Engineered binding molecules to minimize existing interactions

Abstract

The present disclosure is directed to modifying binding molecules to minimize existing binding interactions, including binding molecules engineered to minimize or mitigate background reactivity in the sample matrix caused by drug-nonspecific binding interactions. [Selection diagram] Figure 4

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 695,988, filed July 10, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on July 8, 2019, is titled "REGE-004_SeqList.txt" and is 32,813 bytes in size.

[0003] The present disclosure is directed to modifying binding molecules to minimize existing binding interactions, including binding molecules engineered to minimize or mitigate background reactivity in the sample matrix caused by drug non-specific binding interactions. [Background technology]

[0004] Biological therapy is a valuable means of removing, supplementing, or replacing components of a subject's immune system to treat disease. Exogenous biological substances have the potential to induce an immune response from the subject being treated. The immune response may be triggered by the administration of the biological therapy. However, the subject may have components of the subject's serum proteins that generate a signal prior to the introduction of the biological therapy, thereby resulting in high background or noise levels when tested in an anti-drug antibody (ADA) assay.

[0005] The standard assay employed during the development and investigation of biological therapeutics is the anti-drug antibody (ADA) assay. This assay is used to detect whether the subject's immune system has produced antibodies against the administered biological therapeutic. To be a valid assay, the signal-to-noise ratio must be such that meaningful data can be observed and analyzed. In some subjects, there is already a high background (noise) without the administration of any biological therapeutic, such that the detection of true ADA occurring under treatment is obscured by background signals and the valid ADA assay is diluted.

[0006] The compositions and methods of the present invention provide an avenue for reducing or eliminating the existing drug non-specific background reactivity present in some human serum or plasma samples observed in some ADA assays. It should also be understood that this drug non-specific binding may or may not interfere with the ability of the treatment to be effective, or may or may not remain in circulation. Summary of the Invention

[0007] The present disclosure is directed to binding molecules that have been engineered to mitigate pre-existing drug non-specific background reactivity in a subject's sample either before or after administration of the binding molecule. The present invention is directed to modifying binding molecules to minimize pre-existing binding interactions, including binding molecules engineered to minimize or mitigate background reactivity in the sample matrix caused by drug non-specific binding interactions.

[0008] The present disclosure further provides binding molecules that are specific for one or more specific targets. In one aspect, the present disclosure is directed to binding molecules that are specific for IL4Rα (interleukin 4 alpha) or IL13R (interleukin 13). In another aspect, the present disclosure provides binding molecules that are directed to IgG4 antibodies or fragments thereof. A binding molecule as understood herein is a molecule that specifically interacts with a specific target. Examples of such binding molecules include, but are not limited to, antibodies (including monoclonal antibodies) and fragments thereof, engineered antibodies, fusion proteins, and other similar antigen-binding molecules known to those skilled in the art. In one aspect, the target is IL4Rα. In another aspect of the present invention, a non-natural binding molecule is disclosed that includes the C-terminal heavy chain sequence LSPG (SEQ ID NO: 21) or an antigen-binding portion thereof, which is engineered to minimize or reduce background reactivity in the sample matrix caused by drug non-specific binding interactions.

[0009] The present disclosure provides a non-naturally occurring binding molecule comprising a C-terminal heavy chain sequence, SEQ ID NO:21, or an antigen-binding portion thereof, which mitigates interactions with existing serum proteins, thereby reducing high background signals during ADA analysis.

[0010] In certain embodiments of the invention, the binding molecule comprises a CH domain sequence, or an antigen-binding portion thereof, selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In certain embodiments, the binding molecule comprises a CH domain sequence, or an antigen-binding portion thereof, selected from the group consisting of SEQ ID NO:7, and SEQ ID NO:8. In certain embodiments, the binding molecule comprises a CH domain comprising the amino acid sequence of SEQ ID NO:7.

[0011] In one embodiment of the invention, the binding molecule comprises a truncated CH domain (REGN-E), the sequence of which is SEQ ID NO:22.

[0012] In certain embodiments of the non-natural binding molecule of the present disclosure, the binding molecule comprises a V H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS L CDR2 region: V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region and a C selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 H Contains 3 domains.

[0013] In one embodiment of the invention, the binding molecule comprises the amino acid sequence of SEQ ID NO:9. H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS L CDR2 region: V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region, and a C H Contains 3 domains.

[0014] The present disclosure provides a non-naturally occurring binding molecule, the binding molecule comprising a V H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS L CDR2 region: V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region and a C sequence comprising the amino acid sequence of SEQ ID NO:5, or SEQ ID NO:6, or SEQ ID NO:7, or SEQ ID NO:21, or SEQ ID NO:22 H Contains 3 domains.

[0015] In certain embodiments of the invention, the binding molecule comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0016] In one embodiment of the invention, the binding molecule comprises an IgG4 C H Includes 1 region.

[0017] In one embodiment of the invention, the binding molecule comprises an IgG4 C H Includes 2 areas.

[0018] In certain embodiments of the non-natural binding molecules of the present disclosure, the binding molecule comprises a hinge region. In certain embodiments, the hinge region comprises the sequence of APEFLG (SEQ ID NO: 17).

[0019] In certain embodiments of the non-natural binding molecules of the present disclosure, the binding molecule comprises an IgG4 constant region comprising the amino acid sequence of SEQ ID NO:8.

[0020] The present disclosure provides a capture reagent comprising (a) a solid support to which a first component is operably attached, and (b) at least one capture reagent to which a second component is operably attached, the capture reagent comprising a first non-natural binding molecule, such as a monoclonal antibody, the first non-natural binding molecule being a monoclonal antibody, the first non-natural binding molecule being a monoclonal antibody, H A sequence encoding the CDR1 region, V H A sequence encoding the CDR2 region, V H A sequence encoding the CDR3 region, V L A sequence encoding the CDR1 region, V L A sequence encoding the CDR2 region, V Lat least one capture reagent comprising a sequence encoding a CDR3 region and a sequence encoding a heavy chain constant region, the heavy chain constant region comprising the sequence of SEQ ID NO:21; and (c) at least one detection reagent to which a detectable label is operably linked, the detection reagent comprising a second non-natural binding molecule, such as a monoclonal antibody, the sequence encoding the detection reagent being identical to the V H A sequence encoding the CDR1 region, V H A sequence encoding the CDR2 region, V H A sequence encoding the CDR3 region, V L A sequence encoding the CDR1 region, V L A sequence encoding the CDR2 region, V L and at least one detection reagent comprising a sequence encoding a CDR3 region, and wherein the first component and the second component selectively bind to one another.

