WO2005094259A2 - Domaine de liaison de leptine, compositions et methodes associees - Google Patents

Domaine de liaison de leptine, compositions et methodes associees Download PDF

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WO2005094259A2
WO2005094259A2 PCT/US2004/008675 US2004008675W WO2005094259A2 WO 2005094259 A2 WO2005094259 A2 WO 2005094259A2 US 2004008675 W US2004008675 W US 2004008675W WO 2005094259 A2 WO2005094259 A2 WO 2005094259A2
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leptin
binding domain
free
receptor binding
sample
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PCT/US2004/008675
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WO2005094259A8 (fr
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Arieh Gertter
Radha Krishna
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Arieh Gertter
Radha Krishna
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the present invention relates generally to leptin binding domain compositions and methods thereto.
  • the compositions are useful for detecting the presence of leptin in a sample and distinguishing free leptin from bound leptin in multiple species. More specifically, the present invention relates to methods for determining free leptin in a sample from an individual by assaying a sample for the binding of leptin to an avian leptin receptor binding domain, and detecting the bound leptin by using a labeled anti-leptin antibody.
  • Human leptin is a 16 kDa, 146 amino acid residue, non-glycosylated polypeptide that was described based on the genetic mapping of a recessive mutation that caused severe obesity in mice (1-2). Abolished transcription or production of an inactive obesity (ob) gene product as found to be responsible for producing the obese (ob/ob) mouse, which is characterized by severe obesity, hyperphagia, hyperglycemia, hyperinsulinemia and insulin resistance, hypothermia, and infertility (3-5).
  • the obesity gene product later known as leptin, is produced by the adipose tissue across a wide range of animal species (1, 2) and shows high interspecies conservation, with the human leptin sharing up to 84% and 87% homology with rat and mouse leptin, respectively (1, 6).
  • a number of recent reports have proposed adipose tissue as an important endocrine gland, and have identified leptin as a pleiotrophic hormone affecting many different organs and tissues in the body (9-11).
  • leptin in pathophysiology of multiple endocrine feedback loops, including reproductive, hematopoiesis, and adrenal cortex function, as well as immune system function, have been reported (8, 9, 11-13).
  • leptin has been shown to modulate insulin activity in hepatocytes in vitro (38).
  • Leptin modulates ovarian steroidogenesis in vitro (39, 40) and affects angiogenesis, acting in some tissues as a positive angiogenic factor (41), whereas it is angiostatic in adipose tissues (42).
  • leptin In rat ovary, leptin attenuates apoptosis and thus enhances sexual maturation (43). Leptin also regulates several functions in the pituitary cells (44). Leptin circulates in serum as a free form or bound to leptin-binding proteins, such as a soluble form of leptin-binding receptor (35). The majority of leptin is in the bound form in lean individuals, but in the free form in obese individuals (35). In addition, leptin levels are also influenced by the stage of puberty and gender in both adults and children.
  • Blood leptin levels are about 2-3 fold higher in men than in women (3, 4, 15 -18); its secretion is pulsatile (19) and follows a circadian rhythm, with the highest levels achieved during the night (19, 20). Obesity in man, in contrast to obesity in mice, is associated with a significant increase in circulating leptin levels (14). Fat mass is the main determinant of leptin to the extent that its circulating levels are exponentially correlated with body mass index (BMI) and percent body fat (2-4, 14). The high sensitivity of leptin to changes in body fat is responsible for the observed wide variations in plasma leptin, which could range from 0.03 to over 100 ng/mL (15-18).
  • the main target of leptin's action is located in the brain, and as leptin is produced in adipose tissue, it has to be transferred through the blood-brain barrier. This transfer is mediated mainly through the short form of the leptin receptor located in the choroid plexus (55, 56).
  • the leptin receptor is a member of the cytokine family of receptors and is responsible for mediating the biological activity of leptin (3, 4).
  • four different mRNA splice variants of the leptin receptor have been so far identified (3, 4). Accordingly, secreted leptin may circulate in both free (unbound) as well as in complex forms bound to a number of different binding proteins.
  • the latter reportedly includes a soluble splice variant of the receptor that has no transmembrane domain as well as soluble leptin receptor generated by the proteolytic cleavage of the membrane- anchored receptors and possibly other unidentified leptin binding proteins (3, 4, 22-23).
  • leptin association with binding proteins is thought to increase leptin biovailability and half-life as well as possibly contributing to the state of leptin resistance (2, 3, 4, 19).
  • leptin As the balance of free and complex leptin is influenced by a complex array of variables, including several hormones and growth factors (3, 4), accurate determination of leptin sub- • fractions could be of significant value in advancing our understanding of pathophysiology and potential diagnostics and therapeutic (10) applications of leptin. Progress in leptin research was complemented by the discovery and cloning of the leptin receptor (3, 4, 21). While polynucleotides and polypeptides of chicken leptin receptor are reportedly provided in Horev, et al. (57) and WO 01/30963, the particular binding domain for the avian species was not provided.
  • This invention relates to methods and assays for the detection of free leptin levels in a sample from an individual.
  • Data is presented that demonstrates the ability of avian or chicken leptin binding domain to bind effectively to leptin from multiple species.
  • Production of the chicken leptin binding protein domain (CLBD) was based on considerations of its potential diagnostic and/or therapeutic value, which was in turn grounded on structural and functional similarities that exists among leptin and leptin receptors in various species (1-4, 6). Recognizing the importance of a more specific approach to free leptin determination, we recently embarked on a systematic evaluation of CLBD.
  • the present application provides the first inter-species combination of a non-immunological solid-phase binder (CLBD) with an immunological detection reagent (goat anti-leptin antibody) for specific determination of free leptin.
  • CLBD non-immunological solid-phase binder
  • an immunological detection reagent goat anti-leptin antibody
  • the methodology is applicable for use in multiple animal species, based on the cross-reacting nature of the various binding reagents.
