阅读说明：本技术 用于检测抗药抗体的方法 (Method for detecting anti-drug antibodies ) 是由 徐卫锋 R·C·佩鲁特拉 于 2019-08-02 设计创作，主要内容包括：在某些实施方案中,本发明提供了一种用于检测样品中的抗药抗体(ADA)的方法,其包括：a)在高温下预处理所述样品以解离所述样品中的ADA:药物免疫复合物；b)通过基质从所述样品中分离出所述ADA；c)使用缓冲液从所述基质中回收所述ADA；以及d)在基于细胞的测定或体外测定中检测所述ADA。(In certain embodiments, the present invention provides a method for detecting an anti-drug antibody (ADA) in a sample, comprising: a) pretreating the sample at elevated temperature to dissociate ADA: drug immune complexes in the sample; b) isolating the ADA from the sample by matrix; c) recovering the ADA from the matrix using a buffer; and d) detecting the ADA in a cell-based assay or in vitro assay.)

1. A method for detecting an anti-drug antibody (ADA) in a sample, comprising:

a) pretreating the sample at elevated temperature to dissociate ADA: drug immune complexes in the sample;

b) isolating the ADA from the sample by matrix;

c) recovering the ADA from the matrix using a buffer; and

d) detecting the ADA in a cell-based assay or in vitro assay.

2. The method of claim 1, wherein the elevated temperature is between 60 ℃ and 68 ℃.

3. The method of claim 1, wherein the elevated temperature is about 60 ℃, 61 ℃,62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, or 68 ℃.

4. The method of claim 1, wherein the sample is pretreated at the elevated temperature for a time between about 30 minutes and 2 hours.

5. The method of claim 1, wherein the ADA is sensitive to acid treatment.

6. The method of claim 1, wherein the drug has a lower thermal stability than the ADA.

7. The method of claim 1, wherein the drug is selected from an antibody or fragment thereof, a nucleic acid, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, or a small molecule compound.

8. The method of claim 1, wherein the sample is a biological sample selected from the group consisting of: body fluids, mucous secretions, saliva, blood, whole blood, plasma, and serum.

9. The method of claim 1, wherein the ADA is isolated from the sample by contact with a biotinylated drug followed by contact with a streptavidin-coated substrate.

10. The method of claim 1, wherein the ADA is isolated from the sample by a matrix coupled to the drug.

11. The method of any one of claims 9-10, wherein the substrate is a magnetic bead.

12. The method of claim 1, wherein the recovered ADA is detected in a cell-based assay.

13. The method of claim 12, comprising: i) adding the recovered ADA to a cell in the presence of the drug; and ii) detecting the ADA by measuring a decrease in activity of the drug on the cell.

14. The method of claim 1, wherein the recovered ADA is detected in an in vitro assay.

15. The method of claim 14, comprising: i) contacting the recovered ADA with the drug labeled with a detectable label; and ii) detecting the ADA by measuring the detectable label.

16. The method of claim 15, wherein the detectable label is a label selected from the group consisting of: radioisotopes, enzymes, fluorescent labels, chemiluminescent labels and electrochemiluminescent labels, and substrates for enzymatic detection reactions.

17. The method of claim 1, wherein the drug is a domain antibody.

18. The method of claim 1, wherein the drug is pegylated.

19. The method of claim 1, wherein the drug is rolizumab.

20. The method of claim 1, wherein the elevated temperature is about 62 ℃.

21. The method of claim 1, wherein the sample is serum.

22. The method of claim 1, wherein the sample is from a subject that has been treated with the drug.

Background

Biotherapeutics are foreign antigens and may potentially induce immune responses, leading to the formation of anti-drug antibodies (ADAs), which in turn may lead to a wide range of side effects. Neutralizing antibodies (nabs) belong to a subset of ADAs, which can bind to the pharmacologically active region of a therapeutic agent to inhibit or completely neutralize its clinical efficacy. Preferably, the neutralizing activity is characterized based on a functional NAb assay of the cell. However, cell-based NAb assays are generally susceptible to drug interference as well as interference with numerous serum factors (including, but not limited to, growth factors and disease-related cytokines). BEAD Extraction and Acid Dissociation (BEAD) has been successfully applied to remove circulating drugs and/or other interfering factors from human serum samples, thereby enriching ADA/NAb. However, the strong acids used in the extraction procedure may cause irreversible denaturation of NAb and result in an underestimation of NAb measurements. Therefore, there is a need to develop novel methods for detecting neutralizing anti-drug antibodies.

Disclosure of Invention

In certain embodiments, the invention provides a method for detecting an anti-drug antibody (ADA) in a sample. Such a method comprises: a) pretreating the sample at elevated temperature to dissociate ADA: drug immune complexes in the sample; b) isolating the ADA from the sample by matrix; c) recovering the ADA from the matrix using a buffer; and d) detecting the ADA in a cell-based assay or in vitro assay. Optionally, the elevated temperature is between 60 ℃ and 68 ℃ (e.g., about 60 ℃, about 61 ℃, about 62 ℃, about 63 ℃, about 64 ℃, about 65 ℃, about 66 ℃, about 67 ℃, or about 68 ℃). Optionally, the sample is pretreated at the elevated temperature for a time between about 30 minutes and about 2 hours (e.g., about 30-60 minutes), such as about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

In certain particular embodiments, the ADA is sensitive to acid treatment. In certain particular embodiments, the drug has a lower thermal stability than the ADA.

Optionally, the drug is selected from an antibody or fragment thereof, a nucleic acid, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, or a small molecule compound. Optionally, the sample is a biological sample selected from the group consisting of: body fluids, mucous secretions, saliva, blood, whole blood, plasma, and serum. Optionally, the sample is from a subject that has been treated with the drug.

