NZ788165A - Methods for detecting neutralizing antibodies to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) analog - Google Patents

Abstract

The present disclosure is directed to methods (e.g., in vitro methods) for detecting the presence of neutralizing antibodies to PTH or PTHrP analog in a sample. The in vitro method comprises the steps of obtaining a sample from a subject; contacting the sample with a cell; measuring cyclic adenosine monophosphate (cAMP) levels; and detecting the presence of neutralizing antibodies when cAMP levels are reduced relative to a negative control sample without neutralizing antibodies. An in vitro method of detecting the presence of neutralizing antibodies in a sample from a subject treated with Abaloparatide, is also provided. Further provided herein is a kit for carrying out the methods described herein comprising components required to carry out the obtaining, contacting, measuring and detecting steps and instructions for use. e monophosphate (cAMP) levels; and detecting the presence of neutralizing antibodies when cAMP levels are reduced relative to a negative control sample without neutralizing antibodies. An in vitro method of detecting the presence of neutralizing antibodies in a sample from a subject treated with Abaloparatide, is also provided. Further provided herein is a kit for carrying out the methods described herein comprising components required to carry out the obtaining, contacting, measuring and detecting steps and instructions for use.

Description

METHODS FOR DETECTING NEUTRALIZING ANTIBODIES TO PARATHYROID E (PTH) AND PARATHYROID HORMONE-RELATED PEPTIDE (PTHRP) ANALOG RELATED APPLICATIONS This application is a divisional of New Zealand Application No. 777212, filed 10 January 2020, is related to , and claims the benefit of priority to U.S. Provisional Application No. 62/791,267, filed on 11 January 2019. The contents of this priority application are hereby incorporated by reference .

BACKGROUND Despite the benefits, immunogenicity can arise from protein therapeutics such as Abaloparatide. Abaloparatide is a yroid hormone-related peptide (PTHrP) (1-34) analog which acts as a PTH1 receptor (PTH1R) t. Activation of the PTH1R activates the cyclic adenosine monophosphate (cAMP) signaling pathway in target cells, which results in ses in bone mineral density and bone mineral content. TYMLOS® (Abaloparatide) Injection Product Label (4/28/2017).

Of the patients receiving Abaloparatide for 18 months, 49% developed anti-Abaloparatide antibodies, 68% of which developed neutralizing antibodies to Abaloparatide. Of these patients tested for cross-reactivity, 2.3% and 0% developed reactivity to PTHrP and parathyroid e (PTH), respectively. Of the patients that developed reactivity to PTHrP, 43% developed neutralizing antibodies to PTHrP.

Detection of antibodies, such as neutralizing antibodies, can also be used to monitor the development of potential immunogenicity in ts treated with PTH and/or PTHrP analog. r, current detection methods suffer from a number of drawbacks including the level of sensitivity, the level of specificity as well as the lengthy duration of the assays. Sensitive and specific assays are needed to detect and monitor the presence of neutralizing dies to PTH and PTHrP.

SUMMARY The present disclosure is directed to methods (e.g., in vitro cell-based assays) for the detection of neutralizing antibodies (NAb) to PTH or PTHrP analog.

A first aspect es an in Vitro method for detecting the presence of neutralizing antibodies to PTH or PTHrP in a sample that includes the steps of: obtaining the sample from a subject; contacting the sample with a tion of cells or a cell and a predetermined amount of PTH or PTHrP, wherein the cell or cells comprise a receptor for PTH or PTHrP; measuring cyclic adenosine monophosphate (CAMP) levels; and detecting the presence of neutralizing antibodies when CAMP levels are reduced relative to a negative l sample without neutralizing dies. In some ments, the contacting step comprises incubating the cell or cells with the serum sample. In certain embodiments, the method further comprises preincubation of the serum sample with a predetermined amount of PTH or PTHrP prior to the contacting step. In a particular embodiment, the preincubation is for a period of at least 30 minutes. In certain embodiments, the predetermined amount of PTH or PTHrP is at least 100, 200, 300, 400, or 500 pg/mL. In a specific embodiment, the predetermined amount of PTH is about 500 pg/mL. In another specific embodiment, the predetermined amount of PTHrP analog is about 600 pg/mL.

In certain embodiments, the method further comprises incubation of the cell or cells with a cell permeable CAMP-specific phosphodiesterase inhibitor prior to the contacting step. In a particular embodiment, the CAMP-specific phosphodiesterase tor is 4-(3-Butoxy methoxybenzyl)—2—imidazolidinone.

In some embodiments, the measuring step is performed by a competitive immunoassay. In n ments, the competitive immunoassay is an electrochemiluminescent detection . In certain embodiments, the cell or cells are lysed prior to the measuring step. In certain embodiments, the measuring of CAMP levels is performed using the Mesoscale Discovery Multi- Array 96-well CAMP Plate.

In some embodiments, the cell or population of cells are rat epithelial cell line U1V1R-106.

In certain embodiments, the method r comprises serum-starving the U1VIR—106 cell or cells for a period of time prior to the contacting step. In certain embodiments, the period of time ranges from about 4 hours to about 48 hours, about 4 hours to about 24 hours, about 4 hours to about 16 hours, about 4 hours to about 12 hours, or about 6 hours to about 12 hours.

In some ments, the sample is a human sample. In certain embodiments, the human sample is a human serum sample. In certain ments, the sample is from the subject treated with a PTHrP analog. In a specific embodiment, the PTHrP analog is Abaloparatide. In another specific embodiment, the PTHrP analog is Teriparatide.

Another aspect provides a method of detecting the presence of neutralizing antibodies after aratide treatment, the method comprising the steps of: obtaining a serum sample from a subject d with Abaloparatide, contacting the serum sample with a cell or tion of cells, wherein the cell or cells comprise a receptor for PTH or PTHrP; measuring cyclic adenosine monophosphate (cAMP) levels; and detecting the presence of neutralizing antibodies when cAMP levels are d relative to a negative control sample without neutralizing dies. In certain embodiments, the method further comprises discontinuing treatment with Abaloparatide when neutralizing antibodies are detected in the serum sample.

In yet another aspect, the disclosure provides a kit for carrying out the methods described herein comprising components required to carry out the obtaining, contacting, measuring and detecting steps and instructions for use.

BRIEF PTION OF THE DRAWINGS is a graph depicting ary meter logistic fit of PTH e Dilutions in % PHS. is a graph depicting an exemplary PTHrP analog Dose Response Curve.

DETAILED DESCRIPTION A. Definitions The term “antibody” refers to a full antibody, e.g., an antibody comprising two heavy chains and two light chains, or to an antigen—binding fragment of a full antibody, and encompasses any ptide comprising an antigen—binding site (e. g., site binding to PTH or PTHrP analog Abaloparatide) regardless of the source, species of origin, method of production, and characteristics. As a non-limiting example, the term “antibody” es human, orangutan, mouse, rat, goat, sheep, and chicken dies. The term includes, but is not limited to, polyclonal, onal, mono-specific, poly-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies. The term “antibody” also es, but is not limited to, antibody fragments produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation, and those produced recombinantly. Among these fragments are Fab, Fab', F(ab')Zf FV, scFV, Fd, dAb, and other antibody fragments that retain the antigen-binding function. The antibody or fragment thereof may be any of the known antibody isotypes and their conformations, for example, IgA, IgG, IgD, IgE, IgM monomers, IgA dimers, IgA trimers, or IgM pentamers.

The term “neutralizing antibody”, as described herein refers to any antibody or fragment thereof capable of binding to and interfering with at least one biological activity of PTH or PTHrP analog for which the antibody is specific. The neutralizing dy may inhibit (i.e., eliminate or reduce) one or more ties of PTH or PTHrP analog without inhibiting other activities of PTH or PTHrP.