  • the Free Leptin Receptor-Mediated Enzyme-Linked Immunoassay (RMEIA) is based on a receptor/antibody assay configuration, which is highly compatible with small as well as large-scale manual and/or fully automated applications.
  • the present inventors describe herein subcloning of an avian subdomain, its expression in a prokaryotic host, and its subsequent purification and characterization. Further, the present
  • a leptin binding domain protein having a sufficient binding affinity for human leptin that the binding domain may be used as an antibody mimic in an immunoassay for leptin.
  • a composition comprising an avian leptin receptor binding domain having an amino acid sequence SEQ ID NO: 8 bound in a protein complex to a leptin protein.
  • the chicken leptin receptor binding domain binds to leptin at the same site as the mammalian leptin receptor binding domain, and therefore the avian domain is an agent for competition binding to human leptin in the presence of the human domain, and the avian domain is provided for binding free leptin in biological fluids such as plasma and serum.
  • Another embodiment of the present invention provides a method for detecting a level of free leptin in a sample from an individual, comprising contacting the sample with an avian leptin receptor binding domain of SEQ ID NO:8 for a time sufficient to allow binding between the free leptin and the leptin receptor binding domain to form a bound complex, wherein said receptor binding domain is bound to a solid phase; washing the solid phase with a first wash buffer; contacting the solid phase with an antibody having binding specificity to leptin, wherein said antibody is coupled with a detectable label; washing the solid phase with a second wash buffer; and detecting said label remaining with said solid phase, thus detecting the level of free leptin in the sample.
  • kits for an assay of a level of free leptin in a sample from an individual comprising an avian leptin receptor binding domain comprising SEQ ID No. 8, wherein said domain is bound to a solid phase; an antibody having binding specificity for leptin; and a detectable label coupled with the antibody, wherein the free leptin in the sample binds to the avian leptin receptor binding domain and the antibody binds to the free leptin, thus allowing specific detection of the free leptin in the sample.
  • a method of assaying a test compound for agonist or antagonist activity for the binding of a leptin with a leptin binding domain comprises a) measuring a level of interaction between the avian leptin receptor binding domain and the mammalian leptin in the absence of the test compound; b)
  • test compound has agonist activity, and wherein when the level measured in step b) is less than the level in step a), the test compound has antagonist activity.
  • avian leptin receptor binding domain for detection rather than a leptin receptor specific antibody
  • Advantages to using avian leptin receptor binding domain for detection rather than a leptin receptor specific antibody include use of a much smaller molecule for detection.
  • An additional advantage of the present invention is that using avian or chicken leptin binding domain for detection allows assaying for free leptin levels in multiple species using the same assay.
  • FIG. 1 illustrates a SDS-PAGE analysis of recombinant LBD on a 15% gel.
  • Lane 1 molecular mass markers (172, 111, 79.6, 61.3 (the strongest band), 49, 36.4, 24.7, 19.2, 13.1, 9.3 kDa);
  • lane 2 IPTG-induced bacteria;
  • lane 3 inclusion bodies;
  • lanes 4-6 pooled 100, 125, and
  • FIG. 2 illustrates a purification of hLBD extracted and refolded from inclusion bodies on a Q-Sepharose column.
  • the column (2.5 x 7 cm) was equilibrated with 10 mM Tris-HCl, pH 9.0, at 4 °C.
  • the dialyzed solution of refolded protein was applied to the column at a rate of 120 ml/h. Elution was carried out using a discontinuous NaCl gradient in the same buffer at 120 ml/h, and 5-ml fractions were collected. Protein concentration was determined by absorbance at 280 nm. Every fifth tube was assayed for hLBD content by gel filtration in a SuperdexTM75 HR column (see text). Tubes 51-75, 78-104, and 110-135 were pooled (pools 100, 125, and 150 mM, respectively).
  • FIG. 3 illustrates a circular dichroism (CD) spectram of purified recombinant leptin- binding domain in 65 mM sodium carbonate buffer, pH 7.5.
  • FIG. 4 illustrates a gel filtration of complexes of hLEP and on a Su ⁇ erdexTM75 HR 10/30 column.
  • Complex formation was carried out during a 20- to 30-min incubation at room temperature in TN buffer using various hLEP.LBD molar ratios and then ali ⁇ uots (200 ⁇ l) of the incubation mixture were applied to the column, pre-equilibrated with the same buffer.
  • the initial hormone concentration (2 ⁇ M) was constant in all cases in the upper row, whereas in the lower row the LBD concentration was held constant (4 ⁇ M).
  • FIG. 5 illustrates a competition of unlabeled human leptin (Q), ovine leptin (A), and chicken leptin (T) with 125 I-human leptin (80,000 cpm/tube) for binding to LBD (__) and to homogenate of BAF/3 cells (B).
  • the specific binding (%) in experiments performed with human, ovine, and chicken leptins and their mutants were, respectively, 7.3% in _4, and 8.1% in B, and the nonspecific binding was respectively, 5.4 and 14%.
  • FIG. 6 illustrates an association and dissociation kinetics between LBD and hLEP linked covalently to carboxy-methylated dextran through amino groups.
  • FIG. 7 illustrates an inhibition of human ( ⁇ )-, ovine (&)-, chicken ( ⁇ )-, and interleukin-3 (Q)-stimulated proliferation of BAF/3 cells transfected with the long form of human leptin receptor.
  • Synchronized cells were grown for 48 h in the presence of human, ovine, or chicken leptin (0.57 nM) or interleukin-3 (6 nM) and various concentrations of LBD. The number of cells was determined subsequently by the thiazolyl blue method (see text). Full lines and IC 50 values were calculated using the PRIZMA curve-fitting program (Author, A. (1994) GraphPad TM
  • FIG. 8 illustrates a schematic representation of the human leptin-LBD 1:1 complex.
  • the amino- and carboxyl-terminal domains of LBD are denoted as Dl and D2, respectively.