In certain particular embodiments, the ADA is isolated from the sample by contact with a biotinylated drug, followed by contact with a streptavidin-coated substrate. Alternatively, the ADA is isolated from the sample by a matrix coupled to the drug. For example, the substrate is a magnetic bead.

In certain particular embodiments, the recovered ADA is detected in a cell-based assay. For example, such assays include: i) adding the recovered ADA to a cell in the presence of the drug; and ii) detecting the ADA by measuring a decrease in activity of the drug on the cell.

In certain particular embodiments, the recovered ADA is detected in an in vitro assay. For example, such assays include: i) contacting the recovered ADA with the drug labeled with a detectable label; and ii) detecting the ADA by measuring the detectable label. For example, the detectable label is a label selected from the group consisting of: radioisotopes, enzymes, fluorescent labels, chemiluminescent labels and electrochemiluminescent labels, and substrates for enzymatic detection reactions.

In certain particular embodiments, the agent is an antibody fragment, such as a domain antibody. In certain particular embodiments, the drug is pegylated.

In certain particular embodiments, the drug is lulizumab (i.e., a pegylated anti-CD 28 domain antibody). For example, the drug is pretreated at an elevated temperature of about 62 ℃. For example, the ADA is detected in the serum sample.

Drawings

Figure 1. in the case of cells from continuous culture or freshly thawed cells, lurlizumab inhibits T cell activation and IL-2 driven luciferase reporter production in a dose-dependent manner in a jurkat.ca and Raji two-cell bioassay. A) Cells from continuous culture. Different color lines indicate different cell numbers per well (unit:. times.103). B) Thawed frozen cells (fz) perform better when compared to fresh cells (fs) from continuous culture. Curves were generated from duplicate wells using Softmax.

Figure 2.a). NAb PC rescues IL-2 production inhibited by lurlizumab. Red: rabbit polyclonal Ab, EC50 was 2.22; blue color: mouse mAb clone 13H4, EC50 was 1.45. B) NAb PC curves in the presence of different amounts of lullizumab (mouse mAb clone 13H 4). (blue, red and green lines represent 0.2, 0.25 and 0.3. mu.g/mL of lucizumab with EC50 of 0.53, 0.96 and 1.09, respectively).

Figure 3 ten individual SLE sera were diluted 10-fold in assay media and then added to the cell assay. Error bars represent standard deviation from duplicate wells.

Figure 4 thermal stability of rolizumab and anti-rolizumab NAb PC. A) Differential Scanning Fluorescence (DSF) measured on a UNcle platform to compare the melting temperatures of different proteins. Vertical orange bars indicate 62 ℃. B) Lullizumab; C) anti-lullizumab NAb PC was heated at different temperatures for 30 minutes, cooled, and then added to the cell-based assay. NAb activity was tested in the presence of 0.25. mu.g/mL drug. The original values were normalized to 0 μ g/mL groups to show relative activity. Only rabbit pAb is shown here, with similar results for mAb clone 13H 4. D) pH stability of NAb PC. Rabbit pAb or mouse mAb clone 13H4 NAb PC were treated at different pH for 60 minutes, neutralized, and then added to the cell assay. The original values were normalized to pH 7.0 to show relative activity.

FIG. 5 determination of assay sensitivity. NAb PC was incorporated into pooled normal human serum, BEHD extracted and run in a cell-based assay. Error bars indicate standard deviation from 6 sets of data.

Detailed Description

The present invention relates to novel methods for the qualitative and/or quantitative detection of ADA from a sample.

In certain embodiments, the invention provides a method for detecting an anti-drug antibody (ADA) in a sample. Such a method comprises: a) pretreating the sample at elevated temperature to dissociate ADA: drug immune complexes in the sample; b) isolating the ADA from the sample by matrix; c) recovering the ADA from the matrix using a buffer; and d) detecting the ADA in a cell-based assay or in vitro assay. Optionally, the elevated temperature is between 60 ℃ and 68 ℃ (e.g., about 60 ℃, about 61 ℃, about 62 ℃, about 63 ℃, about 64 ℃, about 65 ℃, about 66 ℃, about 67 ℃, or about 68 ℃). Optionally, the sample is pretreated at the elevated temperature for a time between about 30 minutes and about 2 hours (e.g., about 30-60 minutes), such as about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

In certain particular embodiments, the ADA is sensitive to acid treatment. In certain particular embodiments, the drug has a lower thermal stability than the ADA. Optionally, the drug is selected from an antibody or fragment thereof, a nucleic acid, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, or a small molecule compound. Optionally, the sample is a biological sample selected from the group consisting of: body fluids, mucous secretions, saliva, blood, whole blood, plasma, and serum. Optionally, the sample is from a subject that has been treated with the drug.

In certain particular embodiments, the ADA is isolated from the sample by contact with a biotinylated drug, followed by contact with a streptavidin-coated substrate. Alternatively, the ADA is isolated from the sample by a matrix coupled to the drug. For example, the substrate is a magnetic bead.

In certain particular embodiments, the recovered ADA is detected in a cell-based assay. For example, such assays include: i) adding the recovered ADA to a cell in the presence of the drug; and ii) detecting the ADA by measuring a decrease in activity of the drug on the cell.

In certain particular embodiments, the recovered ADA is detected in an in vitro assay. For example, such assays include: i) contacting the recovered ADA with the drug labeled with a detectable label; and ii) detecting the ADA by measuring the detectable label. For example, the detectable label is a label selected from the group consisting of: radioisotopes, enzymes, fluorescent labels, chemiluminescent labels and electrochemiluminescent labels, and substrates for enzymatic detection reactions.

In certain particular embodiments, the agent is an antibody fragment, such as a domain antibody. In certain particular embodiments, the drug is pegylated.