The terms “cut point” or “assay cut point”, refer to the level of response (e.g., reduction of CAMP levels or reduced ion of cAMP by PTH or PTHrP) at or below which a sample is defined to be negative and above which it is defined to be positive for lizing activity towards PTH or PTHrP analog. The cut point can be a fixed cut point or a variable one to account for the variable nature of cell based assays. Cut point is typically tied to a statistical measure of a control sample (e. g, negative control sample with no neutralizing antibodies for PTH or PTHrP analog).

For example, the statistical measure can be a standard deviation, a standard error, a mean, a , a median absolute ion, a fit parameter, or the like.

“Specificity”, as determined in the assays described herein, establishes that only the positive control shows a neutralizing response of sed cAMP induction and any other non-specific immunoglobulin doesn't show this response. “Selectivity” is the ability of the assay described herein to differentiate and detect the specific decrease in either cAMP levels or cAMP induction in the presence of other components present in the sample (interfering nces). Selectivity can vary between test samples due to the heterogeneous and polymorphic nature of s.

The term “subject” refers to an animal. In some embodiments, the animal is a mammal, including but not limited to a human, abovine, or a rodent. In other embodiments, the mammal is a human.

B. Assays for the measurement ofne utralizing antibodies against PTH and PTHrP analog The disclosure is based on the development of specific and selective assays for the ement of neutralizing antibodies against PTH and/or PTHrP analog. Neutralizing antibodies can be detected using various cell-based systems. In these cell-based assays, neutralizing antibodies inhibit the ability of the therapeutic agent to te a biological process in the target cell (e.g., induction of CAMP by PTH). Neutralizing antibodies can be detected using cell-based systems involving abiological functional readout, such as measuring levels or induction activity of a biomarker.

In an aspect, an in vitro method for detecting the presence of neutralizing dies to PTH or PTHrP in a sample is provided. The method includes: (i) obtaining a sample from a subject (ii) contacting the sample with a population of cells and an predetermined amount of PTH or PTHrP, wherein the cells comprise a receptor for PTH or PTHrP analog such as PTHlR; (iii) measuring cyclic adenosine monophosphate (CAMP) levels; and (iv.) ining the presence of neutralizing antibodies when CAMP levels are reduced relative to a negative control sample without neutralizing antibodies.

In some embodiments, the samples of this disclosure may be any bodily fluid capable of containing neutralizing antibodies t PTH or PTHrP analogs such as Abaloparatide.

Examples e, but are not d to, blood, serum, lymph, plasma, synovial fluid, cerebrospinal fluid, lachrymal fluid, biopsy or tissue sample, cell suspension, saliva, oral fluid, mucus, amniotic fluid, colostrums, mammary gland secretions, urine, sweat and tissue culture medium.

In some embodiments, the disclosure provides a method for the detection of neutralizing antibodies by measuring CAMP level by a competitive immunoassay. In some embodiments, the competitive immunoassay is an electrochemiluminescent ion method.

For example, the competitive immunoassay to validate a cell-based assayin post- usal women for the detection of neutralizing dies (NAb) to the PTH or PTHrP analog may be carried out as follows. The human serum sample, which may or may not contain potentially neutralizing dies, is first preincubated with predetermined amounts of PTH or PTHrP analog for at least 30 minutes. Serum starved rat epithelial cell UMR 106 cells are harvested by trypsinization and resuspended at 106 cells/mL in assay medium containing 133.5 uM of 4-(3-Butoxymethoxybenzyl)—2-imidazolidinone, acell—permeable cAMP-speciflc phosphodiesterase inhibitor. About forty microliters of the cell suspension are added to the cAMP assay plates that y have about twenty microliters of samples and/or controls. The cells in the cell suspension may or may not be lysed. If the cells are lysed, the cells may be lysed while still d to the culture plates. Lysis is carried out in presence of commonly known lysis buffers, preferably using lysis buffer while being incubated at room temperature for a time period of about 5 minutes to about 30 minutes, preferably about 10 minutes.

In some embodiments, the samples of this disclosure may be d at multiple dilutions to obtain an accurate quantitation of neutralizing ty present in the sample. In other embodiments, the samples of this disclosure may be assayed ted to obtain an accurate quantitation of neutralizing activity present in the sample. In some embodiments, the samples of this disclosure may also be diluted to avoid erence from non-specific background components of the samples. For example, proteins found at high concentrations in the serum may, in some circumstances, non-specifically interact with ents of the assay and reduce the sensitivity of the assay. Sample dilution may reduce or eliminate non-specific binding and thereby increase the signal-to—noise ratio of the assay.

In some embodiments, the samples of this disclosure may be assayed at on factors such as, for example, 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:80, 1:100, 1:32, 1:640, 1:500, 121000, 121280, 122560 or 1:5000. In other embodiments, the samples of the disclosure may be assayed at a further serial dilution of the d sample.

After a minimum of 30 minutes at room temperature with shaking, TAG cAMP detection reagent cale ry, MSD Multi—Array 96-well CAMP Kit) diluted 1:200 in MSD Lysis Buffer is added to the assay plates. Reagents were used as provided in kit and prepared as per manufacturer's instructions. Plates are incubated at room temperature for an additional 1 to 2 hours with shaking. One hundred microliters of 2X MSD Read Buffer T are then added to the plates and plates are read immediately on an MSD 6000 or S6000 Sector Imager.

The drug-spike assay involved treatment of the rat epithelial cell line IHVIR-106 in the ce of human serum which may or may not contain neutralizing dies (NAb), followed by measurement of the ability of the predetermined amount of PTH or PTHrP analog to induce ar cyclic adenosine monophosphate (CAMP) by itive immunoassay. In certain embodiments, the serum sample is preincubated with a predetermined amount of PTH or PTHrP analog prior to the contacting step. While not being bound by theory, any PTH or PTHrP analog neutralizing antibodies present in the sample will interact with and neutralize PTH or PTHrP analog and neutralize it during the preincubation step. Thus, when the mixture of the preincubated serum and PTH or PTHrP analog is incubated with the population of cells, neutralized PTH or PTHrP analog will not induce PTHlR receptor and hence will result in the reduction in the levels of CAMP.

In some embodiments, the predetermined amount or tration of PTH or PTHrP analog is at least 100, 200, 300, 400, or 500 pg/mL. In some embodiments, the predetermined amount of PTH is about 500 pg/mL. In some embodiments, the predetermined amount of PTHrP analog is about 600 pg/mL. In some embodiments, the samples were ted in the presence of a final concentration of 500 pg/mL of PTH or PTHrP analog. In other embodiments, the samples were evaluated in the presence of a final concentration of at least 100 pg/mL, at least 200 pg/mL, at least 300 pg/mL, at least 400 pg/mL, at least 500 pg/mL or at least 600 pg/mL of PTH or PTHrP analog.

In some embodiments, the s were evaluated in the presence of a final concentration of 500 pg/mL of PTH. In some embodiments, the samples were evaluated in the presence of a final concentration of 600 pg/mL of PTHrP analog.

Figures 1 and 2 show a curve fit from a single qualification run and is representative of the PTH and PTHrP dose response observed, tively. The dotted lines te the EC2O and EC3O of the PTH and PTHrP responses in the presence of 25% pooled human serum as olated from the curve fit, respectively. As represented in Figures 1 and 2 the dose response curve and neutralization of PTH or PTHrP induced cAMP induction represents the robust endpoint.

The cAMP levels, can be measured using any method known in the art. For example, the CAMP can be measured using an ELISA assay to detect PTHlR levels or activity. In some ments, the measuring CAMP level is performed using the Mesoscale Discovery Multi—Array 96-well cAMP Plate.