  • Tyr-441 and Phe-500 which may be crucial for leptin binding, are labeled and shown in red.
  • FIG. 9 illustrates gel filtration analysis of complexes at various molar ratios between ovine, human and chicken leptins and chLBD. The calculated MW of the chLBD in all experiments was the same.
  • FIG. 10 illustrates inhibition of binding of 1251-hLep to cliLBD by ovine, human and chLeptin.
  • FIG. 11 illustrates a high-performance liquid chromatography (HPLC) profile of free leptin.
  • HPLC high-performance liquid chromatography
  • a fresl serum sample containing 200 ng/mL soluble leptin receptor was pre-incubated overnight with 400 ng/mL exogenous leptin, fractionated as in Figure 11, and fractions were assayed for soluble leptin receptor and free leptin. Arrows mark the elution peak of the gel filtration molecular weight markers.
  • FIG. 13 illustrates a comparison of free leptin RMEIA with total leptin ELISA.
  • RMEIA leptin standards were prepared in normal goat serum.
  • FIG. 14 illustrates a comparison of free leptin RMEIA with total leptin ELISA in samples with total leptin of less than 25 ng/mL.
  • the figure shows the expanded relationship between the corresponding sample values depicted in the lower region of Figure 3.
  • RMEIA leptin standards were prepared in normal goat serum. Values are means of duplicate measurements.
  • FIG. 15 illustrates a relationship between free leptin and soluble leptin receptor.
  • the present invention is directed to a composition
  • a composition comprising an avian leptin receptor binding domain having an amino acid sequence SEQ ID NO: 8 bound in a complex to a leptin protein.
  • a representative example of avian leptin receptor binding domain is chicken leptin receptor binding domain.
  • the leptin protein is mammalian leptin, wherein the mammalian leptin may be human, rat, mouse, ovine, porcine, or bovine leptin.
  • Another aspect of the present invention is directed to a method for detecting a level of free leptin in a sample from an individual, comprising contacting the sample with an avian leptin receptor binding domain of SEQ ID NO:8 for a time sufficient to allow binding between the free leptin and the leptin receptor binding domain to form a bound complex, wherein said receptor binding domain is bound to a solid phase; washing the solid phase with a first wash buffer; contacting the solid phase with an antibody having binding specificity to leptin, wherein said antibody is coupled with a detectable label; washing the solid phase with a second wash buffer; and detecting said label remaining with said solid phase, thus detecting the level of free leptin in the sample.
  • the detectable label include a label that is radiolabeled, chemiluminescent, electroluminescent, fluorescent, enzyme-labeled, or bioluminescent.
  • the solid phase is a micro-titre well plate.
  • the avian leptin receptor binding domain is chicken leptin receptor binding domain.
  • the individual is a mammal, wherein said mammal may possibly be human, rat, mouse, ovine, porcine, or bovine.
  • the sample is a human serum or plasma sample.
  • the individual has a condition or a disease related to the level of free leptin in the sample.
  • kits for an assay of a level of free leptin in a sample from an individual comprising an avian leptin receptor binding domain comprising SEQ ID No. 8, wherein said domain is bound to a solid phase; an antibody having binding specificity for leptin; and a detectable label coupled with the antibody, wherein the free leptin in the sample binds to the avian leptin receptor binding domain and the antibody binds to the free leptin, thus allowing specific detection of the free leptin in the sample.
  • the avian leptin receptor binding domain is chicken leptin receptor binding domain.
  • the individual is a mammal; representative examples include mammals that are human, rat, mouse, ovine, porcine, or bovine.
  • Other aspects include a sample that is a human serum or plasma sample, and wherein the solid phase is a micro-titre well plate.
  • the detectable label may be radiolabeled, chemiluminescent, electroluminescent, fluorescent, enzyme-labeled, or bioluminescent.
  • Another embodiment of the present invention provides a method of assaying a test compound for agonist or antagonist activity for an avian leptin receptor binding domain-leptin complex, comprising a) measuring a level of interaction between the avian leptin receptor binding domain and the mammalian leptin in the absence of the test compound; and b) measuring a level of interaction between the avian leptin receptor binding domain and the mammalian leptin in the presence of the test compound, wherein when the level measured in step b) is greater than the level in step a), the test compound has agonist activity, and wherein when the level measured in step b) is less than the level in step a), the test compound has antagonist activity.
  • the present invention provides recombinant leptin receptor binding domains for use in the elucidation of leptin-leptin receptor interactions, for screening assays, and for diagnostic assays.
  • a recombinant ⁇ 200 amino acid fragment of the ECD of human and chicken leptin receptors were shown to possess the ability to bind human, ovine and chicken leptins and to form stable 1:1 complexes.
  • the chicken leptin receptor binding domain exclusively binds to free leptin even when it is in the presence in a mixture of free-leptin and bound leptin (such as in an in vivo condition) because of the exclusive specificity to the binding domain of leptin.
  • a protein such as an polyclonal or monoclonal antibody or a binding domain of leptin receptor may be labeled with a detectable substance and the protein detected or localized based upon the presence of the detectable substance.
  • detectable substances include, but are not limited to, radioisotopes (e.g., .sup.3H, .sup.l4C, .sup.35S, .sup.1251, .sup.13 II), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as lummol; enzymatic labels (e.g., horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity
  • Indirect methods may be employed in which a primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the primary antigen.
  • a primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the primary antigen.
  • the second antibody may be goat anti-rabbit gammaglobulin labeled with a detectable substance as described herein.
  • the protein is localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • the leptin receptor binding domain may be attached to a solid support.
  • solid support is meant a non-aqueous matrix to which the domain protein of the present invention can adhere.
  • solid supports include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol or silicones.
  • the solid phase may comprise the well of an assay plate or a purification column, for example.
  • an “antagonist" of leptin-binding domain interaction is meant an agent having inhibitory activity for the binding of leptin and a leptin receptor binding domain.