In certain particular embodiments, the drug is rolizumab (i.e., a pegylated anti-CD 28 domain antibody, also referred to as BMS-931699). For example, the drug is pretreated at an elevated temperature of about 62 ℃. For example, the ADA is detected in the serum sample.

In order that the invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

An "anti-drug antibody" or "ADA" is an antibody that specifically binds to any region of a drug. For example, the anti-drug antibody may be an antibody or fragment thereof, which may be directed against any region of the drug antibody, such as the variable domain, constant domain or sugar structure of an antibody. Such anti-drug antibodies may appear as an immunogenic response in a patient during drug therapy. The ADA may be of any human immunoglobulin isotype (e.g., IgM, IgE, IgA, IgG, IgD) or IgG subclass (IgG1, 2,3, and 4). ADAs include ADAs from any animal source, including, for example, human or non-human animal (e.g., veterinary) sources.

For the purposes of this specification, the term "Nab" or "neutralizing antibody" refers to a subset of ADA that can bind to the pharmacologically active region of a therapeutic drug to inhibit or completely neutralize its clinical efficacy.

In the context of the present invention, the term "patient" refers to any subject, preferably a mammal, more preferably a human, having a disease or suspected of having a disease. As used herein, the term "subject" refers to any animal (e.g., a human or non-human animal subject). In some cases, the subject is a mammal. In some cases, as used herein, the term "subject" refers to a human (e.g., a man, a woman, or a child). In some cases, as used herein, the term "subject" refers to a laboratory animal for animal model studies.

As used herein, the term "biological sample" or "sample" refers to a sample obtained or derived from a patient, which comprises patient-derived immunoglobulins and thus may be referred to as an immunoglobulin sample. For example, the biological sample comprises a material selected from the group consisting of: body fluids, blood, whole blood, plasma, serum, mucus secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage (BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor), lymph fluids, lymph node tissue, spleen tissue, bone marrow, and immunoglobulin rich fractions derived from one or more of these tissues. In some embodiments, the sample is or comprises serum, or is an immunoglobulin-rich fraction derived from serum or blood. The sample is or may be derived from a body fluid or body tissue. In some embodiments, the sample is obtained from a subject that has been exposed to a drug (e.g., repeatedly exposed to the same drug). In other embodiments, the sample is obtained from a subject that has not been recently exposed to a drug, or is obtained from the subject prior to scheduled administration of a drug.

As used herein, rolizumab is an anti-human CD28 receptor antagonist Vk domain antibody (dAb) in modified form with a 40kDa branched polyethylene glycol (PEG) that is being developed for Subcutaneous (SC) treatment of autoimmune and inflammatory diseases (e.g., systemic lupus erythematosus). For example, lullizumab has been cited in U.S. patent nos. 8,168,759, 8,454,959, and 9,085,629.

As used herein, "CD 28 activity" is an activity involving binding to or resulting from CD80, CD86, and/or another ligand to CD28, and includes, but is not limited to, CD 28-mediated activation of cell signaling. CD28 activity also includes induction of T cell proliferation and T cell secretion of cytokines, e.g., interleukin 2 (IL-2).

By "domain antibody" is meant a folded polypeptide domain that comprises the sequence characteristics of an immunoglobulin variable domain of the heavy (VH) or light (VL) chain of an antibody and that specifically binds an antigen. Thus, a "domain antibody" includes intact antibody variable domains as well as modified variable domains, e.g., where one or more loops have been replaced by sequences not unique to the antibody variable domain; or an antibody variable domain that has been truncated or comprises an N-terminal or C-terminal extension; and target antigen specificity of folded fragments and full-length domains of the variable domains. "dAb" is used interchangeably with "domain antibody" herein.

As used herein, an entity modified by the term "label" (e.g., an antibody, an anti-drug antibody, a drug, a protein, an enzyme, an antibody fragment, a multidomain biotherapeutic agent (e.g., an antibody drug conjugate), or a related species) includes any entity conjugated to another molecule or an empirically detectable chemical entity (e.g., "detectable label"). Chemical species suitable as labels for the tagging entity include, but are not limited to, enzymes; a fluorescent dye; quantum dots; an optical dye; a luminescent dye; and a radionuclide.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Example 1

1. Introduction to

Biotherapeutic agents (biologic therapeutic or biotherapeutic) have been approved for the treatment of many conditions. Despite many advantages over traditional small molecules, biotherapeutics still have the potential to induce an immune response against themselves, commonly referred to as immunogenicity (1). Immunogenicity is a natural defense mechanism of the human body and generally has a protective effect. As part of the adaptive immune response, when the host encounters foreign proteins or altered self-antigens (such as infectious pathogens, tumor antigens or vaccines), the body may produce antibodies against these foreign/altered self-proteins. However, in the case of biotherapeutics, these drug-specific antibodies, commonly referred to as anti-drug antibodies (ADAs), may induce a wide range of safety-related events, ranging from local infusion reactions to more serious adverse events such as life-threatening hypersensitivity and pure red blood cell aplasia (PRCA) (2, 3). In addition, ADA may lead to a decrease in drug efficacy (4-6).

The reduction in drug efficacy induced by ADA is due to accelerated drug clearance or a specific subset of ADA (neutralizing ADA or NAb) that reduces therapeutic efficacy by preventing drug binding to its target or by steric inhibition of downstream signaling following binding. Health authorities currently recommend that cell-based functional NAb assays be performed as much as possible to characterize the neutralizing potential of ADA (7). In conventional cell-based functional NAb assays, a fixed amount of a drug designated as a systemic drug is added to the cells as a control; any statistically significant signal change due to the presence of NAb means that there is neutralizing activity in the sample. In the presence of circulating drugs from therapy, and depending on the molar ratio of NAb to drug in the sample, NAb may complex with the drug and no longer bind to systemic drugs in the bioassay; this results in reduced or false negative NAb detection. High levels of mAb therapeutics in patient samples are particularly problematic for cell-based functional NAb bioassays (8).