[0021] In certain embodiments of the disclosed assays, a first non-natural binding molecule comprises a sequence encoding a heavy chain variable region and a sequence encoding a light chain variable region, and a second non-natural binding molecule comprises a sequence encoding a heavy chain variable region and a sequence encoding a light chain variable region of the first non-natural binding molecule.

[0022] In certain embodiments of the disclosed assay, the first non-natural binding molecule comprises a sequence encoding a heavy chain constant region comprising SEQ ID NO:7.

[0023] In one embodiment of the disclosed assay, the first non-natural binding molecule comprises the amino acid sequence of SEQ ID NO:9. H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS L CDR2 region: V comprising the amino acid sequence of SEQ ID NO: 14 L Includes the CDR3 region.

[0024] In certain embodiments of the disclosed assay, the first non-natural binding molecule comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0025] In one embodiment of the present invention, the C of an IgG4 antibody, such as dupilumab, H 3 domains, IgG1 C H In a further embodiment, the C domain of an IgG4 antibody, such as dupilumab, is modified to C3 domain. H The 3 domain is truncated. In one embodiment, the truncation occurs at serine 444.

[0026] In certain embodiments of the disclosed assays, the detection reagent comprises dupilumab. In certain embodiments, the second non-natural binding molecule comprises dupilumab.

[0027] In certain embodiments of the disclosed assays, the first component comprises streptavidin, hi certain embodiments, the second component comprises biotin.

[0028] The present disclosure provides an assay comprising: (a) a solid phase to which a first component is operably bound; (b) at least one capture reagent to which a second component is operably bound, the capture reagent comprising a non-natural binding molecule of the present disclosure or a composition of the present disclosure; and (c) at least one detection reagent to which a detectable label is operably bound, the at least one detection reagent comprising dupilumab, wherein the first component and the second component selectively bind to each other. In some embodiments, the first component comprises streptavidin. In some embodiments, the second component comprises biotin. In some embodiments, a binding molecule that is not specifically bound to a sequence of the variable region of dupilumab does not bind the at least one capture reagent. In some embodiments, a binding molecule that is specifically bound to a sequence of the variable region of dupilumab binds to at least one capture reagent and at least one detection reagent.

[0029] The present disclosure provides a method for determining the level of immunogenicity of a biological therapy in a subject, the method comprising: (a) contacting a biological sample from a subject with an assay of the present disclosure under conditions suitable for allowing at least one binding molecule in the biological sample to bind to at least one capture reagent and to at least one detection reagent, the subject having been administered the binding molecule therapy prior to the contacting step; (b) detecting a signal from the at least one detection reagent; and (c) identifying the level of immunogenicity of the subject as high if the signal from (b) exceeds a threshold value, or (d) identifying the level of immunogenicity of the subject as low if the signal from (b) is below the threshold value.

[0030] Certain embodiments of the invention are directed to a method of determining the level of immunogenicity of a biologic therapy, the biologic therapy comprising a binding molecule described herein. In one aspect, the biologic therapy comprises dupilumab.

[0031] In certain embodiments of the invention, a method for determining the level of immunogenicity of a biological therapy is disclosed, wherein the threshold is a predetermined value. In one aspect, the threshold is a safety threshold.

[0032] In certain embodiments of the invention, the amount of the biological therapy is a therapeutically effective amount, which is an amount of a therapeutic agent, e.g., a binding molecule of the invention, sufficient to achieve its intended purpose when administered to a subject.

[0033] In some embodiments of the invention, the level of immunogenicity is a baseline level, hi some aspects, the level of immunogenicity is a subsequent or post-treatment level.

[0034] In other embodiments of the invention, the subject is a participant in a clinical trial. In one aspect, the subject is a patient undergoing treatment. In another aspect, treatment has just begun and the level of immunogenicity is at a baseline level. In yet another aspect, treatment is ongoing and the level of immunogenicity is at a subsequent level. In yet another aspect, treatment is terminating and the level of immunogenicity is at a final level. In another aspect, the subject is a healthy individual.

[0035] In yet another embodiment of the present invention, the subject is an inflammatory disease or disorder, an autoimmune disease or disorder, an allergic disease or disorder, an immune disease or disorder, or a benign proliferative disease or disorder. In one aspect, the subject is atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, ABPA (allergic bronchopulmonary aspergillosis), bullous pemphigoid, chronic obstructive pulmonary disease (COPD), HFE (hand and foot eczema), prurigo nodularis, or any type 2 inflammatory reaction, or a combination thereof. The subject may be any disease or medical condition.

[0036] The present disclosure provides a non-naturally occurring monoclonal antibody comprising a C-terminal heavy chain sequence, or an antigen-binding portion thereof, comprising a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13. The C-terminal heavy chain sequence is H The C-terminal heavy chain sequence may be a 3 domain sequence. The C-terminal heavy chain sequence may comprise SEQ ID NO: 7. The C-terminal heavy chain sequence may comprise SEQ ID NO: 13. The C-terminal heavy chain sequence may comprise SEQ ID NO: 8. H The C-terminal heavy chain sequence may comprise a C domain sequence comprising SEQ ID NO:22. H It may include a domain sequence.

[0037] C consisting of a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13 H It is a non-naturally occurring monoclonal antibody comprising three domains or antigen-binding portions thereof.

[0038] A non-naturally occurring monoclonal antibody comprising a C-terminal heavy chain sequence consisting of a sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:22, or an antigen-binding portion thereof.

[0039] A non-naturally occurring monoclonal antibody, the antibody comprising the amino acid sequence of SEQ ID NO:9. H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS L CDR2 region: V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region and a C sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:13 H 3 domains, or antigen-binding portions thereof.

[0040] The aforementioned antibody comprises a V H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS L CDR2 region, V comprising the amino acid sequence of SEQ ID NO: 14 L It may further comprise a CDR3 region.

[0041] The present disclosure provides a non-naturally occurring monoclonal antibody, the antibody comprising a V H CDR1 region: V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region: V comprising the amino acid sequence of SEQ ID NO:11 H CDR3 region: V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region: V containing the amino acid sequence of LGS LCDR2 region: V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region and an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:13 H 3 domains, or antigen-binding portions thereof.

[0042] The aforementioned antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, or an antigen-binding portion thereof. The aforementioned antibody may comprise an IgG4 C H The aforementioned antibody may comprise an IgG4 C region comprising the amino acid sequence of SEQ ID NO:2. H IgG4 C H The two regions may include a hinge region. The hinge region may include the sequence of APEFLG (SEQ ID NO: 17).

[0043] The antibodies of the present disclosure can mitigate high background signals during immunogenicity analysis.