  • the binding may be inhibited by an effect on the interaction between leptin and binding domain, or by an effect on leptin or binding domain that affects the interaction between leptin and binding domain.
  • an "agonist" of leptin-binding domain interaction is meant an agent having enhancing or stimulatory activity for the binding of the leptin-binding domain complex.
  • the binding may be stimulated by an effect on the interaction between leptin and binding domain or by an effect on leptin or binding domain that affects the interaction between leptin and binding domain.
  • Identification of an antagonist or an agonist is made by allowing leptin and binding domain to interact in the presence of a test agent. A decrease or increase in leptin-binding domain interaction relative to the interaction when the test agent is absent indicates that the test agent has an effect on the binding interaction.
  • leptin-binding domain protein compositions of the present invention conservative amino acid substitutions, such as Glu/ Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn, would be considered equivalent since the chemical similarity of these pairs of amino acid residues would be expected to result in functional equivalency.
  • Amino acid substitutions that conserve the biological function of the leptin-binding domain would conserve such properties as hydrophobicily, hydrophilicity, side-chain charge, or size.
  • Functional equivalency is determined by the interaction of the equivalent bound complex as compared to the native bound complex.
  • leptin-binding domain derivatives, or analogs thereof, e.g., by glycosylation, acetylation, phosphorylation, amidation, fatty acylation, sulfation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, or the like.
  • free'' leptin leptin that is not bound to leptin-binding proteins.
  • bound leptin leptin that is bound to one or more leptin-binding proteins, including but not limited to the soluble form of leptin-binding receptor.
  • Example 1 Purification and Characterization of Leptin Receptor Binding Domain Materials— Ovine leptin (fraction SP), chicken leptin, and human leptin (hLEP) were prepared as described (Gertler, A. et al. (1998) FEBS Lett. 442, 137-140; Raver, N. et al (1998) Protein Expression Purif. 14, 403-408; Raver, N. et al; (2002) Gen. Comp. Endocrinol. 126, 52- 58); pET29a expression vector was purchased from Novogene Inc. (Madison, WI).
  • Restriction enzymes used in the molecular biology experiments were from Fermentas (Vilnius, Lithuania) and New England Biolabs (Beverly, MA). DNA primers were ordered from Invitrogen. Lysozyme, urea, arginine, radioimmunoassay-grade bovine serum albumin, Triton X-100, RPMI 1640 medium, interleukin-3 (EL-3), isopropyl ⁇ -D-thiogalactopyranoside (IPTG), and 3-(4,5- dimethyIthiazoI-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue) were purchased from Sigma, fetal calf serum was from Biolab Co.
  • CM5 sensor chip _V-hydroxysuccinimide, -V-ethyl-iY (3-dimethylaminopropyl)- carbodiimide hydrochloride, ethanolamine hydrochloride, and HBS-EP running buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, and 0.005% (v/v) surfactant P20, pH 7.4) were purchased from Biacore, AB (Uppsala, Sweden). All other chemicals were of analytical grade.
  • LBD Expression Plasmid A DNA insert encoding the LBD fragment, consisting of amino acids 428-635 of the human leptin receptor (SEQ ID NO:l provides the nucleotide sequence and SEQ ID NO:2 provides the amino acid sequence), was prepared by PCR using the following primers: the 5'-sense primer, 5'-
  • GGAATTCCATATGATTGATGTCAATATCAATATCTC-3 1 (SEQ ID NO:3) containing an Ndel restriction site (underlined) and the antisense 3'-end primer, 5'- CATAGGAAGCTTTCAATCCATGACAACTGTGTAGGCTGG-3' (SEQ ID NO:4) containing a stop codon (bold letters) followed by a HindH site (underlined).
  • the resulted PCR product was cloned into the pGEM-T vector, sequenced to ensure lack of mutations, digested with NdeVHind ⁇ , and subcloned into the pET29a plasmid, predigested with the same restriction enzymes. The expression plasmid was then transformed into BL21 cells.
  • LBD LBD—BL21 cells (500 ml) were grown in a 2.5-liter flask in Terrific Broth (TB) medium at 37 °C to an _4 60 o of 0.9, and IPTG was then added to a final concentration of 1 mM. Cells were grown for an additional 4 h and then harvested by centrifugation at 16,000 x g for 10 min and frozen. The bacterial pellet from 3 liters of culture was thawed on ice and resuspended in lysis buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8) containing 0.5 mg lysozyme/ml.
  • lysis buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8) containing 0.5 mg lysozyme/ml.
  • Inclusion bodies were then prepared as described previously and frozen (Gertler, A. et al. (1998) FEBS Lett. 442, 137-140). Subsequently, inclusion bodies obtained from 3 liters of bacterial culture were solubilized in 600 ml of 4.5 M urea, pH 11.5, in the presence of 10 mM cysteine. After 1 h of stirring at 4 °C, the solution was diluted with 2 vol of 0.75 M L-Arg to a final concentration of 0.5 M and stirred for an additional 10 min, and then the clear solution was dialyzed against 5 x 10 liters of 10 mM Tris-HCl, pH 9.
  • the protein was then applied to a Q-Sepharose column (2.5 x 6 cm) pre-equilibrated with 10 mM Tris-HCl, pH 9.
  • the breakthrough fraction (which contained no LBD) was discarded, the absorbed protein was eluted in a stepwise manner by increasing concentrations of NaCl in the same buffer, and 5-ml fractions were collected. Protein concentration was determined by absorbance at 280 nm.
  • Determination of the Amino-terminal Sequence Automated Edman degradation technique was used to determine the amino-terminal protein sequence. Degradation was performed on an ABI Model 470A gas-phase sequencer (Foster City, CA) using the standard sequencing cycle. The respective phenylthiohydantoin derivatives were identified by reverse phase-high pressure liquid chromatography analysis, using an ABI Model 120A phenylthiohydantoin analyzer fitted with a Brownlee 2.1 -mm inner diameter phenylthiohydantoin-Cis column.