The present methods can determine if PTH or PTHrP analog neutralizing antibodies are or are not present in the serum sample in an amount ent to significantly neutralize PTH or PTHrP analog. In certain embodiments, an assay cut point can be calculated to determine when PTH or PTHrP analog neutralizing antibodies are present in the sample. The method may further comprise determining an assay cut point based on a negative control of pooled human serum, correlating the assay cut point with the presence of neutralizing antibodies, and comparing the amount of cAMP reduction in the population of cells to the assay cut point. For example, when a measured amount of cAMP reduction in the sample is less than of the assay cut point, then the serum sample does not contain appreciable quantities of the neutralizing antibodies and when a detected amount of cAMP reduction in the sample is higher than of the assay cut point, then the serum sample contains appreciable ties of the neutralizing antibodies.

In some embodiments, the responses induced by positive and negative control samples are determined to ensure that the assay is oning ly. Negative controls are typically pooled human serum samples from a subject that has not been d to the PTH and/or PTHrP analogs.

In some instances, the negative control samples may be pooled serum samples from untreated subjects.

In some embodiments, the positive controls include serum samples from subjects d with a PTH and/or PTHrP analogs. In other embodiments, the control includes serum samples from subjects spiked with a surrogate neutralizing antibody (SPC). In some embodiments, the serum samples are pooled. In some embodiments, the serum samples are pooled from individual disease state serum samples from post-menopausal women. In other embodiments, the serum is human serum obtained from individual disease state serum samples from post-menopausal women.

Additional positive controls may include frozen samples of pooled human serum with different dilutions of SPC, for example, SPC dilutions of 1:120, 1:240, 1:500, and 1:800 for the high ve control (HPC), mid positive control (MPC), low positive control-l (LPCl) and low ve control-2 (PC2), respectively.

In some embodiments, the cell or population of cells of this sure may be any cells that express PTHlR and allows the induction of cAMP signaling, resulting in activation of the PTHlR and the CAMP signaling y. In some embodiments, the assays of this disclosure may use one cell or a population of cells. In some embodiments, the cell or population of cells is rat epithelial cell line UMR-106.

Cells are grown at any y appropriate for normal cell growth when used in the assays of this disclosure. The number of cells used to achieve an riate density is determined in part by the size and e area of the plate used in the assay. Cells may be used in the assay at any density. In some embodiments, the cells may be used in the assays at the following cell densities: at least 10% confluent, at least 25% confluent, at least 50% confluent, at least 80% confluent, at least 900 confluent, or at least 99% confluent.

In certain embodiments, the method ses serum starving the UMR-106 cell or cells for a period of time prior to contacting them with the sample duiing the contacting step. The cells may be serum starved for a period of time ranging from about 4 hours to about 48 hours, about 4 hours to about 24 hours, about 4 hours to about 16 hours, about 4 hours to about 12 hours, or about 6 hours to about 12 hours.

In some embodiments, the sample is a human sample. In some embodiments, the sample is a human serum sample.

In some embodiments, the sample is from the subject treated with a PTHrP analog. In some embodiments, the PTHrP analog is Abaloparatide. In other ments, the PTHrP analog is Teriparatide.

Detection of antibodies, such as neutralizing antibodies, can also be used to monitor the development of potential immunogenicity in patients treated with PTH or PTHrP analog. For e, neutralizing antibodies in patients treated with PTHrP analog for osteoporosis could be important in detecting and minimizing the s of adverse reactions, optimizing drug dosage and efficacy of treatment. In an aspect, described herein is a method for detecting the presence of neutralizing dies after Abaloparatide treatment. The method comprises obtaining a sample (e.g., pooled or individual human serum sample) from a subject treated with Abaloparatide, contacting the sample with a cell or population of cells, wherein the cells comprise a receptor for PTH or PTHrP, ing cyclic adenosine monophosphate (CAMP) levels, and detecting the presence of neutralizing antibodies when CAMP levels are reduced relative to a negative control sample t neutralizing dies.

The assay described herein provides a convenient and reliable alternative to actual clinical trials that may quickly ascertain r adverse irnmunogeneic events are likely based on ial anti-PTH or anti—PTHrP analog antibody production. In some embodiments, the methods of the disclosure can be used to diagnose the onset of adverse irnmunogenic events post- Abaloparatide treatment. In some embodiments of the , the treatment with Abaloparatide is discontinued when neutralizing antibodies are detected in the serum sample. In other embodiments, when the serum sample does not contain lizing antibodies, the method further comprises centinunig the treatment of the subject with Abaloparatide. In yet another ments of the method, the dosage of Abaloparatide is varied (decreased or increased) when neutralizing dies are detected in the serum sample.

In some embodiments, the samples may also have tested positive in a different primary neutralizing antibody assay and are now being ted to the assay as a confirmatory assay for the presence of neutralizing antibodies. In some other embodiments, the samples are prescreened with an immunoassay, such as an ELISA assay. In yet other embodiments, the samples are prescreenedwith a cell-based assay, such as, for example, the downregulation of a reporter gene.

The reporter gene may be the luciferase gene. The luciferase gene may be linked to a promoter of a gene encoding PTHlR.

In some embodiments, antibody concentrations of anti-PTH or anti-PTHrP analog are determined any one of or combination of immunodiagnostic methods based on detection of complex antigen-antibody, including, for example, enzyme-linked immunosorbent assay (ELISA), receptor binding assay, radio-immunoprecipitation, biosensor-based assay, immunofluorescence, Western blot, irnmunodiffusion, and immunoelectrophoresis. In a particular embodiment, antibody concentrations of anti-PTH or anti-PTHrP analog are ined by ELISA using polyclonal or onal antibodies of anti-PTH or anti-PTHrP analog, as standards.

C. Kits The reagents described herein may be provided in kit . A kit may include, for ce, some or all of the components ary to carry out the assays described herein. For instance, the kit may comprise control compositions (e.g., control human serum samples Without neutralizing antibodies t PTH or PTHrP analog), test cells (e.g., 6 cells affixed to a solid support, and / or frozen), buffers, labeling reagents (e.g., labeled antibodies such as goat anti- mouse IgG , streptavidin-HRP conjugates, allophycocyanin, B- phycoerythrin, R- phycoerythrin, peroxidase, and / or other detectable labels), instructions to carry out the assay and any other necessary or useful components. The components of the kit may be provided in any suitable form, including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit may also include a solid support containing one or more test cells (e.g., microorganisms) in any suitable form. The kits may also include other reagents and / or instructions for carrying out assays such as, for example, competitive inhibition assay, MSD cAMP assay, flow cytometric is, ELISA, irnmunoblotting (e.g., n blot), in situ detection, immunocytochemistry, immunhistochemistry, and / or visualization of data. Kits may also include components such as ners (e. g., tubes) and / or slides pre- formatted to containing l samples and / or reagents with additional space (e. g., tubes, slides and / or space on a slide) for experimental samples. The kit may also comprise one or both of an apparatus for handling and/or storing the sample obtained from the dual and an apparatus for obtaining the sample from the subject (i.e., a needle, lancet, and collection tube or vessel). Other embodiments are also provided as would be understood by one of ordinary skill in the art.

EXAMPLES Example 1. Anti-PTH Assay Validation and ation A study was undertaken to validate a cell-based assay in enopausal women for the detection of neutralizing antibodies (NAb) to the PTH peptide. The assay involved treatment of the rat epithelial cell line 6 in the presence of human serum which may or may not contain neutralizing antibodies (NAb), followed by measurement of the ability of PTH to induce cellular cyclic adenosine monophosphate (CAMP) by competitive immunoassay. The ion of cAMP was performed using a competitive electrochemiluminescent assay, where lizing antibodies to PTH resulted in decreased induction of CAMP by PTH and an increased assay signal.

Materials Reagents used for PTH assay validation are shown in Table 1 and Table 2, below.