In addition, clinical serum samples may contain matrix components (growth factors, cytokines, etc.) which often directly affect the cells and may affect the readout of the functional assay (regardless of the presence or absence of NAb). While small perturbations from interfering factors in functional biometric readings may be tolerable, inter-subject and temporal variability may not allow accurate characterization of the presence of nabs in a sample.

BEAD Extraction and Acid Dissociation (BEAD) sample pretreatment procedures have been modified and optimized to address drug and matrix interference by using acid dissociation drug/ADA immune complexes, followed by addition of excess biotinylated drug to compete for ADA binding. The biotinylated drug/ADA complex is then captured by streptavidin-coated magnetic beads, while serum factors and drugs are removed by washing (9). Although it has been successfully applied to item (10), biotherapeutics conjugated to polyethylene glycol (PEG) may not be compatible with such acid dissociation-based extraction, as is the case with lullizumab. Rolizumab is an immunoglobulin light chain variable region (vk) domain antibody (dAb) that is an anti-human clade 28(CD28) receptor antagonist modified with a 40 kilodalton (kDa) branched PEG. Alternatively, we take advantage of the lower thermostability of the domain Ab by heating the sample at 62 ℃, which not only destroys the drug/ADA immune complex, but also selectively denatures the domain Ab drug. As a first step in the disruption of the drug/ADA complex, an acid is used in the bed process; after neutralization, excess circulating drug competes with biotin-drug for ADA binding, which requires much larger amounts of biotin-drug to overcome the drug. Thus, irreversible denaturation of the domain Ab drug at 62 ℃ is superior to the acid-based BEAD method, as much less biotin-drug is required. In addition, the use of heat instead of strong acid to eliminate the first step of acid treatment can retain acid sensitive NAb species.

2. Materials and methods

2.1. Reagent

RPMI-1640, heat-inactivated Fetal Bovine Serum (FBS), G418, HEPES, and sodium pyruvate were purchased from Gibco/Life Technology (Greenland, N.Y.). NeoLite luciferase reporter assay systems were purchased from PerkinElmer (Walthermer, Mass.) and Hi-Sur Mag streptavidin beads from OceanNanotech (san Diego, Calif.). Pooled human sera, individual normal human sera, and individual lupus-afflicted human sera were purchased from bioreclaimation (hickwier, n.y.). Jurkat T cells and Raji B cell lines were originally obtained from ATCC, and Jurkat T cells were further modified to generate jurkat.ca cells stably expressing IL-2 driven luciferase. The neutralization positive control was a proprietary monoclonal mouse affinity purified antibody to the drug product.

2.2. Cell culture

Stably transfected Jurkat cells (Jurkat. CA) and Raji cells expressing IL-2 driven luciferase were expanded in vented cap cell culture flasks (BD Falcon, Franklin lake, N.J.) at 37 ℃, 5% CO2, and 95% Relative Humidity (RH). The growth medium for both cells was RPMI 1640 with 10% heat-inactivated FBS. CA cell line growth medium also contained 400. mu.g/mL G418, HEPES and sodium pyruvate. The freezing medium for both cell lines was pure FBS supplemented with 10% DMSO. After thawing, cells were washed once and resuspended in bioassay media (RPMI-1640 medium supplemented with 10% FBS) and used directly in the bioassay.

2.3. Preparation of test controls for BEAD Extraction and Acid Dissociation (BEAD) procedure

Different concentrations of NAb PC were incorporated in pooled or individual healthy human serum with or without 5 μ g/ml drug product and incubated at room temperature for 4 hours with rotation to allow immune complexes to form. Drug products at different concentrations were also incorporated into human serum and prepared in the same manner. The samples were then aliquoted and frozen at-70 ℃ until use.

2.4. BEAD Extraction and Acid Dissociation (BEAD) and BEAD extraction and thermal dissociation (BEHD) procedures

We extracted ADA (9) with a slight modification of the previously disclosed solid phase or BEAD extraction and acid dissociation (bed) procedure. Briefly, 100 μ L of the human serum samples prepared above and the control samples were first mixed with an equal volume of 400mM glycine-HCl (pH 2.0) and incubated on a shaker (Labnet Orbit P4, woodbrique, nj) at 1200rpm for 60min at Room Temperature (RT). Each sample was then neutralized with 28. mu.L of 1.8M Trizma base (pH 8.8) containing 50. mu.g/mL of biotinylated drug and incubated for 90min at 1200rpm on a shaker. Alternatively, 100 μ L of control and sample were added to a KingFisher deep well 96 well polypropylene plate, covered with a plate sealer, and incubated for 40-60min in Eppendorf Thermomixer R set at 62 ℃ and shaken at 400 rpm. After cooling the deep-well plate for about 15 minutes, 28 μ Ι _ of biotinylated drug (diluted in 1% BSA in DPBS, 50 μ g/mL) was then added and the sample plate was incubated overnight at 2 ℃ -8 ℃ with shaking at 1000 rpm. ADA dissociated from the drug product and bound to the biotin-drug from the acid or heat treatment was then immobilized on 250 μ g streptavidin-coated magnetic beads (25 μ L added at 10mg/mL and incubated at 1000-. The bead complexes were then captured by magnetic plate using a KingFisher magnetic particle processor (Thermo Scientific, Waltherm, Mass.), washed twice with 600 μ L PBST and eluted with 60 μ L2 × RPMI-1640(pH 2.3). 50 μ L of the final eluted solution was transferred to a new 96-well polypropylene plate containing 22 μ L of 100mM HEPES (pH 8.2).