  • SDS-PAGE SDS-PAGE was carried out according to Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685) in a 15% polyacrylamide gel under reducing and non-reducing conditions. Gels were stained with Coomassie Brilliant Blue R. Gel filtration chromatography was performed on a SuperdexTM75 HR 10/30 column with 0.2-ml aliquots of the Q-Sepharose column-eluted fractions using 25 mM TN buffer (Tris-HCl buffer, pH 8, containing 150 mM NaCl). Freeze-dried samples were dissolved in H 2 O.
  • Laemmli Laemmli, U. K. (1970) Nature 227, 680-685
  • Gels were stained with Coomassie Brilliant Blue R.
  • Gel filtration chromatography was performed on a SuperdexTM75 HR 10/30 column with 0.2-ml aliquots of the Q-Sepharose column-eluted fractions using
  • CD spectra and Extinction Coefficients The CD spectra in millidegrees were measured with an AVTV model 62A DS circular dichroism spectrometer (Lakewood, NJ) using a 0.020-cm rectangular QS Hellma cuvette. The spectrometer was calibrated with camphorsulfonic acid. The absorption spectra were measured with an AVIV model 17DS UV-visible ER. spectrophotometer using a 1.000-cm QS cuvette and correction for light scattering. Lyophilized protein was dissolved in water, dialyzed against 50 mM phosphate buffer, pH 7.5, for 20 h, and then centrifuged at 11,000 x g for 10 min.
  • the CD measurements were performed at 25.0 °C as controlled by thermoelectric Peltier elements to an accuracy of 0.1 °C.
  • the CD spectra were measured in five repetitions resulting in an average spectrum for each protein. Standard deviation of the average CD signal at 222 nm was in the 5% range.
  • the CD data were expressed in- degree cm 2 /dmol per mean residue, based on a molecular mass of 24.6 kDa calculated for the protein from the 208 amino acids.
  • the protein concentration was determined by the Biuret method (Goa, J. (1953) Scand. J. Clin. Lab. Invest.
  • the amino-terminal sequence of the purified LBD was (Met)-Ala-Ile-Asp-Val-Asn-Ile-Asn-Ile-Ser-Xaa-Glu (SEQ ID NO:5), as predicted from the primary structure (Haniu, M. et al. (1998) J. Biol. Chem. 273, 28691-28699), with an additional Met residue.
  • the unidentified amino acid at position 10 is most likely Cys, which could not be identified by the present method.
  • the results of the CD analysis are presented in FIG. 3.
  • the secondary structure calculations revealed the contents of a- helices, ⁇ -strands, ⁇ -turns, and unordered fortos to be (mean ⁇ S.D.) 6.6 ⁇ 0.4, 37 ⁇ 1.2, 25 ⁇ 1.0, and 31 ⁇ 1.6%, respectively, indicating strong similarity to the structure observed in the ECDs of hGH, human prolactin, and rat prolactin receptors (De Vos, A. M. et al (1992) Science 255, 306- 312; Somers, W. et al (1994) Nature 372, 478-481; Elkins, P. A. et al (2000) Nat. Struct. Biol. 7, 808-815).
  • Example 2 Determination of Complex Stoichiometry Complexes between LBD and hLEP were prepared at various molar ratios in TN buffer. After a 20- to 30-min incubation at room temperature, 200- ⁇ l aliquots were applied to a SuperdexTM75 HR 10/30 column. To determine the molecular mass of the complex, the column was calibrated with several pure proteins.
  • Binding Assays Radiolabeled human 125 I-leptin served as a ligand, and other (human, ovine, and chicken) nonlabeled leptins served as competitors.
  • the experiments were conducted using either recombinant LBD or homogenates of BAF/3 cells stably transfected with the long form of hLEP receptor. In the latter case, the cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum in the presence of IL-3 to minimize leptin- receptor down-regulation until a concentration of 10 6 cells/ml was reached. Then the cells were spun and stored at -70 °C.
  • reaction buffer (12.5 mM sodium barbiturate, pH 8.6, buffer containing 0.1% (w/v) bovine serum albumin, 7.5 mM EDTA, 150 mM NaCl, and 0.1% (w/v) Triton X-100), and homogenized with a Polytron for 30 s at 10,000 rpm on ice.
  • Each tube contained 150 or 200 ⁇ l of reaction buffer in the case of the assay with the cells or recombinant LBD, respectively, 100 ⁇ l of 125 I-hLEP (100,000 cpm for cells or 180,000 cpm for binding domain assays), and 100 ⁇ l of different leptin solutions (providing 0-5000 ng/tube) in the reaction buffer, and the reaction was started by addition of 150 ⁇ l of cell homogenate or 100 ⁇ l of LBD (20 ng). The tubes were incubated for 24 h at room temperature. Then the leptin ⁇ eceptor complex was precipitated by adding 250 ⁇ l of 1% (w/v) bovine immunoglobulin and 500 ⁇ l of 20% (w/v) polyethylene glycol.
  • the surface was activated for 7 min with a mixture of _V-hydroxysuccinimide (0.05 M) and _V- ethyl-iV (3-dimethylamino ⁇ ropyl)-carbodiimide hydrochloride (0.2 M).
  • hLEP was injected at a concentration of 50 ⁇ g/ml in 10 mM acetate, pH 3.5, until the desired level (1000 resonance units) was achieved.
  • Ethanolamine (1 M, pH 8.5) was injected for 7 min to block the remaining activated groups.
  • a control surface was prepared by activating the carboxyl groups and then blocking the activated groups by ethanolamine as described.
  • the LBD resuspended in HBS-EP buffer
  • was passed at different concentrations 31.25, 62.5, 125, and 250 nM
  • Regeneration of the surface after each interaction was performed by using a 10- ⁇ l pulse of 10 mM glycine buffer, pH 2.