[0044] The present disclosure provides an assay comprising: (a) a solid phase to which a first component is operably bound; (b) at least one capture reagent to which a second component is operably bound, the at least one capture reagent comprising a first non-natural monoclonal antibody of the present disclosure; and (c) at least one detection reagent to which a detectable label is operably bound, the at least one detection reagent comprising a second non-natural monoclonal antibody of the present disclosure, wherein the first component and the second component selectively bind to one another.

[0045] The detection reagent may include dupilumab. The second non-naturally occurring monoclonal antibody may include dupilumab. The first component may include streptavidin. The second component may include biotin.

[0046] The present disclosure provides an assay comprising: (a) a solid phase to which a first component is operably attached; (b) at least one capture reagent to which a second component is operably attached, the at least one capture reagent comprising a non-native monoclonal antibody of the disclosure; and (c) at least one detection reagent to which a detectable label is operably attached, the at least one detection reagent comprising dupilumab, wherein the first component and the second component selectively bind to one another. The first component may comprise streptavidin.

[0047] The second component may include biotin.

[0048] In some embodiments, the at least one capture reagent does not bind an antibody that does not specifically bind to a variable region sequence of dupilumab. In some embodiments, the at least one capture reagent and the at least one detection reagent bind to an antibody that specifically binds to a variable region sequence of dupilumab.

[0049] The present disclosure provides a method for determining the level of immunogenicity of a monoclonal antibody therapy in a subject, the method comprising: (a) contacting a biological sample from a subject with any of the assays of the present disclosure under conditions suitable for allowing at least one antibody in the biological sample to bind to at least one capture reagent and to at least one detection reagent, the subject having been administered the monoclonal antibody therapy prior to the contacting step; (b) detecting a signal from the at least one detection reagent; and (c) identifying the level of immunogenicity of the subject as high if the signal from (b) exceeds a threshold value, or (d) identifying the level of immunogenicity of the subject as low if the signal from (b) is below the threshold value.

[0050] Monoclonal antibody therapy may include the antibodies of the present disclosure.

[0051] The monoclonal antibody therapy can include dupilumab. The threshold can be a predetermined value. The threshold can be a safety threshold. The amount of monoclonal antibody therapy can be a therapeutically effective amount. The level of immunogenicity can be a baseline level. The level of immunogenicity can be a subsequent level.

[0052] The subject may be a participant in a clinical trial. The subject may be a patient undergoing treatment. Treatment may be beginning and the level of immunogenicity may be at a baseline level. Treatment may be ongoing and the level of immunogenicity may be at a subsequent level. Treatment may be ending and the level of immunogenicity may be at a final level. The subject may be a healthy individual. The subject may have an inflammatory disease or disorder, an autoimmune disease or disorder, an allergic disease or disorder, an immune disease or disorder, or a benign proliferative disease or disorder. The subject may have atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

[0053] Any of the above aspects can be combined with any of the other aspects.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification, the singular also includes the plural unless the context clearly indicates otherwise, for example, the words "a", "an" and "the" are understood as singular or plural, and the word "or" is understood as inclusive. For example, "an element" means one or more elements. It will be understood that throughout this specification, the word "comprising" or variations such as "comprise" or "comprising" are intended to include the indicated elements, integers or steps, or groups of elements, integers or steps, but are not intended to exclude other elements, integers or steps, or groups of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the word "about." [Brief description of the drawings]

[0055] [Figure 1]FIG. 1 is a plot characterizing the specificity of pre-existing reactivity in baseline patient samples with competing antibody constructs in an anti-drug antibody (ADA) assay. The y-axis is percent inhibition from 0 to 100 in increments of 25. The x-axis is the specific competing antibody constructs used in this bridging assay, listed from left to right as dupilumab, REGN-A, wild-type IgG4 (wt IgG4), wild-type IgG1 (wt IgG1), wild-type IgG2 (wt IgG2), wild-type IgG3 (wt IgG3), REGN-B, and REGN-C. The six patient samples (S1-S6) are represented, in order, by circle, square, triangle, asterisk, diamond, and inverted triangle. The antibody reagents referred to in this graph were used at 200 μg / mL as competitive inhibitors in the ADA confirmatory assay format. A high percentage of inhibition in this assay indicates that the competitor was able to inhibit the pre-existing signal in the samples, suggesting that the competitor molecule contains a region to which pre-existing reactivity can bind, whereas a lower percentage of inhibition indicates that the competitor molecule does not contain a region to which pre-existing reactivity can bind. [Diagram 2] Figure 2 illustrates the three key constructs and their structural differences compared to dupilumab. REGN-B, REGN-C and REGN-E are molecular analogs of dupilumab. All three constructs are IgG4k constructs with the same specificity and similar affinity to anti-IL4R as dupilumab, and possess the same CPPC hinge region mutant sequence present in dupilumab. [Diagram 3] 3 is a diagram illustrating a comparison of the amino acid sequences and CH3 domains of wild-type IgG4, IgG1, and IgG2 subtypes. The leucine at position 445 is the third from the C-terminus of the CH3 domain sequence, as indicated by the arrow. [Figure 4]FIG. 4 is one embodiment showing a diagram depicting drug-specific bridging (panel A, ADA assay #1), non-drug-specific bridging due to pre-existing reactivity in the current ADA assay (panel B), and no non-drug-specific bridging due to the use of REGN-C as the capture reagent (panel C, ADA assay #2). Streptavidin on the plate is shown as a grey cross, biotin moieties as small black squares, and ruthenium labels as stars. In panels A and B, biotinylated REGN-D (dupilumab) capture reagent (black bifurcated structure) is shown, while in panel C, biotinylated REGN-C is shown (black and white checkered bifurcated structure). In panels B and C, pre-existing reactivity is represented by smaller bifurcated structures with dashed outlines. [Figure 5A] FIG. 5A is a plot showing the assay signal from a subset of patient baseline samples from the original ADA assay (assay #1) using biotinylated REGN-D as the capture reagent as illustrated in panel A of FIG. 4. The y-axis is signal / noise ratio from 0 to 100 on a logarithmic scale. The x-axis ranks individual patient samples by signal / noise ratio. Cut points are indicated by dashed lines. [Figure 5B] Figure 5B is a plot showing the assay signal of the same patient baseline samples from a modified ADA assay (assay #2) using biotinylated REGN-C as the capture reagent as illustrated in panel C of Figure 4. The y-axis is the signal / noise ratio from 0 to 100 on a logarithmic scale. The x-axis shows the individual patient samples in the same order as shown in Figure 5A. The cut points are indicated by dashed lines. [Figure 6]Figure 6A is a plot showing the assay signal from a subset of patient baseline samples from a drug-specific bridging anti-drug antibody (ADA) assay similar to that shown in Figure 4, using intact dupliumab as the capture and detection reagent. Figure 6B is a plot showing the assay signal from the same subset of patient baseline samples tested in Figure 6A, using a modified bridging ADA assay with REGN-B (Figure 2, a human IgG4 mAb in which the entire CH3 of IgG4 has been changed to the IgG1 CH3 domain) as the capture and detection reagent. Figure 6C is a plot showing the assay signal from the same subset of patient baseline samples tested in Figure 6A, using a modified bridging ADA assay with REGN-E (Figure 2, a human IgG4 mAb in which the CH3 domain has been truncated at the stop codon after serine 444) as the capture and detection reagent. [Figure 7] FIG. 7 is a series of plots characterizing the specificity of pre-existing reactivity in baseline patient samples using competing antibody constructs in an anti-drug antibody (ADA) assay. The y-axis is percent inhibition from 0 to 100 in increments of 25. The x-axis is the specific competing antibody construct used in the bridging assay, from left to right: dupilumab, REGN-F, and REGN-F(L445P) in the top plot, or dupilumab, REGN-G, and REGN-G(L445P) in the bottom plot. Six patient samples (S1-S6) are represented, respectively, by circles, squares, triangles, asterisks, diamonds, and inverted triangles. The antibody reagents referred to in this graph were used at 200 μg / mL as competitive inhibitors in an ADA confirmatory assay format. High percent inhibition in this assay indicates that the competitor was able to inhibit the pre-existing signal in these samples, suggesting that the competing molecule contains the region to which the pre-existing reactivity binds. A lower percent inhibition indicates that the competitor molecule does not contain a region to which the pre-existing reactivity can bind. [Figure 8]Figure 8 is a series of plots showing assay signals from a subset of patient baseline samples from a drug-specific bridging anti-drug antibody (ADA) assay similar to that shown in Figure 4, with REGN-F used as both the capture and detection reagent (top panel) or with REGN-F(L445P) used as the capture reagent in combination with REGN-F as the detection reagent. [Figure 9] Figure 9 is a series of plots showing assay signals from a subset of patient baseline samples from a drug-specific bridging anti-drug antibody (ADA) assay similar to that shown in Figure 4, with REGN-G used as both the capture and detection reagent (top panel) or REGN-G used as the capture reagent in combination with REGN-G(L445P) as the detection reagent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] It is to be recognized that this disclosure is not limited to the compositions and methods described herein, as well as the experimental conditions described, which may vary, and since the scope of the present disclosure will be limited only by the appended claims, it is to be understood that the language used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, any compositions, methods, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned are incorporated herein by reference in their entirety.

