP (1:500 dilution; Advanced Biomatrix # 5276-UG) was used according to the manufacturer’s protocol. Samples were imaged on a Zeiss Axioscan 7.

Statistical analysis. The data are expressed as means ± SEM. Differences between groups were assessed using ANOVA. Statistical significance was determined at P < 0.05 using either a one-tailed or two-tailed Student’s t-test or a two-way ANOVA with Bonferroni’s post hoc test, as appropriate. Detailed statistical information, including the exact sample size (n), measures of central tendency, dispersion, precision (mean ± SEM), and significance levels, are provided in the figures and figure legends. All statistical analyses were conducted with GraphPad Prism 10.4.1.

TABLE 1 - NUCLEOTIDE SEQUENCES FOR ANTIBODY VARIABLE REGIONS

TABLE 2 - PROTEIN SEQUENCES FOR ANTIBODY VARIABLE REGIONS

TABLE 3 - CDR HEAVY CHAIN AA SEQUENCES

TABLE 4 - CDR LIGHT CHAIN AA SEQUENCES

TABLE 5 - CDR HEAVY CHAIN DNA SEQUENCES

TABLE 6 - CDR LIGHT CHAIN DNA SEQUENCES

TABLE 7 - BINDING AFFINITIES OF ANTLLEPTIN ANTIBODIES DETERMINED USING OCTET 96-RED INSTRUMENT

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

VII. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

WHAT IS CLAIMED IS:

1. A method of detecting leptin in a sample comprising:

(a) contacting a sample with an antibody or antibody fragment comprising clone- paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and

(b) detecting leptin in the sample by detecting binding of the antibody or antibody fragment to leptin in the sample.

2. The method of claim 1 , wherein the sample is a body fluid.

3. The method of claim 1 or claim 2, wherein the sample is blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces.

4. The method of any one of claims 1-3, wherein detection comprises ELISA, RIA, lateral flow assay or Western blot.

5. The method of any one of claims 1-4, further comprising performing steps (a) and (b) a second time and determining a change in leptin levels as compared to the first assay.

6. The method of any one of claims 1-5, wherein the antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

7. The method of any one of claims 1-5, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1.

8. The method of any one of claims 1-5, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone- paired sequences as set forth in Table 1.

9. The method of any one of claims 1-5, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

10. The method of any one of claims 1-5, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

11. The method of any one of claims 1-5, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

12. The method of any one of claims 1-11, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

13. A method of treating a subject comprising delivering to the subject an antibody or antibody fragment comprising clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

14. The method of claim 13, wherein the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.

15. The method of claim 13, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, 90%, or 95% identity to clone- paired sequences from Table 1.

16. The method of claim 13, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

17. The method of claim 13, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80%, 90% or 95% identity to clone- paired sequences from Table 2.

18. The method of any one of claims 13-17, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, Fiab'h fragment, or Fv fragment, or wherein the antibody is a chimeric antibody or a bispecific antibody.

19. The method of any one of claims 13-18, wherein the antibody or antibody fragment is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

20. The method of any one of claims 13-19, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

21. The method of any one of claims 13-20, wherein the subject exhibits one or more of liver fibrosis, diabetes, and metabolic syndrome.

22. The method of claim 21, wherein the delivering results in one or more of increased sensitivity to insulin, reduction in fat mass and/or weight loss, substance weight loss over time, reduced weight gain over time, improvement of diabetic phenotype, improved rate of survival, and reduced liver fibrosis.

23. The method of any one of claims 13- 19, wherein the subject has cancer, such as leukemia, meningioma, adenocarcinoma, multiple myeloma, uterine cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, liver cancer, breast cancer, thyroid cancer and gallbladder cancer.

24. The method of claim 23, wherein the delivering results in one or more of reduced tumor burden, reduced tumor size, cancer cell death, cancer remission, delay of cancer progression, and/or increase in patient survival.

25. The method of any one of claims 21-24, wherein the subject is treated with a second therapy, such as insulin, GLP-1 receptor antagonists, SGLT2 inhibitors, biguanides, sulfonylureas, insulin secretagogues, chemotherapy, radiotherapy, immunotherapy, or surgery.

26. A monoclonal antibody or antibody fragment, wherein the antibody or antibody fragment comprising clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

27. The monoclonal antibody or antibody fragment of claim 26, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.

28. The monoclonal antibody or antibody fragment of claim 26, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.

29. The monoclonal antibody or antibody fragment of claim 26, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1.

30. The monoclonal antibody or antibody fragment of claim 26, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

31. The monoclonal antibody or antibody fragment of claim 26, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

32. The monoclonal antibody or antibody fragment of any one of claims 26-31 , wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, Fiab'p fragment, or Fv fragment.

33. The monoclonal antibody or antibody fragment of any one of claims 26-31, wherein the antibody is a chimeric antibody, or is bispecific antibody.

34. The monoclonal antibody or antibody fragment of any one of claims 26-33, wherein the antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase halflife and/or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

35. The monoclonal antibody or antibody fragment of any one of claims 26-34, wherein the antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody.

36. A hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment comprises clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

37. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone- paired sequences from Table 1.

38. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 1.

39. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone-paired variable sequences from Table 1.

40. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

41. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 2.

42. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

43. The hybridoma or engineered cell of any one of claims 36-42, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, Fiab'h fragment, or Fv fragment.

44. The hybridoma or engineered cell of any one of claims 36-43, wherein the antibody is a chimeric antibody or a bispecific antibody.

45. The hybridoma or engineered cell of any one of claims 36-43, wherein the antibody or antibody fragment is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

46. The hybridoma or engineered cell of any one of claims 36-45, wherein the antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody.

47. A vaccine formulation comprising one or more antibodies or antibody fragments comprising clone -paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

48. The vaccine formulation of claim 47, wherein at least one of the one or more antibodies or antibody fragments is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.

49. The vaccine formulation of claim 47, wherein at least one of the one or more antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.

50. The vaccine formulation of claim 47, wherein at least one of the one or more antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1.

51. The vaccine formulation of claim 47, wherein at least one of the one or more antibodies or antibody fragments comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

52. The vaccine formulation of claim 47, wherein at least one of the one or more antibodies or antibody fragments comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

53. The vaccine formulation of any one of claims 47-52, wherein at least one of the one or more antibody fragments is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

54. The vaccine formulation of any one of claims 47-52, wherein at least one of the one or more antibodies is a chimeric antibody or is bispecific antibody.

55. The vaccine formulation of any one of claims 47-54, wherein at least one of the one or more antibodies or antibody fragments is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

56. The vaccine formulation of any one of claims 47-55, wherein at least one of the one or more antibodies or antibody fragments further comprises a cell penetrating peptide and/or is an intrabody.

57. A vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment according to any one of claims 26-34.

58. The vaccine formulation of claim 57, wherein the expression vector(s) is/are Sindbis virus or VEE vector(s).

59. The vaccine formulation of claim 57 or claim 58, wherein the vaccine is formulated for delivery by needle injection, jet injection, or electroporation.

60. The vaccine formulation of claim 57, further comprising one or more expression vectors encoding a second antibody or antibody fragment, such as a distinct antibody or antibody fragment of claims 26-34.