Table 1 z*l;(s¥i!~‘mnm UM RR} (35 Rat Osteosamom;W orking Ciel? AT’CC- PKN CRL? 6113} , Lm i: I"? ‘ 6i465(}75, , ”WML “ a:$5 Bank (WCB); Passagefiéxfi l—‘amthyroiai Homx‘me Pept.ide{P’l‘I~{} (Vi-.34} Pam-anipflflmnmcwtitaifiEN {355- %5_(ES-13was; {Human} 82» AME-2TH 5““wa i.{1“34} {Human PhD-enixPhammcwticaiS {Sir-0554}??? g '1 5514 spccatxcn'y 3 29.84 pge'mlj“ 2‘ * High antibodypofiitivc CBHtIIfliéK EMT), me‘édfiflmAnfi—PTH Amfmfi ,, _ _ : 1:135 Ennis-11,1250} ngme nifimfigfiym Labs RPQML‘Y “W593«n Mid stairway positive 0011:1111le MPG}? I’PsEamé§m§“1\léii*PT3‘i i‘tntibmfi: W23Mas, i (fiNNG’S;' 1:250 Dilution, 319:4 ngfml; 3* BmfigilyHK-Lahs ' Low an ifhodv positive com‘mHSlX LPCl')‘ P33133135 fiomAminPTHAlli-EMF AM; “5thM .. . ‘ ' " .. ~ if 3' 1:3{30 Bxlntmnfififi t . . .. _ W Si! BiuAgilym LShS Low dv positive control {4X LPCEL PreparedfmmAmi—PTH Antmody M V. ‘ _ " ‘ " - -- - ’ RPLM: ‘lHNNGE3‘ . 1:400 3321mm, my} ngxmt. . nthAgslytxx Labs ‘3 a Mes-10382111: Discmeryt‘MSfl}Kil MSD MultinArray ghnwell 3A MP Hate, t ,r _ . M 11kar. .. I. common-ant N [{lflflTDB *Mue meat I};apart-age amnfmm rm the WC}? vé'ak'ware Inhaler!immrremfy and shank!mad P4 as ‘igmssags? c1! falmw ** Concentration determined by quantitative ELISA Table 2 iptinn S-ourcei‘fefldflr‘ Catalan-g Number §§§§§:10 S Phosphate ed Saline GilmofiLifi: logies 14 l 90 'l‘i" LE EX tees; whoa-"Life 3‘20hmlo :ies 13004 0.4% Trip-am Blue Solution Gibmeife. Technoiogies 15250 Dulhecco‘s Modified Eagle Medium A. .. i . , . . , t P . . r,’bm we “dumb?”_.,.- at “Q3 65*a, s.

{DMEM’}, m, high ilucose Fetal Bovine Serum {'FBS)» Silica-"Life ”fechnologies 15000 Pauli: illinvStreptomyc in {Penffitrep}: 10,000 {Jibwiil'e logiesT‘ - \ .:‘ t; -\ gm 1:140‘ 5";22l un1its,-"111LPe11; 10,000 1: Km}; Stre _ 200 mM L-Jlttlafi'lifie ‘L- rltit} Gibwfiife Technologies 25030—08} Phenol Reanree DMEMi 1X, tz‘ xii—gamma? Gibcchife Technologies El 063 % Bovine Serum Albumin {BS5A} Sigma $95746 3f.) mg R0 2:20—17:24 (MW; gi‘stss} 2AM?— RAJ} Magma '3 Toms 8415 specific phosphodiesterase inhibitor Dimemyl Sutfoxide {BMSD} Sigma '52-'88 3:52:12: Lulmn: Flasks {_ 5 u m- v; 1th vented Coming 4.306“ Sterile 96~deep well Plate with lid MP Emmedicgals | ?6~223-05 MSD Milli-ivArray Qfivwel'ch'MP' Kit components {not inc Ending plates} — TAG Labeled MM? Mean-Scale Dimmer}: (15359} — RBEAE-B — Read Buffer T, with surfac $2111“?in — R§2TC—2 ~ MM? Lysis Buffer a Rfifififiw l — Blocker A ' R93BA—4 To generate controls, pooled human serum (PHS), pooled from individual disease state serum s from post-menopausal women (placebo controls); and individual e state serum samples from post-menopausal women (placebo controls) were sourced from a clinical study.

Frozen controls included: 0 Frozen Negative control (NC) = Pooled Human Serum (PHS) 0 Frozen 4X high positive control (4X HPC) = Human serum pool spiked with 1:175 dilution of surrogate antibody positive l (SPC) 0 Frozen 4X mid ve control (4X MPC) = Human serum pool spiked with 1:250 dilution of SPC 0 Frozen 4X low positive control 1 (4X LPCl) = human serum pool spiked with 1:300 dilution of SPC 0 Frozen 4X low positive control 2 (4X LPC2) = human serum pool spiked with 1:400 dilution of SPC Each frozen control is diluted 1:4 for final assay SPC dilutions of 1:700, 1:1000, 1:1200 and 1:1600 for the high positive control (HPC), mid positive control (MPC), low positive control-1 (LPCl) and low positive control-2 (PC2), respectively.

IflVIR-106 cells are maintained in growth medium (UMR-GM, Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum, 1% Pen-Strep (10k units Penicillin- 10k ug/mL Streptomycin) and 1% L-glutamine) in 75-150 cm2 tissue culture flasks until ready for use. Cells are split at a ratio between 1:4 and 1:20 when growth reaches 2 70% confluence for routing culture maintenance. Prior to initiating an assay, cells are plated in sub- culturing flasks (25 cm2 to 150 cm2) at a density of 106 5 cm2 (example 5e6 cells for T-75).

The following day, flasks are starved with assay medium (UMR—Am, Phenol Red-Free DMEM, containing 1% Bovine Serum Albumin). The following day validation samples and antibody controls are pre-incubated with PTH peptide for a minimum of 30 minutes. The validation samples and controls are adjusted such that the final assay concentration of serum is equal to the assay minimum required dilution of 1:4. Twenty microliters of s and/or controls are placed on a MSD cAMP assay plate. The d UMR-106 cells are harvested by trypsinization and resuspended at 106 cells/mL in assay medium ning 133.5 uM of utoxy methoxybenzyl)—2-imidazolidinone, acell-permeable CAMP-specific phosphodiesterase inhibitor (R0 20-1724, MW 278.35, R&D Systems/Tocris Catalog 0415). Forty microliters of the cell suspension are added to the CAMP assay plate. After a minimum of 30 s at room temperature with shaking, TAG CAMP detection reagent (Mesoscale Delivery, MSD Multi-Array 96-well cAMP Kit) diluted 1:200 in MSD Lysis Buffer is added to the assay plates. Plates are incubated at room ature for an additional 1 to 2 hours with g. One hundred iters of 2X MSD Read Buffer T are then added to the plates and plates are read immediately on an MSD 6000 or S6000 Sector Imager.

Results PTH Drug Concentration Relative light units (RLU) were read from assay plates on a Meso Scale Discovery (MSD) Sector 6000 electrochemiluminescent reader. Data were exported from the MSD database to permit further analysis. Calculations for establishing the assay cut point, including removal of outliers, were performed in JMP® software v12.01 (SAS, Cary, NC). Outlier ination proceeded stepwise. During stepwise outlier mination, the standard ration of the JMP whisker and box plot was used to declare outliers. cally, replicates outside of whiskers (the interquartile range of the replicates (IQR) plus or minus 1.5 times the IQR) were determined to be outliers.

The assay cut point was established using 64 individual diseased-state placebo control serum samples ed by the sponsor. Samples were run in groups of 32, as singlets, three times within a total of six runs. The six runs were performed by three analysts over five days and produced 192 data points. A minimum of eight replicates of the negative control (NC) was included on each plate. All data points were normalized as specified above.