[0058] As used herein, the term "human IL4R" (hIL-4R) is intended to refer to a human cytokine receptor that specifically binds interleukin-4 (IL-4), IL-4Rα (SEQ ID NO: 18). The term "human interleukin-13" (hIL-13) refers to a cytokine that specifically binds the IL-13 receptor, and "hIL-13 / hIL-13R1 complex" refers to a complex formed by hIL-13 binding to the hIL-13R1 complex, which complex binds the hIL-4 receptor and elicits biological activity.

[0059] As used herein, the term "binding molecule" is intended to refer to a molecule that specifically interacts with and binds to a particular target. Targets can include biological molecules or small (chemical) molecules. Target molecules may define antigens or antigenic portions. Examples of binding molecules include, but are not limited to, antibodies (including monoclonal antibodies, bispecific antibodies, and antibody fragments), fusion proteins, and other antigen-binding molecules known to those skilled in the art.

[0060] As used herein, the term "antibody" is an example of a binding molecule, also called an immunoglobulin, that typically contains four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or V H ) and a heavy chain constant region. The heavy chain constant region is made up of three domains, C H 1. C H 2 and C H Each light chain comprises a light chain variable region (LCVR or V L ) and a light chain constant region. The light chain constant region contains one domain (CL1). H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which incorporate regions that are more conserved, called framework regions (FRs). H and V LEach is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies may include antibodies of the subclasses IgG1, IgG2, IgG3, or IgG4. Antibodies may also include a combination of regions from different subclasses. IgG4 antibodies may include, but are not limited to, dupilumab and cemiplimab.

[0061] Other examples of "binding molecules" include, but are not limited to, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and pentaspecific antibodies. In some embodiments, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and pentaspecific antibodies may comprise an Fc portion of an antibody. In some embodiments, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and pentaspecific antibodies may comprise an IgG4 backbone. Another example of a "binding molecule" includes an antibody-drug conjugate (ADC). In some embodiments, an ADC may comprise an IgG4 backbone. Another example of a "binding molecule" includes a bispecific T cell conjugate (BiTE). In some embodiments, a BiTE may comprise an IgG4 backbone. Another example of a "binding molecule" includes a TRAP fusion protein. In some embodiments, a TRAP fusion protein has an IgG4 backbone. Another example of a "binding molecule" includes a fynomer.

[0062] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-4Rα). It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L 1 and C H(ii) a F(ab')2 fragment, which is a bivalent fragment containing two F(ab') fragments linked by disulfide bridges in the hinge region; (iii) a V H and C H (iv) an Fc fragment containing one domain; (iv) a V of one arm of an antibody L and V H (v) an Fv fragment containing the V H (vi) CDRs, and (vii) dAb fragments, which contain the V domain of the Fv fragment. L and V H Although the V L and V H The regions can be linked, using recombinant methods, by synthetic linkers that allow them to be made into a single contiguous chain that pairs to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as, for example, diabodies, are also encompassed (see, e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448).

[0063] As used herein, a "neutralizing" or "blocking" antibody is intended to refer to an antibody whose binding to hIL-4Rα results in inhibition of the biological activity of hIL-4 and / or hIL-13. This inhibition of the biological activity of hIL-4 and / or IL-13 can be assessed by measuring one or more indicia of the biological activity of hIL-4 and / or hIL-13 known in the art, such as, for example, intracellular activity induced by hIL-4 and / or by IL-13 and hIL-4 binding to hIL-4Rα (see Examples below).

[0064] "CDRs" or complementarity determining regions are hypervariable regions dispersed within more conserved regions called "framework regions" (FRs). In various embodiments of the disclosed anti-hIL-4Rα antibodies or fragments, the FRs may correspond to human germline sequences or may be naturally or artificially modified.

[0065] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. An antigen may have one or more epitopes. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues within a polypeptide chain. In some circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0066] The term "immunogenicity" refers to the ability of an antigen or immunogen to induce an immune response in the human or animal body. Protein therapeutics have the potential to elicit adverse immune responses that can interfere with the pharmacokinetics and efficacy of the drug. This immune response can take the form of the production of anti-drug antibodies (ADA).