  • the experiment was done using the kinetics Wizard of the Biacore control software, which corrects automatically for refractive index changes and nonspecific binding by subtraction of the responses obtained for the control surface from the data obtained for the interaction with hLEP.
  • the obtained binding curves were fitted to the association and dissociation phases at all leptin receptor concentrations simultaneously using evaluation software from Biacore. The best fit was obtained for a simple bimolecular interaction (Langmuir model).
  • Example 3 Proliferation Assay BAF/3 Proliferation Assay- The proliferation rate of leptin-sensitive BAF/3 1442-CI4 cells stably transfected with the long form of human leptin receptor was used to estimate self- and antagonistic activity of recombinant LBD, using the thiazolyl blue method as described (Raver, N. et al (2000) Protein Expression Purif. 19, 30-40). To determine antagonistic activity of LBD, human, ovine, or chicken leptin were added to each well (to a final concentration of 0.57 nM) with various concentrations of recombinant LBD. The average absorbance in wells with wild-type leptins after subtraction of the negative control was used as a positive control to calculate percent inhibition caused by LBD.
  • LBD inhibited the proliferation of BAF/3 cells stimulated, respectively, by human, ovine, and chicken leptins in a dose-dependent pattern, but the molar excess required to achieve 50% inhibition in cells stimulated by human, ovine, or chicken leptins was rather large, namely 200, 200, and 600 molar excess, respectively (FIG. 7).
  • the inhibitory effect was, however, very specific, as no inhibition was observed in cells stimulated by IL-3 even at a 10 5 molar excess of LBD.
  • human LBD in low concentrations (nM) blocked leptin induced, but not interleukin-3 -induced proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor, in dose dependent manner, while in high concentrations of LBDs, they were able to induce proliferation by themselves.
  • Example 4 Comparison of Kd values for interaction of human leptin with human leptin receptors
  • the results herein demonstrate production of recombinant LBD, a 208-amino acid fragment of the ECD of human leptin receptor (corresponding to residues 428 to 635 of the full- size WT receptor), which has the ability to bind human and other leptins.
  • a scale-up of its production will enable an increase in yield and the production of material for both structural and in vivo studies.
  • the electrophoretically pure monomeric protein was capable of forming a stable 1:1 complex with hLEP.
  • BD minimal binding domain
  • ECD extracellular domain
  • the ligand-binding determinants of cytokine receptor ECDs consist of six segments denoted L1-L6 (De Vos, A. M. et al (1992) Science 255, 306-312; Livnah, O. et al (1996) Science 273, 464-471). These segments are positioned in three loop regions, L1-L3 situated in ' the amino-terminal domain, L4 in the interdomain linker, and L5 and L6 in two main loops, located in the carboxyl-terminal domain.
  • Previous structural and mutational research with the hGH and hGH receptor ECD system has indicated that the binding epitope consists of many interacting residues, some of which are crucial for ligand binding (Clackson, T.
  • Trp-583 extends the WS motif into the LBD.
  • Two arginine residues (Arg-612 and Arg-573) are sandwiched between each tryptophan pair to form an extended p-cation system.
  • membrane-embedded receptor-soluble system could be useful as a model for mapping of the binding epitope of both receptor and hormone.
  • a short fragment of the receptor with high affinity binding capabilities to the hormone provides a higher potential system for crystallization and subsequent structural studies.
  • extensive mutagenesis and subsequent binding assays would identify the crucial amino acid residues in the binding sites and may provide a platform for the design of small molecules and/or peptidic high affinity binders of leptin receptor.
  • Example 5 Chicken Leptin Binding Domain (chLBD)
  • the present inventors have prepared LBD from chicken receptor from DNA provided by Dr. Miri Einat (Agricultural Research Organization, the Volcani Center, P.O. Box 6, 50250 Beit Dagan, Israel) and characterized binding and functional properties thereof. Such properties were compared between the recombinant human (amino acids 428 to 635) and chicken (amino acids 419 to 624) leptin receptor-binding domains (LBD). Primers used for cloning the chicken receptor binding domain were designed based on the sequence of the human receptor binding domain and the known sequence of the complete chicken receptor gene.
  • the amino acid sequence of the chicken leptin receptor binding domain is provided at SEQ ID NO: 8 and the nucleotide sequence that encodes the chLBD is provided at SEQ ID NO:7.
  • the LBDs were subcloned, expressed in prokaryotic host, refolded and purified as at least 95% monomers, revealed by SDS-PAGE and gel filtration under non denaturative conditions. Ten to twenty milligram preparations of the chicken lepin receptor binding domain are readily prepared.
  • antibodies used in the present invention may be monoclonal or polyclonal in nature. Antibodies may be raised against recombinant human leptin or leptin/leptin-binding protein complex purified from human sera. Polyclonal antibodies could be raised in various species including but not limited to mouse, rat, rabbit, goat, sheep, donkey, horse, using standard immunization and bleeding procedures. Animal bleeds with high titres may be fractionated by routine selective salt- out procedures such as precipitation with ammonium sulfate and specific immunoglobulin fraction separated by successive affinity chromatography on Protein-A-Sepharose and leptin- Sepharose columns according to standard methods.
  • the purified polyclonal as well as monoclonal antibodies must be then characterised for specificity and lack of cross-reactivity with related molecules as much as possible. This could be easily performed by standard methods using labelled leptin (e.g., with radioisotopes or biotin) as tracer in competition with increasing levels of unlabeled potential cross-reactants for antibody binding. In some cases, further purification may be required to obtain highly specific antibody fraction or for selection of higher affinity antibody fraction from a polyclonal pool. In the case of monoclonal antibodies, care should be taken to select antibodies with good binding characteristic and specificity not only for the immunogen, but also for the native circulating molecules, particularly when recombinant molecule or peptide antigen are used for immunization.