The ing equation was d: RLU ofSample Normalized Value {NEHmRLU} Miran RLU of Negative. Control Standard Deviation 1‘: {r g; _, Standard} (lm‘isnkm of individual xii-easuremmts ‘31: {3V _— >400‘_ .

Mean of men‘s-1:11:81 measurements Dbserved % Recovery ==m X' 3.08 where % CV is percent coefficient of variance and RLU is the Relative Light Units A drug final assay concentration of 500 pg/mL was established during the development and qualification of the assay in human serum and was obtained by spiking into the assay at a 12X concentration of 6 ng/mL. Figure 1 shows a curve fit from a single cation run and is representative of onal runs performed during qualification. The dotted lines indicate the EC20 and EC30 of the PTH response in the presence of 25% pooled human serum as interpolated from the curve fit. The d concentrations interpolated were 436 and 728 pg/mL, respectively for the EC30 and the EC20.

Statistical Method All statistical analyses were completed using JMP Statistical Discovery Software (Version 12.01; SAS Institute, Inc, Cary, NC, USA)). Statistical methods used for the analyses are consistent with procedures recommended by Shankar et al., 2008.

The study design for unspiked samples was two groups of samples measured over three runs, for a total of six runs. The design allowed for evaluation of mean effect due to group. The design also allowed for the estimation of random variation attributable to samples nested in groups and run number.

A linear mixed effects analysis ofvariance ) model was used to investigate systematic (fixed) and random sources of variation in reported count values for disease state samples with no inhibitor present. Statistical analyses were performed on normalized results per plate by dividing the mean response of the sample by the mean of the NC plate. Group was defined as fixed effects in the ANOVA model, with least squares means compared at the 0.05 significance level to assess systematic differences in the mean response among levels of these factors. Random effects were defined in the model for sample, and group was nested within run and the residual. bution of the sample best linear unbiased predictor (BLUP) values was then examined to identify samples as biological statistical outliers using the r box-plot in JMP. The distribution ofANOVA conditional residual values was evaluated to identify “analytic” statistical outliers using again the outlier box-plot procedure within JMP’s bution platform. All normalized result values for samples identified by this criterion were d and the statistical analysis was repeated until no r outliers were identified.

The linear mixed s ANOVA of the normalized values identified 25 values as outliers.

Seven values were fied as ‘analytic’ outliers. Six samples were fied as a ‘biologic’ statistical outlier, which excluded 18 results (6 samples x 3 runs = 18). All statistical outliers were excluded from the cut point assessment g 167 values for analysis.

The linear mixed effects ANOVA e data revealed no statistically significant difference between sample groups (p-value = 0.2082). The difference between samples accounted for 14.6% of the total variability. Most of the variability in the method was associated with the analytical components (47.2% due to run and 38.2% due to residual variation).

Sensitivity To evaluate sensitivity, an ultrahigh positive control sample was prepared by g the SPC [29.84 ug/mL] into 25% PHS at a 1:20 dilution. The 1:20 dilution was then serially diluted 2-fold, resulting in eight total dilutions of the SPC of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280 and 12560, this corresponds to a tration range of 1492 ng/mL to 12 ng/mL in neat matrix. The samples were evaluated at the 1:4 MRD and in the presence of a final concentration of 500 pg/mL of PTH. The sensitivity was determined from four independent runs as the lowest concentration of the antibody dilution curve that was consistently detected as positive (above the cut point) based on the mean normalized value. Assay sensitivity data are shown in Table 3.

Table 3: Antibody Sensitivity Runs? 15 Tr- -:iI.“3 " . $3 =1 E?“ aim-fig» fitter»; tfim’iij’w 4FFish-{’3I???f}“1‘33?{I'fli‘tliiigflffi‘fighzfiigléffg{lflffi'fi £535er MRiquafifiafti iiairzggrs‘a’mrmx‘f m‘ {yum R‘u‘m..§' 3 \ s The SPC concentration range evaluated only achieved negative scoring (below the cut point) in one of four independent runs (Run #2) for the individual data points and the four parameterlogistic regression (4PL) fitted response. The three other runs ed ve (above the cut point) even at the lowest SPC concentration evaluated (12 . As shown in Table 3, the normRLU for each SPC concentration were averaged across runs and ed to the assay cut point, and negative scoring was not achieved. Therefore, in three of four runs, and by averaging all four runs, the sensitivity of the assay is S 12 ng/mL.

Spe cificity by Non-Spe cific Immunoglobulin Specificity was assessed by evaluating the reactivity of commercial human IgGin the assay.

Three human IgG concentrations 10, 1.0 and 0.1 ug/mL were spiked into neat human serum pool (PHS) and evaluated in the presence of a final concentration of 500 pg/mL PTH. A11 specificity samples were below the assay cut point of 1.234 and were ered negative. Both the 1.0 and 0.1 ug/mL samples were within the acceptance criteria of30% ofthe negative control, the IgG 10 ug/mL was 60% (40% less than negative control); however, there was no impact as the sample tested negative in the assay and a ve result qualitatively demonstrates that the sample had no activity in the assay. The results are summarized in Table 4.

Table 4: Specificity Res ults {Emiflifiitm Meat: iz‘ {git H} gtgf‘i’flL Mgggfiyg {git} 1’35 ugml Igfi (ll pgfimL Negative (imamflflfi(I) Ht: :2:p.i’c.$‘£=n i!{if a. r33308?: Rua: 4"» .

Specificity by Drug Tolerance Increasing the PTH drug concentration resulted in a rapid decrease in signal in the presence of the SPC. Because the assay cut point was established by a cellular se that was dependent upon a fixed concentration of PTH, it is expected that the assay would have limited drug tolerance.

To evaluate this limit, the HPC,LPC1 and LPC2 were treated with 750, 1000 or 2000 pg/mL of PTH and compared to samples treated with nominal PTH at 500 pg/mL in a single run.

The signal response decreased for each control with increased drug concentration for each level of controls, except for LPC2 1.5x, which has no impact on the interpretation of the results. The results are shown in Table 10. The controls remained positive in the presence of sing concentrations of PTH up to 1000 pg/mL. The results are in Table 5.

Table 5: Drug Tolerance Results Sample Name Drug Assay Mean NormRLU CV (% ) Result Concentration ControlHPC nominal Drug 500 pg/mL 4.106 HPC 1.5x l Drug 750 pg/mL 3.755 HPC 2x l Drug 1000 pgme 2.772 HPC 4x nominal Drug 2000 pgme 1.008 ControlLPCl nominalDrug 500 pgi’mL 2.916 Datapresentedarefiom Run 8 ivity Selectivity was assessed with atotal of 10 individual placebo human serum samples. Each sample was tested unspiked as well as spiked with the anti-PTH, SPC stock at the HPC and LPC1 dilution levels of 1:175 (170.1 ng/mL) and 1:300 (99.5 ng/mL), respectively, and evaluated in the assay with controls at the MRD of 1:4. Samples were positive for the NAb when spiked with concentrations of antibody equal to the level of the HPC and LPC1 (Table 11). One sample, Sample , was also positive unspiked with anormalized mean of 1.863. Reference controls (NC, HPC, LPC1 and LPC2) were run on each assay plate as assay controls.

Example 2. Anti-PTHrP Assay Validation and Calibration A study was undertaken to validate a cell-based assay in post-menopausal women for the detection of neutralizing antibodies (NAb) to the PTHrP peptide. The assay involved treatment of the rat epithelial cell line UMR—106 in the presence of human serum which may or may not contain neutralizing antibodies (NAb), followed by measurement of the ability of PTHrP to induce cellular cyclic adenosine monophosphate (cAlVIP) by competitive immunoassay. The ion of CAMP was performed using a itive electrochemiluminescent assay, where neutralizing antibodies to PTHrP resulted in decreased ion of CAMP by PTHrP and an sed assay signal.