[0067] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned, including appropriate nucleotide insertions or deletions, with another nucleic acid (or its complementary strand), there is nucleotide sequence identity of at least about 95%, more preferably at least about 96%, 97%, 98% or 99%, of the nucleotide bases as measured by any well-known sequence identity algorithm, such as, for example, FASTA, BLAST or Gap, as described below.

[0068] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, for example, by the GAP or BESTFIT programs, using default gap weights. Typically, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functionality of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) fatty-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. In certain embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood table, as disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood table.

[0069] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences by measuring the similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their variants. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the regions of most overlap between the query sequence and the search sequence and the percentage of sequence identity (Pearson (2000) supra). Another algorithm for comparing disclosed sequences to a database containing a large number of sequences from various organisms is the computer program BLAST, specifically BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0070] Methods for producing human antibodies include, for example, those described in U.S. Pat. No. 6,596,541, Green et al. (1994) Nature Genetics 7:13-21), and U.S. Pat. Nos. 5,545,807 and 6,787,637.

[0071] Rodents can be immunized by any method known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory; Malik and Lillehoj (1994) Antibody Techniques, Academic Press, CA). Antibodies of the present disclosure are typically prepared using VELOCIMMUNE® technology (U.S. Patent No. 6,596,541). Transgenic mice in which the heavy and light chain variable regions of endogenous immunoglobulins have been replaced with the corresponding human variable regions are inoculated with the antigen of interest, and lymphoid cells (e.g., B cells) are collected from the mice expressing the antibody. The lymphoid cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and combined with constant regions of the desired isotype of heavy and light chains. Such antibody proteins may be produced in cells, such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable regions may be isolated directly from antigen-specific lymphocytes.

[0072] DNA encoding the heavy and light chain variable regions of the antibody may be isolated and operably linked to DNA encoding human heavy and light chain constant regions, and this DNA may be expressed in a cell capable of expressing fully human antibodies. In one embodiment, the cell is a CHO cell.

[0073] Antibodies may be therapeutically useful in blocking ligand-receptor interactions or inhibiting receptor component interactions, rather than by cell killing via fixation of complement (complement-dependent cytotoxicity) (CDC) and by involvement of antibody-dependent cellular cytotoxicity (ADCC). The constant region of the antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0074] Human immunoglobulins can exist in two forms with hinge heterogeneity. In the first form, the immunoglobulin molecule contains a stable four-chain construct of about 150-160 kDa, in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, composed of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification. The frequency of occurrence of the second form in the various intact IgG isotypes is due to, but not limited to, isotype-related structural differences in the hinge region of the antibody. In fact, a single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form relative to the levels typically observed with a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure provides methods for improving the yield of desired antibody forms, for example in production, by using C H 2 or C H The present invention also includes antibodies having one or more mutations in the hinge region of SEQ ID NO:3.

[0075] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As described below, the antibody is characterized and selected for desired properties, including binding affinity to hIL-4Rα, ability to block binding of hIL-4 to hIL-4Rα, and / or selectivity for human proteins. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody of the present disclosure, e.g., wild-type or modified IgG4 or IgG1 (e.g., SEQ ID NOs: 4, 19, 20, and 23). The constant region selected can vary depending on the particular application, but the variable region has high affinity antigen binding properties and target specificity.

[0076] Immunogenicity assays The present disclosure provides a non-natural binding molecule, such as a monoclonal antibody or antigen-binding portion thereof, comprising the C-terminal heavy chain sequence LSPG (SEQ ID NO: 21). In certain embodiments, the C-terminal heavy chain is a human C H 3 domains. In some embodiments, the human antibody is of the IgG4 class. In some embodiments, the heavy chain sequence comprises SEQ ID NO: 7. In some embodiments, the heavy chain sequence comprises SEQ ID NO: 8.

[0077] The present disclosure provides an immunogenicity assay, including an anti-IL-4Rα binding molecule, such as an antibody or antigen-binding fragment thereof, of the present disclosure. The immunogenicity assay of the present disclosure can take the form of an anti-drug antibody (ADA) assay. The ADA assay of the present disclosure can be an ADA binding assay or can be directed to an enzyme-linked immunosorbent (ELISA) assay. In an ADA bridging assay, a biotinylated form of the binding molecule of interest binds to streptavidin on a plate. An antibody-like binding molecule, when present in the sample, then binds to both the biotinylated binding molecule and a labeled form of the same binding molecule, forming a bridging interaction with a detectable signal from the label. Suitable labels will be known to those skilled in the art. Exemplary labels include ruthenium, horseradish peroxidase, alkaline phosphatase, and fluorophores. The ADA bridging assay can include titration of samples that undergo a bridging reaction to generate a standard curve, and the use of an unlabeled cold competitive antibody to inhibit the reaction.

[0078] FIG. 4 shows a drug-specific bridging ADA assay in panel A, a non-drug-specific bridging assay due to pre-existing reactivity in the current ADA assay in panel B, and an ADA assay without non-drug-specific bridging due to the use of REGN-C as a capture reagent in panel C. The pre-existing reactivity is seen in panels B and C as smaller bifurcated structures with dashed outlines. It should be understood that the molecular analogs REGN-A, B, C, D, and E of the assay reagents of the disclosed embodiments are interchangeable, thereby allowing one skilled in the art to design various permutations of the described assay. In one aspect, to reduce background, one skilled in the art may combine REGN-D with any of the following reagents: REGN-B, REGN-C, or REGN-E, in a sequence order of "capture reagent" + "detection reagent" (flipped as D+B, D+C, D+E, or B+D, C+D, or E+D) to form new combinations. In another embodiment, one skilled in the art may make the following pairs to reduce background signal: B+B, C+C, or E+E.

[0079] In certain embodiments of the present disclosure, the level of immunogenicity (or ADA) is determined using a binding molecule, such as an antibody (or an antigen-binding portion thereof). In certain embodiments, the level of immunogenicity is assessed with dupilumab using an IgG4 antibody. One issue that typically needs to be addressed in these immunogenicity studies is high background signal, which is often associated with the IgG4 antibody used. To mitigate this high background, the present disclosure provides an IgG4 antibody that includes a heavy chain sequence of any of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:7, or SEQ ID NO:8, resulting in a lower background signal.

[0080] In certain embodiments of the disclosure, a binding molecule, e.g., an antibody or an antigen-binding portion thereof, comprises one or more Vs selected from the group consisting of: (1) SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L(2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) a C sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:7, and SEQ ID NO:8 H 3 or CH sequence, and the binding molecule comprises: (1) one or more V selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) a C not selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:7, and SEQ ID NO:8 H In one embodiment, the CH domain sequence of (3) may include SEQ ID NO:5 and SEQ ID NO:6.