  • Monoclonal antibodies may be prepared according to the well established standard laboratory procedures "Practice and Theory of Enzyme Immunoassays" by P. Tijssen (In Laboratory Techniques in Biochemistry and Molecular Biology, Eds: R.H. Burdon and P.H. van Kinppenberg; Elisevier Publishers Biomedical Division, 1985), which are based on the original technique of Kohler and Milstein (Kohler G., Milstein C. Nature 256:495, 1975). This is usually performed by removing spleen cells from immunized animals and immortalizing the antibody producing cells by fusion with myeloma cells or by Epstein-Barr virus transformation, and then
  • Procedures for antibody (500 ng/100 uL/well) coating to microwells were as previously described (29-31). The same procedure was also used for CLBD coating to microwell, except that the effect of coating concentration and volume as well as using coating buffer pH in the 2.6 to 9.1 range were examined.
  • the coating buffers with pH in the 2.6 to 6.5 range were made by titrating 0.1 M citric acid with 0.2 M dibasic sodium phosphate. Coating buffers pH 6.5, pH 8.5 and pH 9.1 were based on 0.2 M sodium phosphate, 0.05 M sodium borate, or 0.1 M sodium carbonate, respectively. Procedures for antibody conjugation to bio tin or HRPO have been also described (29-31).
  • diluent #1 (50 mM Sodium Phosphate, pH 7.4 containing, 0.005 M sodium EDTA, and 1 g bovine serum albumin (BSA), 9 g NaCl, 1 mL Trasylol, and 2.5 mL Proclin-300 per litre] and diluent #
  • 29 Buffer A Fifty mM Trisma Maleate, pH 7.0, containing 0.001 M EDTA and 9 g NaCl, 5 g BSA, 0.5 mL Tween-20, and 2.5 mL Proclin-300 per litre.
  • Buffer B Fifty mM Sodium Borate, pH 8.5, containing 9 g NaCl, 5g BSA, 0.5 mL Tween-20, and 2.5 mL Proclin-300 per litre.
  • the anti-leptin antibodies were purified using standard antibody purification schemes. Both monoclonal and polyclonal antibodies were purified by affinity chromatography over Protein-A columns and if necessary, by affinity chromatography over a gel column containing immobilized leptin. To evaluate the impact of assay design on performance, both one-step (simultaneous incubation of sample plus detection antibody or CLBD-biotin tracer) and two-step (sequential incubation of sample and the detection antibody or CLBD-biotin tracer) configurations were assessed. Based on such experimentation, assay designs involving solid-phase capture receptor and liquid-phase detection antibody appeared more promising.
  • the capture receptor in this case CLBD, may be linked to various supports by the standard non- covalent or even covalent binding methods, depending on the analytical as well as clinical requirements of the assay.
  • the solid-support might be in forms of test tubes, beads, microparticles, filter paper, membranes, glass filter, magnetic particles, silicon chip, or materials and approaches known to those skilled in the art.
  • the use of microparticles, particularly magnetizable particles that have been directly coated with the receptor (magnetic particles- capture receptor) or particles that have been labelled with a universal binder (e.g., avidin or anti- receptor antibody) are ideal for significantly shortening the assay incubation time. This along with other alternative approaches known to others may allow for assay completion within minutes without limiting the required sensitivity of the assay.
  • the use of magnetizable particles or similar approaches would also allow for convenient automation of the technology on the widely available immunoanalyzers. Obviously, the assay sensitivity could be improved by using
  • the antibody used for leptin detection may be either directly labelled to a reported molecule, or detected indirectly by a secondary detection system.
  • the latter may be based on several different principles, including antibody recognition by a labelled anti-species antibody or other forms of immunological or non-immunological bridging and signal amplification detection systems (e.g., the biotin-streptavidin technology).
  • the signal amplification approach may be used to significantly increase the assay sensitivity and improve low levels reproducibility and performance.
  • the label used for direct or indirect antibody labelling may be any detectable reporter molecule. Examples of suitable labels are those widely used in the field of immunological and non-immunological detection systems. These may include fluorophores, luminescents, metal complexes and radioactive labels, as well as moieties that could be detected by other means (e.g., electrical) or suitable reagents such as enzymes and their various combinations and substrates.
  • the assay design may be homogeneous or heterogeneous, depending on particular application of the assay and the need for speed, sensitivity, accuracy and convenience.
  • the detail discussion on helpful designs may be found in various immunoassay books and literatures, including "Practice and Theory of Enzyme Immunoassays" by P. Tijssen (In Laboratory Techniques in Biochemistry and Molecular Biology, Eds: R.H. Burdon and P.H. van Kinppenberg; Elisevier Publishers Biomedical Division, 1985),
  • the assay buffer was assay buffer B (0.05 mol/L borate, pH 8.5, 9 g/L NaCl, 1 g/L bovine serum albumin (BSA), 50 ml/L normal goat serum, 0.5 ml Tween 20, 5 mL/L proclin 300).
  • the standard matrix was 50 mM sodium phosphate, pH 7.4 containing, 0.005 M sodium EDTA, and 1 g bovine serum albumin (BSA), 9 g NaCl, 400 mL goat serum, 1 mL Trasylol, and 2.5 mL Proclin-300 per litre.
  • the wells were washed xl prior to use or stored for up to 2 days in the blocking buffer at 4 °C.
  • the stopping solution was 0.2 mol/L sulfuric acid in deionised water.
  • the composition of the coating and blocking buffers as well as the wash solution were as described previously (29, 30).
  • Coupling of the detection antibodies to HRP was performed as described (29, 30).
  • the coupling reaction involved activation of the enzyme with sulfo-SMCC and its subsequent conjugation to the anti- leptin antibody, which had been activated by 2-iminothiolane.
  • the stock HRP-conjugated antibody solution was diluted at least 1000-fold prior to use.
  • Free Leptin standards were prepared by diluting recombinant human leptin in the standard matrix buffer
  • leptin reference standard values of about 0.5 to 100 ng/mL.
  • the standards were freshly prepared prior to use.
  • the quality control samples used were fresh serum samples containing various levels of leptin.