Materials ts used for PTH assay validation are shown in Table 6 and Table 7, below.

Table 6.

L Bzatrhfigat Ra: 13mt gs, Snares:.

Number, mam—wt3 Rat; 0.1:‘ “mm m“i3 12- wM mgk‘ tun‘ AT‘CC MS} canteen Lens; 61%50?§ RPIEMMESWQE Bank {WCB}; rlbt #4 Parathymid l-Iiormnnemiselamd Hots-in Phaenin I‘lmrmac-eutienie PEN- {156-414 {PTHrP} {E 4343 f'ii-lumam R31; Mme»; AaitbFTHr'E‘ Arsfibudy' {SPCJ {LESSH‘mmm E‘hmnix Illmrtnnemtimis ill—GlSfi-Oé {H TEE-{H Rat, Minnie} {SSE’S ILA $31131,” High antibody prisitive ctmtmi {AlfiKl-ll‘iii}, f‘iregimd Emm Ami-{Fif‘ifirfl‘ Anztihmly Ls)! RP l‘fléaylfifiLMfil EEG} Damian 2.38 L! 7.53311 EH?3£§—iat BinAEfiyfix Lab‘s Mid, antihbdv‘ flesifive c-mm‘fli fixMPC}. Renamed fmm i‘THfl" Amibndv Lat R} ”“5 IéDLMt.. . .. we Qiiution '1 .t3 ngf'mL 313543: BieAgiiyfix Lab's Low antibodv aside“. metro} TU‘L‘IE}. ‘- L t "5» w 3 ; 3untied}.~, -. a, Let V _ Rnn‘PTHrPH flgsmL .. f P (k owimi item RPlTMayttiDilt-im t Erik-‘53 0.25 ttgr‘mL m 17334155: iyflx Labs Lew aniihedy positive centre} {Li’fl‘lfi Premred from F'THIP Antibody Let R? l ”ER-{aiyl éflLMfi-i ngs’mi {a am}; am 51-54th; in Thai at nimgngtix. Lab-s Shamanic 3&2me {MSD} Rit MS!) Mam-Army 96-w‘fiiil EAMP‘ Plate. ZOSQFM? em N7 PKN Ki 50F???) *Nam that the passage numbers on rim WEB winds were initiated inmart‘evtiir and .st‘mrufsf mad F4 m:- “pamaxs * n! thaw " * * tration determined by quantitative ELISA Table 7.

.Bescriptian Smarty?!radar (faxing: Ni;miter fifgg" 1* Phflwhm‘g Bum?“ Sam {'iihmisfiLiife 'zfechmtmgim 1.4190 Try LE Ex .ress Gibmiiife Technologies 12604 04% Trypzm Blue Sohttion GihcofLife ’fec‘hnologies 13250 DuEEemo‘s Modified Eagle Medium (”1“- m‘? “L‘mt‘hhm.. . . . . . .. h y ‘ 3' ‘." ‘ ’ :», 9 art,“ 3 191* Gin: _. 3;.

BMEM}. 3.x. hiihwgiuwse Fetal Bovine Serum (F38) Gibmflife Technologies 1,6000 11iI-1—Streptmnycin {fi’em’Stzrep}: 10,000 (libw’hfi‘ Etchmingm‘a, :2 3,: ., ,_ f5; LNGq _ 33i units-’miEen; 10,0100 :gs‘anStre 201} mM L-zzlutmnine {Light} fiihcofLiefs: Technologies 25030081 Phenol Red.~§x‘ee‘{}h-1EM, 1X, iucose GibcofLife ‘I'ecimolo .‘ies 31063 313.3% Bovine SerumAlbumin {BSA} gas-5%» mg: R0 2?” ' 1 ~' ”2’l! jig“ 1‘?‘19.,(2?8 .35 .1 } ("a“)us - t .. . _ , R831) fistgms. specific odiestxrrasc inhibitor. r" Foam: 041.5 Dhnethyl Squoxide {Iii-MSG} Sigma 82433 :Sszug Lulmic Flasks"r1“ ‘- {"FD3 cm.3 M2in 1th $13de7: Coming, 5130641,. ., Sterile 331} well Plate w ill: lid M}? Biomedimis '76—'33 3415' MSD-Mufti—Army Qfi—xvelicAMP Kit components {' not inc Ending plates} - TAG d cAMP‘ Mes-oiéicaie ery {MSD} « 11311-11393 — Read Buffer T, w ith surfac 1am {4X} cAMP Lygis Bulk? - 116053I. ~ 1310:: ker A — RQSBAA To generate controls, pooled human serum (PHS), pooled from individual disease state serum samples from post-menopausal women (placebo controls); and individual disease state serum samples from post-menopausal women (placebo controls).

Frozen controls included: 0 Frozen Negative control (NC) = Pooled Human Serum (PHS) 0 Frozen 4X high positive control (4X HPC) = Human serum pool spiked with 2.30 ug/mL of surrogate antibody positive control (SPC) 0 Frozen 4X mid positive control (4X MPC) = Human serum pool spiked with 1.15 ug/mL of SPC 0 Frozen 4X low positive control 1 (4X LPC1) = human serum pool spiked with 0.55 ug/mL of SPC 0 Frozen 4X low positive control 2 (4X LPC2) = human serum pool spiked with 0.34 ug/mL of SPC Each frozen control is diluted 1:4 for final assay SPC dilutions of 1:120, 1:240, 1:500, and 1:800 for the high positive l (HPC), mid positive control (MPC), low positive control-1 (LPC1) and low ve control-2 (PC2), respectively.

Methods UMR-106 cells are maintained in growth medium (UMR—GM, Dulbecco’s d Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum, 1% Pen-Strep (10k units Penicillin— 10k ug/mL Streptomycin) and 1% L-glutamine) in 75-150 cm2 tissue culture flasks until ready for use.

Cells are split at a ratio between 1:4 and 1:20 when growth reachesZ 70% confluence for routing culture maintenance. Prior to initiating an assay, cells are plated in lturing flasks (25 cm2 to 150 cm2) ata density of 106 cells/5 cm2 (example 5e6 cells for T-75). The following day, flasks are starved with assay medium (UMR—Am, Phenol Red—Free DMEM, containing 1% Bovine Serum Albumin). The following day validation samples and antibody controls are pre-incubated with PTHrP for a m of 30 minutes. The validation samples and controls are adjusted such that the final assay concentration of serum is equal to the assay minimum required dilution of 1:4. Twenty microliters of s and/or controls are placed on a MSD cAMP assay plate. The starved UTVJR- 106 cells are harvested by trypsinization and resuspended at 106 cells/mL in assay medium containing 133.5 uM of utoxymethoxybenzy1)—2—imidazolidinone, a cell-permeable cAMP-specific phosphodiesterase inhibitor (RO 20-1724, MW 278.35, R&D Systems/Tocris Catalog 0415). Forty microliters of the cell suspension are added to the cAMP assay plate. After a minimum of 30 minutes at room temperature with shaking, TAG cAMP detection reagent (Mesoscale Delivery, MSD Multi—Array 96-well cAMP Kit) d 1:200 in MSD Lysis Buffer is added to the assay plates. Plates are ted at room temperature for an additional 1 to 2 hours with shaking. One hundred microliters of 2X MSD Read Buffer T are then added to the plates and plates are read immediately on an MSD 6000 or S6000 Sector Imager.

Results PTHrP Drug tration Relative light units (RLU) were read from assay plates on a Meso Scale Discovery (MSD) Sector 6000 electrochemiluminescent reader. Data were exported from the MSD database to permit further analysis. Calculations for establishing the assay cut point, including l of outliers, was performed in JMP® software v12.01 (SAS, Cary, NC). Outlier determination proceeded stepwise. During stepwise outlier mination, the standard configuration of the IMP whisker and box plot was used to declare rs. Specifically, replicates outside of whiskers (the interquartile range of the replicates (IQR) plus or minus 1.5 times the IQR) were determined to be outliers.