[0081] In one embodiment of the present invention, the C of an IgG4 antibody, such as dupilumab, H 3 domains, IgG1 C H 3 domain, resulting in a lower background signal (FIG. 6B). In a further embodiment, the C H The 3 domain is truncated, in one embodiment, at serine 444, resulting in a lower background signal (Figure 6C).

[0082] In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: (1) one or more V sequences selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) a C sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:7, and SEQ ID NO:8 H3 or CH sequence.

[0083] In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: (1) one or more V sequences selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) C H 3 or C H C comprising one or more sequences selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:7 and SEQ ID NO:8. H 3 or C H The present invention provides a binding molecule comprising the sequence:

[0084] In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: (1) one or more V sequences selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) C H 3 sequences, comprising one or more sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 21. H 3 sequences.

[0085] In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: (1) one or more V sequences selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) C H C comprising one or more sequences selected from the group consisting of SEQ ID NO:8, SEQ ID NO:21 and SEQ ID NO:22. H The present invention provides a binding molecule comprising the sequence:

[0086] In some aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising:H C comprising one or more sequences selected from the group consisting of SEQ ID NO:8, SEQ ID NO:21 and SEQ ID NO:22. H The present invention provides a binding molecule comprising the sequence:

[0087] In some aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: H 3 sequences, comprising one or more sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 21. H 3 sequences.

[0088] In some aspects, the present disclosure provides an IgG4 C antigen comprising an amino acid substitution at position 445 from proline to leucine. H A binding molecule comprising the domain is provided.

[0089] In some aspects, the disclosure provides binding molecules comprising SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5.

[0090] In some aspects, the disclosure provides binding molecules comprising SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6.

[0091] In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: (1) one or more V sequences selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) C including SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5 H A binding molecule comprising the sequence is provided.

[0092] In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: (1) one or more V sequences selected from the group consisting of SEQ ID NO:12, LGS, SEQ ID NO:14, and combinations thereof. L (2) one or more V selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H (3) C including SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6H A binding molecule comprising the sequence is provided.

[0093] In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO:7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO:7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO:7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO:7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO:7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO:7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO:7. In some embodiments, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 7. In some embodiments, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO: 7. In some embodiments, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO: 7.

[0094] In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO:8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO:8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO:8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO:8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO:8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO:8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO:8. In some embodiments, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 8. In some embodiments, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO: 8. In some embodiments, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO: 8.

[0095] In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 913% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO: 13. In some aspects, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 13. In some aspects, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO: 13. In some aspects, the disclosure provides binding molecules comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO: 13.

[0096] In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21.In some aspects, the disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO:7, wherein the at least one polypeptide sequence comprises SEQ ID NO:21.

[0097] In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21.In some aspects, the disclosure provides a binding molecule comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO:8, wherein the at least one polypeptide sequence comprises SEQ ID NO:21.

[0098] In some embodiments, any binding molecule of the present disclosure comprises a C H In some embodiments, the binding molecule of the present disclosure may exhibit reduced background reactivity in an immunogenicity (ADA) assay compared to a binding molecule comprising the sequence SEQ ID NO: 4. H The antibody may exhibit reduced background reactivity in an immunogenicity (ADA) assay compared to a binding molecule containing the sequence.

[0099] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising the anti-IL-4Rα binding molecules of the present disclosure. The therapeutic compositions according to the present disclosure will be administered with suitable carriers, excipients, and other agents that are included in the formulation to provide improved transport, delivery, tolerance, and the like. A number of suitable formulations can be found in the following formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, etc.), DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0100] The dosage may vary depending on the age and size of the subject to be administered, the target disease, symptoms, administration route, etc. When the binding molecules of the present disclosure are used to treat various symptoms and diseases associated with IL-4Rα in adult patients, it is advantageous to administer the binding molecules of the present disclosure intravenously, usually at a single dose of about 0.01 to about 20 mg per kg of body weight, more typically about 0.02 to about 7 mg, about 0.03 to about 5 mg, or about 0.05 to about 3 mg per kg of body weight. In some embodiments, when the binding molecules of the present disclosure are used to treat various symptoms and diseases associated with IL-4Rα, the administration schedule can be 300 mg once every two weeks (Q2W) and can be extended up to every four weeks (Q4W). The frequency and duration of treatment can be adjusted depending on the severity of the symptoms.

[0101] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and buccal routes. The compositions can be administered by any conventional route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0102] Pharmaceutical compositions can also be delivered in vesicles, for example liposomes (see Langer (1990) Science 249:1527-1533; Treat et al. (1989); Lopez-Berestein, ibid., pp. 317-327 in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365).

[0103] In certain embodiments, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201, supra). In another embodiment, a polymeric material can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, the controlled release system is located near the target of the composition, thereby requiring only a small portion of the systemic dose (see, e.g., Goodson in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984). Other controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.

[0104] The injectable preparations may include dosage forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in, for example, a sterile aqueous medium or an oily medium conventionally used for injections. Examples of aqueous media for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents, which may be used in combination with a suitable solubilizer, such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. Examples of oily media include sesame oil, soybean oil, and the like, which may be used in combination with a solubilizer, such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations thus prepared are preferably filled into suitable ampoules.

[0105] The oral or parenteral pharmaceutical compositions described above are advantageously prepared into a dosage form with a suitable unit dose to correspond to the dosage of the active ingredient. Such dosage forms with a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the binding molecule contained is generally about 5 to 500 mg per dosage form in a unit dose, and in some embodiments, the binding molecule is contained in an amount of about 5 to 100 mg, particularly in the form of an injection, and about 10 to 250 mg in other dosage forms.

[0106] The binding molecules of the disclosure are useful for treating diseases and disorders that are ameliorated, inhibited, or ameliorated by reducing IL-4 activity, such as diseases characterized by aberrant or overexpression of IL-4 or an aberrant host response to IL-4 production.