2.5. Bioassay for detecting neutralizing Activity

IL-2-luciferase bioassays are used to assess the absence, presence or relative levels of anti-therapeutic protein neutralizing antibodies in a sample. Briefly, 30 μ L of neutralized BEAD eluate from section 2.4 was incubated with 15 μ L of 250ng/mL systemic drug in a 96-well half-area whiteboard at room temperature for 20-40 min. Ca cells were thawed, washed and resuspended to a final concentration of 3.0x10 in bioassay medium (10% FBS in RPMI 1640)⁶cells/mL, and 15 μ L was added to the plate and incubated at room temperature for an additional 20 min. In the final step, Raji cells were thawed, washed and resuspended to a final concentration of 1.5X10 in bioassay medium containing 2.5. mu.g/mL anti-human CD3 Ab⁶Individual cells/mL, 15 μ L of which was added to the plate, mixed, and then transferred to 5% CO set at 37 ℃₂And an incubator at 95% humidity for 4 h. After incubation, 75 μ L of Neolite luciferase solution was added to each well, mixed, placed in a dark room temperature incubator shaken at 500rpm, centrifuged, and then read using an ensspire (PerkinElmer, waltham, massachusetts) plate reader with the default 96-well luminescence protocol.

2.6. DSF for protein thermostability

Differential scanning fluorescence with concurrent static light scattering was performed on an unclle platform (Uncariamed Labs Inc.). Briefly, purified protein samples were diluted to 1mg/mL in DPBS buffer (pH 7.2) (thermolfisher) and 9 μ Ι _ of each sample was loaded in triplicate into a microcuvette array. The extent of sample aggregation was determined by on-line dynamic light scattering before the start of the run. Temperature-induced protein unfolding was determined by measuring the change in intrinsic fluorescence using a step gradient of 1 ℃ from 20 ℃ to 85 ℃ (30 sec temperature plateau at each step). The Tm (midpoint of the unfolding curve, corresponding to the melting temperature) was calculated in UNcle software using barycentric fluorescence (in nanometers) as a function of temperature (in degrees celsius). Concurrent static light scattering at 266nm and 473nm was recorded to detect and control aggregate formation during the course of the experiment.

2.7 statistical methods

2.7.1 control accuracy assessment

The variance component method in the context of an analysis of variance (ANOVA) model was used to calculate the estimate of accuracy for the control samples (11). The ANOVA model included factors for the analysts, the day of the assay, and the assay panels within a day. Estimates of inter-analyst, daytime, inter-panel, and intra-panel variances were calculated in the ANOVA model and were each expressed as Standard Deviation (SD) and then as coefficient of variation (CV [% ] ═ 100 × SD/mean). The total standard deviation (total SD) is calculated as the square root of the sum of these variance estimates. Total CV (%) (100 total SD/average) was used to set the acceptability of the plates.

2.7.2 cut Point evaluation

Each lupus patient sample was assayed in 6 different cases, with two assays performed by 3 analysts. NAb determination cut points were calculated using the disclosed method (12). To correct for interplate fluctuations of RLUs across the day, the ratio of patient sample RLUs to plate negative control (NegC) RLU was calculated (average replicates of each). Since the ratio was used for cutpoint evaluation, the correlation between patient sample RLU and NegC RLU from the same plate was calculated and the data plotted. Positive correlation will support the use of a ratio calculation method.

To calculate NAb cut points, the normality of the sample ratio distribution was evaluated. The abnormal values were evaluated based on the individual ratios of each patient sample. Values are considered outliers if they are below the 25 th percentile of the distribution minus 3 times the interquartile range, or values are above the 75 th percentile of the distribution plus 3 times the interquartile range.

After exclusion of outliers and log transformation for normality, NAb determination cut points were calculated as the lower bound of the 99% interval of the single sided parameter of the ratio. The equation used is of the form:

NAb determination cut point EXP (mean value)_{Ratio of}-z total SD_{Ratio of})。

In the equation, "mean value_{Ratio of}"is the average of the log ratios after excluding the abnormal values; "z" is the value of the "z-score" from a normal distribution, corresponding to the area under the lower 1% tail normal curve (2.33); and "Total SD_{Ratio of}"is an estimate of the total standard deviation of the logarithm of the ratio after exclusion of outliers. "EXP" is the inverse logarithm of the expression.

3. Results

3.1. Therapeutic-induced inhibition of IL-2 production in cellular assays

Rolizumab is an anti-human CD28 antagonistic immunoglobulin light chain variable region (V κ) dAb modified with 40kDa branched PEG. It is a potent inhibitor of T cell activation and is a pure antagonist as determined by in vitro agonist, co-stimulation and cross-linking experiments (13-15). As shown in figure 1A, jurkat.ca cells from continuous culture produced high levels of IL-2 promoter driven luciferase reporter when activated by agonistic anti-CD 3 Ab and Raji B cells (which provided CD80/CD86 to engage CD28 on jurkat.ca cells). Rolizumab inhibited T cell activation and luciferase reporter production in a dose-dependent manner (fig. 1A).

To facilitate downstream sample testing, frozen cells were thawed and immediately used in the assay to see if they could be used as a ready-to-use template reagent, eliminating the need for a waiting period of about 3-7 days to allow the frozen cells to recover and begin continuous cell culture maintenance. Much higher raw signals were found in freshly thawed cells when compared to cells from continuous culture (fig. 1B). Ca and Raji cells in different numbers and ratios showed different raw signals, reaction windows, and sensitivities as indicated by EC50 for each curve. 45000 jurkat.ca cells and 22500 Raji cells were selected per well for further assay development and optimization to balance the original signal with the total number of cells required. (FIG. 1B and Table 1).