  • the nominal concentrations of the control samples were established by analyzing the samples in a conventional Leptin ELISA.
  • the intra-assay CVs was determined by replicate analysis of 4 samples in one run; interassay CVs by duplicate measurement of four samples in 9 separate runs. Specificity was assesses by the various approaches described in the Results section. For comparative evaluations, random adult male and female samples were assayed by the present Free Leptin RMEIA and by commercially available ELISAs for total leptin or soluble leptin receptor (DSL, Webster, TX). All values are means of duplicate measurements.
  • the antibody-capture format evaluated in some details involved incubation of sample (50 uL) and assay buffer (50 uL) in antibody coated wells followed by sequential washing and incubation with CLBD-biotin tracer, streptavidin-HRPO, and colorimetric quantification.
  • CLBD-biotin tracer 50 uL
  • streptavidin-HRPO 50 uL
  • colorimetric quantification The uses of strategies commonly known to minimize NSB were found ineffective.
  • the standard matrix should be as close as possible to sample matrix so that differential effects of standard vs sample response are minimized. Accordingly, comparative analysis of signal generated for standards prepared in different buffers and in various animal sera was performed. As expected, the receptor-mediated one- and two-step assay formats responded differently to the various standard matrixes, with the serum-based standards generating a significantly lower binding response than the similarly prepared and tested buffer-based standards (Table 6). In addition, the use of serum- based standards, particularly in a one-step configuration, appeared highly advantageous in terms of NSB signal of the zero-dose standard and signal/dose ratios.
  • the selected RMEIA protocol demonstrated acceptable analytical performance characteristics. As shown in Table 9 and Table 10, the overall intra- and inter-assay imprecision of the assay was similar to levels seen for conventional immunoassays and were in general better than 10%.
  • a serum sample containing high endogenous soluble leptin receptor was fractionated as above and fractions assayed for free leptin and soluble leptin receptor HPLC profile. Results identified elution of soluble leptin receptor immunoreactivity and free leptin reactivity in the expected ⁇ 300 KD (33) and ⁇ 15 KD regions (26), respectively (data not shown). Because of the possibility of low leptin occupancy of the endogenous soluble leptin receptor, the serum sample was overnight incubated with excess recombinant leptin (300 ng/ml) and rechromatographed.
  • the free leptin reactivity detected by the present assay was evident in a single peak eluting in the same molecular weight region as above. Again, no free leptin reactivity in fractions containing the soluble leptin receptor was detectable.
  • the free leptin RMEIA an iinmimofunctional assay, involves a recently validated chicken leptin binding peptide that specifically binds to free (bioactive) leptin as well as allowing for leptin detection by an antibody-directed approach.
  • the combination of receptor binding and immunological detection of bound leptin is an improtant measure of assay specificity.
  • the latter was further demonstrated by complete inhibition of the binding activity in response to leptin pre-incubation with increasing concentrations of CLBD (Table 11).
  • the substantiating specificity data was also indicated by differential mixing of samples with high endogenous soluble leptin receptor with samples containing high free leptin (low receptor) and analyzing the effect on free leptin determinations. Although, a gradual decrease in free leptin levels in response to increasing levels of endogenous soluble leptin receptor addition was observed, the inhibition was not as complete as described above. The lack of complete inliibition (Table 12) is most possibly due to the high occupancy levels of the circulating soluble leptin receptors by endogenous leptin (50-100%), particularly in obese subjects (34).
  • the free leptin measured by the present RMEIA correlated inversely with the corresponding levels of the soluble leptin receptors.
  • Figure 5 the lowest free leptin levels were observed for samples that contained high concentrations of soluble leptin receptors, again exemplifying conditions where determination of free leptin might be of significant benefit. The latter is highly relevant given the finding that nearly 15-50% and 50-100% of the soluble leptin receptors in normal and in obese subjects circulates, respectively, in complex forms with the endogenous leptin (34).
  • RMEIA receptor-mediated enzyme immunoassay
  • the assay involving a recombinant chicken leptin receptor binding protein domain (CLBD) in concert with an enzyme-labeled anti-leptin detection antibody, is considered highly advantageous in expediting leptin investigations at both research and application levels.
  • the method involves capturing the free leptin with CLBD coupled to a solid-phase and detecting the captured free leptin with an antibody coupled to a detection system.
  • the present assay configuration optimized for the various contributing variables could be obviously modified using different approaches known to those skilled in the art.

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Abstract

L'invention concerne des méthodes associées aux domaines de liaison du récepteur de leptine aviaire. Elle démontre que ces compositions sont utiles pour détecter la présence de leptine dans un spécimen et distinguer leptine libre de leptine liée dans des espèces multiples. Elle concerne des méthodes et des trousses servant à déterminer leptine libre dans un spécimen prélevé sur un individu par détermination sur un spécimen de la liaison de leptine à un domaine de liaison du récepteur de leptine aviaire et détection de leptine liée au moyen d'un anticorps anti-leptine marqué.
PCT/US2004/008675 2004-03-22 2004-03-22 Domaine de liaison de leptine, compositions et methodes associees WO2005094259A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007080404A2 (fr) * 2006-01-12 2007-07-19 Asterion Limited Ligands de leptines
WO2020253187A1 (fr) * 2019-06-21 2020-12-24 深圳市亚辉龙生物科技股份有限公司 Immunogène de leptine, cellule d'hybridome, anticorps monoclonal, anticorps polyclonal et leur utilisation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007080404A2 (fr) * 2006-01-12 2007-07-19 Asterion Limited Ligands de leptines
WO2007080404A3 (fr) * 2006-01-12 2008-03-06 Asterion Ltd Ligands de leptines
WO2020253187A1 (fr) * 2019-06-21 2020-12-24 深圳市亚辉龙生物科技股份有限公司 Immunogène de leptine, cellule d'hybridome, anticorps monoclonal, anticorps polyclonal et leur utilisation

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