[0107] The present disclosure encompasses combination therapy, in which an anti-IL-4Rα binding molecule (e.g., an antibody or antibody fragment) is administered in combination with a second therapeutic agent. Coadministration and combination therapy are not limited to simultaneous administration, but include treatment regimens in which an anti-IL-4Rα binding molecule is administered at least once during a course of treatment with at least one other therapeutic agent administered to the patient. The second therapeutic agent can be another IL-4 antagonist, such as another binding molecule, or a soluble cytokine receptor, an IgE antagonist, an anti-asthma drug (corticosteroid, nonsteroid, β-agonist, leukotriene antagonist, xanthine, fluticasone, salmeterol, albuterol) that can be delivered by inhalation or other suitable means. In certain embodiments, an anti-IL-4Rα binding molecule, such as a binding molecule of the present disclosure, may be administered with an IL-1 antagonist or an IL-13 antagonist, such as rilonacept. In some embodiments, anti-IL-4Rα binding molecules, such as those disclosed herein, may be administered in combination with binding molecules targeting cytokines and / or receptors in type 1 or type 2 inflammatory responses. The second agent may include one or more leukotriene receptor antagonists to treat diseases such as allergic inflammatory diseases, e.g., asthma and allergies. Examples of leukotriene receptor antagonists include, but are not limited to, montelukast, pranlukast, and zafirlukast. The second agent may include a cytokine inhibitor, such as one or more of antagonists of TNF (etanercept, ENBREL™), IL-9, IL-5, or IL-17.

[0108] therapeutic use The present disclosure provides compositions and methods for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a binding molecule of the disclosure.

[0109] The present disclosure provides compositions and methods for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the disclosure.

[0110] In certain embodiments of the disclosure, the disease or disorder is a type 1 or type 2 inflammatory disease or disorder.

[0111] In certain embodiments of the present disclosure, the disease or disorder is an autoimmune disease or disorder.

[0112] In certain embodiments of the present disclosure, the disease or disorder is an allergic disease or disorder.

[0113] In certain embodiments of the present disclosure, the disease or disorder is an immune disease or disorder.

[0114] In certain embodiments of the present disclosure, the disease or disorder is a benign proliferative disease or disorder. In certain embodiments of the present disclosure, the disease or disorder is a malignant proliferative disease or disorder.

[0115] In certain embodiments of the present disclosure, the disease or disorder is atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

[0116] In certain embodiments of the present disclosure, the binding molecules of the invention are administered systemically. In one aspect, the binding molecules are administered intravenously or subcutaneously. In another aspect, the binding molecules are administered by injection or infusion. In yet another aspect, the binding molecules are administered by subcutaneous injection.

[0117] In some embodiments of the disclosure, the binding molecule is administered systemically. In one aspect, the binding molecule is administered by subcutaneous injection. In another aspect, the therapeutically effective amount comprises a subcutaneous injection of about 75 mg, 150 mg, 300 mg, or 600 mg. In yet another aspect, the therapeutically effective amount comprises at least one subcutaneous injection, at least two subcutaneous injections, at least three subcutaneous injections, or at least four subcutaneous injections of about 75 mg, 150 mg, 300 mg, or 600 mg. In yet another aspect, the therapeutically effective amount comprises a subcutaneous injection of about 75 mg, 150 mg, 300 mg, or 600 mg administered once every week, once every two weeks, once every four weeks, or chronically as a maintenance dose to control the symptoms of the disease.

[0118] In certain embodiments of the present disclosure, the binding molecule is administered systemically. In one aspect, the binding molecule is administered by subcutaneous injection. In another aspect, the therapeutically effective amount comprises an initial dose of about 600 mg. In yet another aspect, the initial dose comprises a pair of injections of 300 mg each administered at two different injection sites. In yet another aspect, including the aspect in which the therapeutically effective amount comprises an initial dose, the therapeutically effective amount further comprises a maintenance dose of about 300 mg. In another aspect, the maintenance dose is administered every other week.

[0119] In certain embodiments of the disclosed methods, the binding molecule is administered systemically. In one aspect, the binding molecule is administered intravenously at a dose of about 1.0 mg / kg, 3.0 mg / kg, 8.0 mg / kg, or 12.0 mg / kg.

[0120] In certain embodiments of the present disclosure, the binding molecule is administered in combination with a second therapeutic agent. In one aspect, the second therapeutic agent comprises an immunosuppressant. In one aspect, the second therapeutic agent comprises an agonist antibody. In one aspect, the second therapeutic agent comprises an immunoactivator. In another aspect, the second therapeutic agent comprises an IL-1 β-inhibitor, an IL-5 inhibitor, an IL-9 inhibitor, an IL-3 inhibitor, an IL-13 inhibitor, an IL-17 inhibitor, an IL-25 inhibitor, a TNFα inhibitor, an eotixin-3 inhibitor, an IgE inhibitor, a prostaglandin D2 inhibitor, an immunosuppressant, a corticosteroid, a glucocorticoid, a proton pump inhibitor, a nonsteroidal anti-inflammatory drug (NSAID), or a combination thereof.

[0121] In certain embodiments of the present disclosure, the binding molecule is administered in combination with a second therapeutic agent. In one aspect, the second therapeutic agent comprises a corticosteroid. In a particular aspect, the corticosteroid is a topical corticosteroid.

[0122] It should be appreciated that the compositions, formulations, kits, and methods of the present disclosure are not limited to any one disease state and / or medical condition, and may be applied to any disease state and / or medical condition in which a patient exhibits pre-existing responsiveness to an antibody therapeutic.

[0123] [Table 1-1]

[0124] [Table 1-2] EXAMPLES

[0125] Example 1: IgG4 C H The 3 constant region substitution reduces pre-existing immune reactivity in some patient samples. Competitive inhibition studies were performed to characterize the high pre-existing reactivity signals observed in some samples. In these studies, commercially available antibody agents or monoclonal antibodies specifically constructed for this purpose were used. A list of antibody constructs or antibody agents that helped to elucidate the specificity of this pre-existing reactivity is shown in Table 2.

[0126] [Table 2]

[0127] These antibody constructs were used at 200 μg / mL as competitive inhibitors in an anti-drug antibody (ADA) confirmatory assay format. The assay format is shown in FIG. 4, and the results of this assay are shown in FIG. 1 (FIG. 1 and FIG. 4). A high inhibition rate in this assay indicates that a given competitor was able to inhibit the signal of pre-existing reactivity in these samples, suggesting that the competing molecule contains a region to which the pre-existing reactivity binds. A lower inhibition rate indicates that the competing molecule does not contain a region to which the pre-existing reactivity can bind. The agents listed in Table 1 above can be broadly divided into three categories: IgG4 backbone / Fc hinge region constructs, commercially available isotype antibodies, and dupilumab engineered constructs. In initial experiments, we investigated whether the high reactivity in this assay was directed to the CDRs or IgG4 backbone of dupilumab. REGN-A is a human monoclonal antibody with the same IgG4 backbone as dupilumab but different CDR sequences, and does not bind to IL-4Rα.