TABLE 1 original signals generated by IL-2 luciferase in the presence of varying numbers and ratios of Jurkat and Raji cells

3.2. Neutralizing Ab against Lulizumab rescues production of IL-2

A set of potential NAb Positive Controls (PCs) were screened in the cell assay and the most effective clones were selected. As shown in fig. 2A, both rabbit polyclonal ab (pab) and mouse monoclonal ab (mab) PC rescued luciferase reporter expression in a dose-dependent manner in the presence of 0.4 μ g/mL rolizumab (i.e., the systemic drug, which is the final concentration of drug in the cellular assay). For the most sensitive NAb determination, the systemic drug level should be as low as possible while still having good signal-to-noise ratio (S/N) and low Coefficient of Variation (CV). As expected, the higher the drug concentration, the lower the signal of the luciferase reporter in the absence of NAb PC (fig. 2B and table 9). Although 0.3 μ g/mL of the systemic drug has the highest S/N for the entire NAb curve, it should be noted that the lower end of the NAb curve, where the sensitivity of the NAb assay is determined. Therefore, 0.25. mu.g/mL of the systemic drug was selected for further assay optimization; at this systemic drug level, 0.125, 0.25 and 0.5 μ g/mL of NAb consistently had higher S/N than the other two drug concentrations (Table 2).

TABLE 2 NAb curves for different concentrations of drug. Only one of three replicate experiments with similar results is shown here.

3.3. Low recovery of NAb in the presence of lullizumab in the case of BEAD treatment

Rolizumab is being developed for the treatment of autoimmune and inflammatory diseases in which patients often express large amounts of inflammatory cytokines in the circulation (14). As shown in figure 3, when ten individual Systemic Lupus Erythematosus (SLE) sera were diluted 10-fold in assay media and added to the cell-based assay, the luciferase primary signal of one sample (S8) increased by more than 50% and the luciferase primary signal of four samples (S2, S3, S5, and S9) inhibited by more than 50% when compared to the control sample of media only. This indicates that a 10-fold dilution is not sufficient to dilute all the interferents in SLE serum. However, further dilution leads to a decrease in the sensitivity of the NAb assay, as NAb in the sample will also be diluted away. For example, if the assay requires a 20-fold dilution, and if the assay can only detect 0.5 μ g/mL of NAb after dilution, the sensitivity of NAb assays in pure assay serum can potentially be as high as 10 μ g/mL. The expected levels of rillizumab in clinical samples were as high as 5 μ g/mL, meaning that if the sample was diluted and added to the assay without any extraction, NAb below 15 μ g/mL could not be detected (the molecular weight of rillizumab was about 50KDa, and 15 μ g/mL NAb would all form an immune complex with 5 μ g/mL rillizumab at a 1:1 molar ratio and would not bind systemic drugs in the assay). BEAD Extraction and Acid Dissociation (BEAD) was first tested to see if NAb PC could be dissociated from the drug in the sample by acid treatment, then bound to biotinylated drug/BEAD complexes and eluted from streptavidin-coated magnetic BEADs. This extraction will remove not only the excess drug in the sample, but also matrix factors that may interfere with the assay. Although BEAD has been successfully applied to a variety of cell-based functional NAb assays, the NAb activity after BEAD treatment was poor for the rolizumab NAb assay. If we set any cut-point for NAb activity to 1.3, the sensitivity of NAb detection in the presence of 5 μ g/mL drug would be about 8 μ g/mL (table 2 and data not shown). No improvement was achieved after confirming that clone 13H4 was acid stable and optimized each of the key steps of extraction (e.g., increased amount of biotin-drug, increased amount of streptavidin-magnetic beads, and unchanged length of acid treatment at different pH) (data not shown). Although rolizumab has a small protein backbone of 12kDa, it has a branched PEG moiety of 40kDa that occupies a large space equivalent to a 500kDa protein. It is assumed that at low pH, the branched PEG moiety can prevent (e.g., by steric hindrance) the interaction of NAb and the biotinylated rolizumab protein, ultimately resulting in low recovery of NAb.

3.4. Thermostability of lulizumab and Positive control

The non-pegylated rolizumab was small, only 12kDa when compared to the 150kDa NAb PC. This prompted us to examine whether rolizumab could be selectively and irreversibly denatured after heating, leaving intact NAb extracted by biotinylated non-pegylated drug. First, we compared the thermal stability of pegylated or unpegylated lucizumab and a panel of anti-lucizumab NAb PCs prepared in PBS using Differential Scanning Fluorescence (DSF) on the UNcle platform. As shown in figure 4A and table 3, the melting temperatures of both rillizumab with and without PEG moieties were much lower, so most of them had denatured at 62 ℃, while all NAb PCs, including both pAb and mAb, just started to denature at this temperature.

Table 3 mean melting temperatures of lullizumab and different NAb PCs measured by DSF.

To further confirm that the thermostability of lullizumab and NAb PC were different, rabbit pAb #1 and clone 13H4 were heated at different temperatures for 50-60 minutes and then added to the cell-based assay to test for remaining activity. Although the drug heated at 62 ℃ lost significant activity, particularly when the drug concentration was low (about 0.5 μ g/mL or less), all NAb PCs tested retained their activity at all temperatures tested (fig. 4B and 4C). These results indicate that most drugs are irreversibly denatured at 62 ℃, while all NAb PCs tested remain active. Interestingly, when tested for pH stability of NAb PC, the mouse mAb clone 13H4 showed resistance to pH 2.1 (same pH used for the first acid treatment in BEAD), while the rabbit pAb lost nearly 60% of activity after pH 2.1 treatment (fig. 4D). In other projects, similar poor pH stability of polyclonal NAb PC was observed (data not shown). The fact that the same rabbit pAb retained almost all activity when heated at 62 ℃ suggests that heating may be a potential alternative to pH-sensitive ADA/NAb dissociation.