A drug spike assay concentration of 600 pg/mL was established during the development and qualification of the assay in human serum. Figure 2 shows a curve fit from a single qualification run and is representative of the PTHrP dose response ed. The dotted lines indicate the EC20 and EC30 ofthe PTHrP response. The rounded concentrations interpolated were 436 and 728 pg/mL, respectively for the EC30 and the EC20.

Sensitivity To evaluate ivity, an ultrahigh positive control sample was ed by spiking the SPC [68.98 ug/mL] into matrix at a 1:16 dilution (4X) then serially diluting 2-fold, resulting in concentrations of the SPC of 4311, 2156, 1078, 539, 270, 135, 67 and 34 ng/mL in the assay. The dilution samples were evaluated with the assay positive controls at the MRD of 1:4 and in the presence of 600 pg/mL of PTHrP. The ivity was determined as the lowest concentration of the antibody dilution curve that was tently detected as positive based on the mean RLU value, after adjusting for the MRD. Results are shown in Table 8. Antibody Sensitivity for this assay was determined to be an antibody dilution of 1:64 and both of the prepared low positive controls are likely to be below detection.

Table 8: Antibody Sensitivity Nam: (In: mint: 'Pimg (its! aim:- c2}; .M‘RU SH: $E’C (Tmmmsmiiim Mnan REL) Mensa RLU kiwi: RU; {Mating ng-‘mi.. #13115 124345 Positive § 38425 2mm thiw 848.515 E 32.3le Firm? Seenxii‘ivigv was {firms-whites? issued in: like assay MR3 tgf‘é-jbi'xi Raining {ma {mind rm the may: of; rizgnr'tmlzas remiss-‘9 in first {tai‘msptmding -fighter tféifpflmzfisi skim-w, Spe cificity by Non-Spe cific Immunoglobulin Specificity was assessed by evaluating the reactivity of commercial human IgGin the assay.

Three human IgG trations 10, 1.0 and 0.1 pg/mL were spiked into neat human serum pool (PHS) and evaluated in the ce of a final concentration of 500 pg/mL PTH. All specificity samples were below the assay cut point of 1.234 and were considered negative. Both the 1.0 and 0.1 pg/mL samples were within the acceptance ia of30% ofthe negative control, the IgG 10 ug/mL was 60 % (40% less than negative control); however, there was no impact as the sample tested ve in the assay and a negative result qualitatively demonstrates that the sample had no activity in the assay. The results are summarized in Table 4.

Specificity was performed by evaluating the reactivity of commercial human IgG in the assay. Three human IgG concentrations; 10, 1.0 and 0.1 pg/mL were spiked into human serum pool (NC) and ted in the presence of 600 pg/mL PTHrP. Human serum pool (NC) was spiked with 600 pg/mL PTHrP, but no IgG, to serve as the baseline control for this assay. Specificity samples with the highest and lowest IgG content were below the plate cut point of 5378.7 and were considered negative. The results are summarized in Table 9. The mid-IgG content sample 1.0 ug/mL had an atypically high signal of 16,2735 whereas the average HPC signal in this run was only 7697. 8 Table 9: Specificity Res ults iik’s‘ataiitioa EEG 301.19;me Spe y by Drug Tolerance Assay specificity is such that increasing the PTHrP concentration results in a rapid decrease in signal in the presence of the SPC. Because the assay cut point is established at cellular response that is dependent upon a fixed concentration of PTHrP,it is expected that the assay will have limited drug tolerance. To evaluate this limit, the HPC, LPCl and LPC2 were treated on individual assay plates with 900, 1200 or 2400 pg/mL of PTHrP and compared to samples treated with nominal PTHrP at 600 pg/mL. [007 6] As ed, the signal response decreased for each control with increased drug concentration. The HPC control remained positive in the presence of up to 600 pg/mL of PTHrP.

As seen in section 14.5 on assay ivity, the LPC1 and LPC2 controls did not test positive in the nominal concentration of 600 pg/mL of PTHrP. [007 7] The presence of PTHrP in serum samples at levels above the assay concentration of 600 pg/mL may negatively impact the ability of the assay to detect neutralizing dies.

Selectivity ivity was assessed in 10 individual o human serum samples. Each sample was tested unspiked as well as spiked with the SPC stock at the HPC and LPCl dilution levels of 1:30 and 1:125 respectively and evaluated in the assay with controls at the MRD of 1:4. Samples were positive for the NAb when spiked with concentrations of antibody equal to the level of the HPC. Only two of the ten samples tested positive for NAb when spike with concentrations body equal to the level of the low positive l. Three samples tested positive for NAb in the absence of antibody spike.

A reference control (NC, HPC and LPCl) was run on each assay plate for determination of the % ry. The % ry was calculated by dividing the RLU value of the 0, 1:30 or 1:125 SPC dilution spiked sample by the normalized value of the NC, HPC or LPC1, respectively and expressed as a percentage. All samples spiked at the HPC concentration of NAb were within 30% of the RLU value of their respective controls. The LPCl control in Run 6 did not test positive in the assay.

Example 3. Determination of anti-PTH and anti-PTHrP Concentration Anti-PTH or anti—PTHrP IgG concentration was determined in two rabbit polyclonal antibody reagents.

Reagents and Materials: 0 Greiner bio-one high binding microplate 96-well, prod# 655061, L/N E16093KS o Anti-PTH (Ab)(l-34)(Human) — Phoenix Pharmaceuticals, cat# G—055-08, L/N 01553- 0 THrP (Ab)(l-34)(Human, Rat, Mouse) — Phoenix Pharmaceuticals, cat# H056-04, L/N 01736-1. o PTH (l-34)(Human) peptide — Phoenix Pharmaceuticals cat# 055-03, L/N 430926 0 PTHrP (l-34)(Human, Rat, Mouse) peptide - Phoenix Pharmaceuticals cat# 05604, L/N 432088 0 Rabbit IgG biotin — Rockland, cat# 011-0602, L/N 36734 0 Donkey anti-Rabbit IgG (H+L) HRP,prod# 711152, L/N 125015 0 Wash buffer: 1X PBS + 0.05% tween 20 (Fisher, L/N 160170) e in-house buffer L/N 170320-1, Exp: 17 Sep 2017 0 Assay t: 1X PBS + 3% BSA ffymetrix, prod. 10857, L/N 4295530, Exp 08/2021) Covance in-house buffer L/N 170317-3, Exp: 17 Sep 2017 0 Standard: Rabbit IgG whole molecule bioton conjugated, Rockland, prod# 011-0602, L/N 36734, Exp: Mar 2018 o Detecting Antibody: Donkey anti—Rabbit IgG (H+L) peroxidase conjugated, Jackson ImmunoResearch -cat#7ll152,lot# 129517, Exp: 11 Jan 2018 o ABTS peroxidase substrate (l-component) - KPL, prod# 5001, lot# , Exp: 11/2017 Methods ELISA plates (Greiner bio—one high binding microplate l, prod# 655061, L/N E16093KS) were coated with either PTH or PTHrP e solution (lug/mL) at a volume of lOOuL/well in assay coating buffer (PBS). Three columns of each plate were coated with various concentrations of biotin labeled rabbit IgG at avolume of 100 uL/well for the standard curve.