3.5. Heat rather than acid treatment to improve recovery of anti-lurizumab NAb

Since promising thermal stability data showed significant differences between the drug and NAb PC, heating at 62 ℃ was evaluated as an alternative to the ineffective first step of acid dissociation in the BEAD extraction. Various concentrations of rabbit pAb or mouse mAb clone 13H4 in serum were incubated with or without 5 μ g/mL of lullizumab for at least 4 hours at room temperature to ensure immune complex formation. The samples were then incubated in a pre-warmed 62 ℃ heat block for 30 minutes; or treated with 400mM glycine (pH 2.0) for 60 minutes. 28 μ L of biotinylated protein scaffold of rolizumab (50 μ g/mL) prepared in PBS with 1% BSA or 1.8M Tris (pH 8.8) was then added to the cooled or acid treated plates, respectively. After overnight incubation at 4 ℃, streptavidin-coated magnetic beads were added to pull down the biotin-drug/NAb complex. After bead washing, nabs were acid washed off the beads, neutralized, and finally added to the cell-based assay. As shown in Table 4, heating to dissociate the drug/NAb immune complexes has much better recovery of NAb than acid dissociation, we call this new procedure bead extraction and thermal dissociation: (Bead Extraction and Heating Dissociation, BEHD). For rabbit pAb PC, the relative NAb activity achieved at 4. mu.g/mL NAb with heating in both groups with and without drug>1.5; whereas in the acid-treated group, only 16. mu.g/mL of NAb achieved relative NAb activity>1.5. Similarly, 4. mu.g/mL of clone 13H4 achieved relative NAb activity when treated with heat>1.6; and in the acid-treated group, at the same level>1.6 relative NAb activity only in 8-16 u g/mL.

TABLE 4 relative NAb activity after heating and acid treatment. In the case of rolizumab, heating dissociates the drug/NAb complex better than acid. The original values were normalized to 0. mu.g/mL NAb groups to calculate relative NAb activity.

3.6. Assay validation

With optimized cell assays and BEHD extraction, the assays were validated by the following parameters: cut point, accuracy, sensitivity, drug interference, selectivity and stability.

3.6.1 cutting Point

The cutpoints were assessed using serum samples from 50 untreated SLE patients in the study. Each sample was run in duplicate in 2 plates over 2 days by 3 analysts. In addition to SLE patient samples, each plate also contained a control sample. The positive control clone 13H4(PC) was prepared by incorporating anti-lurlizumab NAb into pooled sera from healthy donors. LPC is a low positive control neutralizing antibody of 3. mu.g/mL (LPC-1) or 2.5. mu.g/mL (LPC-2) in serum. Since the method had a pre-treatment for the extraction of BEHD, LPC-1 and LPC-2 were also prepared as LPC-extraction controls (LPC-EXC) in the presence of 5 μ g/mL lucizumab to monitor the extraction of BEHD in each run. HPC is a high positive control neutralizing antibody at 10. mu.g/mL. NegC is the same pooled serum without PC from healthy donors. PC and NegC samples were run as 2 and 3 replicates, respectively, on each plate. The acceptance criterion for each plate was set to have a coefficient of variation CV (%) of 30.0% per PC. In addition, the mean raw Relative Luminescence Unit (RLU) value of NegC must be lower than that of LPC, LPC-EXC, and HPC. At least 4 of the 6 PCs (HPC, LPC, and LPC-EXC) and at least 1 at each level must meet board acceptability. At least 2 of the 3 NegC replicates must meet the% CV standard.

NegC was treated the same as control and patient samples. To account for possible fluctuations of RLUs across the plate, the ratio of sample RLUs to plate NegC RLU (RLU/NegC RLU) was used to calculate the cut-point for SLE patient samples. Strong correlation between SLE sample RLUs across plates compared to NegC RLUs was observed (Pearson "r ═ 0.96), supporting the use of NegC RLUs to correct plate-to-plate RLU fluctuations.

The orthomorphism hypothesis was confirmed by the Shapiro-Wilk test. After removing outliers, the NAb assay cut point based on the ratio of sample RLU/NegC RLU was determined to be 1.61 (99% confidence, table 5). Samples that produced an average RLU/NegC RLU value of ≧ 1.61 are considered neutralized.

TABLE 5 determination of neutralization assay cut-points for samples of Systemic Lupus Erythematosus (SLE) subjects.

·^aStatistical outliers (nominal n ═ 6):

·^ban estimate with a precision of 0.00% indicates that the variation between and/or within the plate and the subject is greater than the variation between days and/or analysts, and an estimate cannot be calculated.

·^cData using logarithmic transformation

3.6.2 precision

Accuracy evaluations (CV [% ]) were performed across all 12 runs of the PC ratio (PC/NegC RLU ratio). See table 6. Each control and donor sample was analyzed separately as described in section 2.7.1. All precision estimates (including the total precision) across all control samples were within 18.4% (% CV). Since the lower bound of the 99% prediction interval on one side of HPC/NegC alone is higher than the cut point of 1.61, the acceptance criteria for HPC/NegC is ≧ 1.94, while all other PCs are ≧ 1.61.

TABLE 6 accuracy assessment and summary of the determination of PC/NegC RLU ratios

^aAn estimate of 0.0% accuracy indicates that the variation between and/or within plates is greater than the day and/or analyst variation, and an estimate cannot be calculated. The average of the plate repeats was used.

^bPrediction interval calculation is performed using the logarithmically transformed data.

3.6.3 selectivity

Selectivity was assessed with 20 individual SLE human serum study samples. Samples were run without and with anti-lullizumab (3 μ g/mL) incorporated at LPC levels. 75% of the unlabeled samples were negative and 100% of the labeled samples were positive under LPC (Table 7).

Table 7 selectivity assessment in SLE serum.

3.6.4 sensitivity

The assay sensitivity was defined as the lowest concentration of affinity purified mouse anti-lullizumab NAb PC that consistently produced results with a cut point equal to or higher than 1.61. The PC antibody was spiked into pooled normal human serum at 40, 20, 10, 4, 2, 1 and 0.5. mu.g/mL. During the cut-point evaluation, a total of 6 curves were drawn by 3 analysts over 2 days. The lowest PC concentration found to test reliably positive in all of these runs was 2.0. mu.g/mL. All 6 curves were analyzed by a four parameter logistic (4PL) function and interpolated at 1.61 using GraphPad prism software. The median value was 1.31, thus setting the estimated sensitivity of the assay to 1.31 μ g/mL (FIG. 5).

3.6.5 drug interference

LPC-1 and NegC at 3. mu.g/mL were spiked with 10, 5, 2.5 and 1. mu.g/mL lullizumab to estimate the drug tolerance of the assay. LPC doped with lullizumab was able to produce a positive reaction at the highest tested concentration (10 μ g/mL) and all NegC samples doped with lullizumab produced negative (non-neutralizing) results, thus determining that more than 10 μ g/mL of drug could be tolerated at LPC-1 levels. (Table 8).

TABLE 8 drug interference determined. In the presence of 10. mu.g/mL drug, NAb PC at LPC level (3. mu.g/mL) could be detected in the sample.

3.6.6 NAb PC stability

The room temperature and freeze-thaw stability of anti-lullizumab NAb PC in human serum was evaluated using HPC, LPC-1 and LPC-1-EXC levels. Three aliquots of each control level were left at ambient room temperature for 24 hours, or subjected to 10 freeze-thaw cycles. Samples were measured and classified relative to cut point. All samples met the screening criteria under each condition, confirming that NAb PC was stable for 24 hours at ambient room temperature, or could be stable for up to 10 freeze-thaw cycles (table 9).

TABLE 9 stability test. The PC was stable when left at room temperature for 24 hours or after 10 freeze-thaw cycles.

4. Discussion of the related Art

One of the major challenges of cell-based functional NAb assays, particularly for mAb biotherapeutics, is the presence of large amounts of drug in patient test samples, which often interferes with the detection of NAb in cell-based assays (16, 17).

In a typical ADA assay, a biotin-conjugated drug and an HRP or ruthenium labeled drug are added to the test sample to compete with the circulating drug for ADA in the sample; as long as sufficient ADA can form a complex with the labeled drug, the sample will be tested as ADA positive in the bridging assay. In other words, in the presence of an excess of labeled drug, it is possible to shift the equilibrium to favor the formation of an ADA-labeled drug complex, rather than an ADA-circulating drug complex (18). However, in cell-based NAb assays, the cells cannot distinguish between the drug in the sample and the added drug (such as biotinylated drug): all drugs present may have a direct or indirect effect on the cells. Thus, if the molar ratio of drug level to NAb exceeds 1:1, NAb will not be detectable unless the excess drug is removed prior to addition to the cell-based assay. The first method for successfully removing large amounts of drug for immunogenicity testing was SPEAD, solid phase extraction and acid dissociation: first incubating a sample containing a drug/ADA immune complex with a biotinylated drug overnight to form a biotin-drug/ADA complex; these complexes are then captured by streptavidin-coated high-binding plates. After washing, ADA (19) was eluted from the biotin-drug using an acid. This original SPEAD method has two limitations: 1) some ADA/NAb PCs have very high affinity and very low dissociation rates, making overnight incubation insufficient for the formation of a new equilibrium, favoring the biotin-drug/ADA complex. 2) In addition, the high binding plates have limited binding capacity, which limits the amount of biotinylated drug added; if the drug in the test sample is too high, only small amounts of ADA/NAb can be recovered with limited amounts of biotinylated drug, resulting in low drug tolerance and low NAb detection. For these reasons, BEAD evolved from SPEAD. An acid dissociation step was added to replace the overnight incubation without acid and the plates were replaced with streptavidin coated magnetic beads with much more binding surface so that more biotin-drug could be added resulting in more efficient competition for ADA/NAb binding (10).

Although the BEAD method has been successfully applied to a variety of NAb assays, we have recently found that up to 40% of the 15 NAb PCs we tested were acid labile and could not withstand strong acid treatment, resulting in low NAb recovery (data in preparation). This means that at least some NAb species in the real test sample may also be acid sensitive and may not be detectable after the bed extraction.

In the current manuscript, we further demonstrate that the new forms of biotherapeutics (e.g., pegylated domain Ab) may not be compatible with the BEAD extraction. In the current case study, we utilized the low melting temperature of rolizumab when compared to the intact 150kDa NAb. The 62 ℃ heating step not only dissociates the NAb/drug complex to replace the strong acid treatment in the bed, but also irreversibly denatures most of the drug in the test sample, leaving the intact NAb extracted with the non-pegylated biotin-lullizumab. Irreversible denaturation of the drug is desirable because a decrease in the amount of functional drug in the sample promotes the equilibrium towards the ADA/biotinylated drug complex, resulting in increased yield of extracted ADA. Notably, the rabbit pAb NAb PC with poor low pH stability survived heating at 62 ℃, suggesting that heating may be a potential alternative to pH sensitive ADA/NAb dissociation.

We were able to validate the assay with good accuracy, precision and sensitivity. The same bead extraction and thermal dissociation method (we name it BEHD) can be applied to any other biotherapeutic agent with a melting temperature sufficiently lower than that of a fully human Ab. To our knowledge, this was the first report of the use of heat to facilitate ADA/NAb extraction in immunogenicity testing.

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Equivalents of the formula

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.