Plates were incubated ght at 2-8°C. After washing the plates with wash buffer (1X PBS + 0.05% Tween 20), 200uL of the blocking buffer (lXPBS + 3% BSA from fymetrix Prod 10857) was added to each well and incubated at room temperature for 1 hour. Plates were washed with wash buffer (1X PBS + 0.05% Tween 20) and anti-PTH or anti—PTHrP antibody samples were added to their designated wells at a volume of 50uL per well using 2-fold serial dilutions. t was added in the three columns used for ping the IgG standard curve. Plates were incubated at 35-37°C for 1 hour. Plates were washed with wash buffer (1X PBS + 0.05% Tween ) and the secondary antibody, (donkey anti—rabbit IgG (H&L), peroxidase conjugated - Jackson ImmunoResearch, #711035152, lot 125015) was added to the entire ELISA plate. The plates were incubated for approximately 1 hour at room temperature and then washed with wash buffer (1X PBS + 0.05% Tween 20). Enzyme activity that was retained on the plates was measured by adding the HRP substrate ABTS (2,2'-Azinobis [3—ethylbenzothiazolinesulfonic acid]- diammonium salt - KPL #506601, lot 150405) at 100 uL per well and incubated for approximately 30 minutes at room temperature. The plates were read at 415nm, with a reference at 570nm. The IgG concentration was derived by comparing the absorbance of the unknown with the standard curve.

Summary Methods Plate Coating /well): o PTH peptide (1-34) ), 1mg/mL diluted to lug/mL in coating buffer 0 PTHrP peptide (1-34) (Human, Rat, Mouse), 1mg/mL diluted to lug/mL in coating buffer 0 Rabbit IgGwhole molecule bioton conjugated, 1mg/mL diluted to 1, .333, .111, .037, .012, .004, and .001 ug/mL in coating buffer (columns 10-12 only). 0 te at 2 to 8C overnight. 0 Wash 3X, 350uL/well in wash buffer (1X PBS + 0.05% tween 20) using BioTek EL406 Blocking (200uL/well): 0 1X PBS + 3% BSA 0 Incubate at room temp. for 1 hour 0 Wash 3X, 350uL/well in wash buffer (1X PBS + 0.05% tween 20) using BioTek EL406 Sample Placement - plate 1 - (50ul/well): 0 Columns 1-3 - PTH (1-34) (Human) d Ab, diluted 1:1,000 then serially diluted by 2 0 s 10-12 - Assay t blanks Sample Placement - plate 2 - (50ul/well): 0 Columns 1-3 - PTHrP (1-34) (Human) antibody, diluted 1:1,000 then serially diluted by 2 0 Columns 10-12 - Assay diluent blanks 0 Incubate at 35-39C for 1 hour. 0 Wash 6X, 350uL/well in wash buffer (1X PBS + 0.05% tween 20) using BioTek EL406 Secondary Ab (lOOuL/well): o Donkey anti-Rabbit IgG (H+L) peroxidase conjugated, diluted 15,000 in assay diluent 0 Incubate at room temp. for 1 hour. 0 Wash 6X, 350uL/well in wash buffer (1X PBS + 0.05% tween 20) using BioTek EL406 Substrate (lOOuL/well): o ABTS peroxidase substrate (l—component) 0 te at room temp. for 30 minutes. 0 Read plate at 415nm, 570nm using BioTek Power Wave HT plate reader, S/N 259240 Data Analysis 0 tation is derived from Beer's Law formulation ofunknown/known X concentration of known X dilution ofunknown 0 The reference point of the standard curve (known) is the point t to the middle ofthe curve (peak o.d. minus blank/ 2). The reference point ofthe unknown is the data point whose absorbance is closest to the reference point of the standard.

Conclusions Standard curves were ed using Rabbit IgG. ELISAs were run to determine the concentration of PTH or PTHrP in two rabbit polyclonal antibody reagents. Data points were selected from each assay correlating to the linear portion of the standard curve using Gen5 software. The results are summarized in Table 10.

Table 10 Anti-PTH antibody IgG THrP antibody IgG concentration (ug/mL) concentration (ug/mL) Anti-PTH (Ab)(l- 34)(Human) — Phoenix Pharmaceuticals, cat# G- 055-08, L/N 4 Anti-PTHrP (Ab)( 1- 34)(Human, Rat, Mouse) — Phoenix Pharmaceuticals, cat# HO4, L/N 01736-

Claims (24)

1. An in vitro method for detecting the presence of neutralizing antibodies to PTH or PTHrP in a sample, the method comprising: obtaining the sample from a subject; contacting the sample with a population of cells or a cell, n the cell or cells se a receptor for PTH or PTHrP; measuring cyclic adenosine monophosphate (CAMP) levels; and detecting the presence of neutralizing antibodies when CAMP levels are reduced relative to anegative control sample without neutralizing antibodies.

2. The method of claim 1, wherein the measuring CAMP levels is med by a competitive immunoassay.

3. The method of claim 2, wherein the itive immunoassay is an electrochemiluminescent detection method.

4. The method of any one of claims 1 to 3, wherein the contacting step comprises incubating the cell or cells with the serum sample.

5. The method of any one of claims 1 to 4, wherein the method further comprises preincubation of the serum sample with a predetermined amount of PTH or PTHrP analog prior to the contacting step.

6. The method of claim 5, wherein the preincubation is for a period of at least 30 s.

7. The method of any one of claims 1 to 6, wherein the cell or cells are lysed prior to the measuring step.

8. The method of any one of claims 1 to 7, further comprising incubation of the cell or cells with a cell ble CAMP—specific phosphodiesterase inhibitor prior to the contacting step.

9. The method of claims 8, wherein the CAMP—specific phosphodiesterase inhibitor is 4—(3- Butoxy-4—methoxybenzyl)—2-imidazolidinone.

10. The method of any one of claims 1-9, wherein the measuring cAMP levels is performe d using the Mesoscale Discovery Multi-Array 96-well cAMP Plate.

11. The method of claim 5, wherein the predetermined amount of PTH or PTHrP analog is at least 100, 200, 300, 400, or 500 pg/mL.

12. The method of claim 5, wherein the predetermined amount of PTH is about 500 pg/mL.

13. The method of claim 5, wherein the predetermined amount of PTHrP analog is about 600 pg/mL.

14. The method of claim 1, wherein the cell or cells are rat lial cell line UMR-106.

15. The method of claim 14, further sing serum-starving the 6 cell or cells for a period of time prior to the contacting step.

16. The method of claim 15, wherein the period of time ranges from about 4 hours to about 48 hours, about 4 hours to about 24 hours, about 4 hours to about 16 hours, about 4 hours to about 12 hours, or about 6 hours to about 12 hours

17. The method of any one of claims 1-16, wherein the sample is a human sample.

18. The method of claim 17, n the human sample is a human serum sample.

19. The method of claim 18, wherein the sample is from the subject treated with a PTHrP analog.

20. The method of claim 19, wherein the PTHrP analog is Abaloparatide.

21. The method of claim 19, wherein the PTHrP analog is Teriparatide.

22. A method of detecting the presence of neutralizing antibodies after Abaloparatide treatment, the method comprising: obtaining a serum sample from a subject treated with Abaloparatide; contacting the serum sample with a cell or population of cells, wherein the cell or cells comprise a or for PTH or PTHrP; measuring cyclic adenosine monophosphate (CAMP) levels; and detecting the presence of neutralizing antibodies when cAMP levels are reduced relative to anegative l sample without neutralizing antibodies.

23. The method of claim 22, further comprising discontinuing treatment when neutralizing antibodies are detected in the serum sample.

24. A kit for carrying out the method of any one of claims 1 or 22 comprising components required to carry out the obtaining, contacting, ing and ing steps and instructions for PTH Dose Response Curve m y. w. my yam: 12mm y {3m fimrfisafieas-imimfl M(-3. 1 PTH rP n.Iaog DO nSe Cu rVe 1..a» $3..“ J.... a: “m. mmWW.W Wmm mW H HU“H . m.. \ an.EGG. .rm.m an.” {a H H. FE {I». 2