[0128] As shown in Figure 1, competitive inhibition experiments with both dupilumab and REGN-A showed significant inhibition of the high assay signal in six baseline samples from the patients examined. Because the CDR region sequences of these two antibodies are different, this inhibition result suggests that the high signal reactivity is not targeted to the CDR portion of dupilumab, but rather to some common antibody backbone sequence. Both dupilumab and REGN-A contain the IgG1 "CPPC" hinge region sequence, which stabilizes the antibody hinge region in these IgG4 molecules. To determine whether the pre-existing immune reactivity is targeted to this CPPC mutation, a commercially available IgG4 kappa antibody with a wild-type (wt) CPSC sequence in the hinge region was examined in competitive inhibition experiments. As can be seen in Figure 1, the wild-type IgG4 antibody also showed significant inhibition similar to dupilumab and REGN-A. This result suggests that the high baseline assay signal is probably directed against the wild-type constant region of the IgG4 molecule, rather than directed against the CPPC hinge region mutation in dupilumab. These results indicate that the high signal observed at baseline is not specific for dupilumab.

[0129] Further experiments were performed to determine whether the high pre-existing reactivity of this assay was directed against constant region sequences that may be common among the various IgG subtypes. Using the same competitive inhibition approach, the effect of three commercially available human IgG1κ, IgG2κ, and IgG3κ antibodies on this pre-existing reactivity was examined. None of these antibodies showed a high baseline signal in these samples (see Figure 1). This suggested that the pre-existing reactivity was likely associated with a region that was unique to the IgG4 constant region sequence and that was not common among any of the IgG subtypes tested.

[0130] A human monoclonal antibody (REGN-B) was constructed, which is an IgG4 C H 3 domains IgG1 C HThis antibody is similar to dupilumab except that it is replaced by a C3 domain (see Figure 2). H REGN-B did not significantly inhibit the high signal in the samples (see Figure 1), possibly due to pre-existing reactivity to the C3 domain of the IgG4 molecule. H These results suggest that the protein targets certain regions within the three domains.

[0131] Dupilumab C H To further identify the regions that may be associated with these high signals within the three domains, we performed a multi-domain analysis of the C H The amino acid sequences of the three domains were aligned (see Figure 3). H Differences at six distinct amino acid positions between the three domain sequences and the C H Differences at five distinct amino acid positions between the three domain sequences were recorded (see FIG. 3).

[0132] An IgG4 construct with a leucine (L) at position 445 substituted with a proline (P) was available and tested in a competitive inhibition assay. This construct did not show significant inhibition of these high assay signals, indicating that the pre-existing reactivity was likely specific to the L445 region of dupilumab. Additional samples with high assay signals were tested in a competitive inhibition assay with this construct with a P substituted for L at position 445, and a similar low level of inhibition was observed. This seemed to confirm that the pre-existing reactivity was specifically targeted to the region around L445. Thus, changing the leucine (present in wild-type IgG4) in dupilumab to a proline (present in the same position in wild-type IgG1, IgG2, and IgG3) suppresses this pre-existing reactivity that leads to a high signal in the ADA assay.

[0133] An IgG4 construct with a leucine (L) to proline (P) substitution at position 445 was available and tested in a competitive inhibition assay. This construct did not show significant inhibition of these high assay signals, indicating that the pre-existing reactivity was likely specific to the L445 region of dupilumab. Additional samples with high assay signals were tested in a competitive inhibition assay with this construct with a P to L substitution at position 445, and a similar low level of inhibition was observed. This seemed to confirm that the pre-existing reactivity was specifically targeted to the region around L445. Thus, changing the leucine (present in wild-type IgG4) in dupilumab to a proline (present in the same position in wild-type IgG1, IgG2, and IgG3) suppresses this pre-existing reactivity that leads to a high signal in the ADA assay.

[0134] A second dupilumab-based human monoclonal antibody construct (REGN-C) was generated. This construct is identical to dupilumab except for the insertion of a point mutation at residue 445 in the antibody sequence that changes leucine to proline (abbreviated as L>P). REGN-C was unable to significantly inhibit the high signal in the baseline samples tested (see Figures 1 and 2). This confirmed that the pre-existing reactivity specifically targets the region around L445 in dupilumab and suggested that the use of this engineered dupilumab in the ADA assay would eliminate most, if not all, of the high level background signal observed in the current ADA assay.

[0135] Further experiments were performed with two additional antibodies, REGN-F and REGN-G, to demonstrate that the leucine to proline modification at residue 445 in the antibody sequence suppresses pre-existing reactivity. As shown in FIG. 7, competitive inhibition experiments with dupilumab, REGN-F and REGN-G demonstrated significant inhibition of the high assay signal in six baseline samples, indicating that dupilumab, REGN-F and REGN-G exhibit high levels of pre-existing reactivity. However, when REGN-F and REGN-G were unable to inhibit the high assay signal, this pre-existing reactivity was suppressed by a leucine to proline substitution at position 445 of REGN-F and REGN-G, referred to herein as REGN-F(L445P) and REGN-G(L445P), respectively. REGN-F, REGN-F(L445P), REGN-G and REGN-G(L445P) were also tested as drugs in a drug-specific bridging anti-drug antibody assay similar to that shown in FIG. 4. As shown in Figures 8 and 9, both REGN-F and REGN-G showed high assay signals when used as capture and detection reagents, which is an indication of high pre-existing reactivity. In contrast, when REGN-F(L445P) as capture reagent was used in combination with REGN-F as detection reagent (Figure 8), or when REGN-G as capture reagent was used in combination with REGN-G(L445P) as detection reagent (Figure 9), the assay signal was significantly reduced, demonstrating that the L445P substitution suppresses pre-existing reactivity. REGN-F and REGN-G contain separate variable domains that are also different from the variable domains of the antibodies tested in the results shown in Figure 1. That is, the results shown in Figures 7-9 demonstrate that pre-existing reactivity is independent of the variable domains and CDRs, but is instead specific to the region around L445. Furthermore, this demonstrates that the L445P substitution can be applied to IgG4 antibodies in general, regardless of the identity of their CDRs, to reduce pre-existing reactivity.

[0136] A region within the dupilumab sequence that is targeted by at least the majority of the existing reactivity was identified. H This was likely due to some substrate configuration in these serum samples that could bridge between labeled dupilumab molecules in the assay by binding at or near L445 in the 3 domain. This indicates that this pre-existing reactivity is not dupilumab drug specific, but can bind to any IgG4 molecule. Furthermore, this result suggests that by using this modified form of dupilumab with the L445P mutation in the ADA assay, most, if not all, of the high level background signal is eliminated.

[0137] Example 2: Development of a modified anti-drug antibody (ADA) assay that reduces background immune reactivity in patient samples A modified ADA assay was developed that uses biotinylated REGN-C (with the L445P mutation) as the capture reagent. Figure 4 illustrates the assay design differences between the current ADA assay and the modified ADA assay using REGN-C. For simplicity, the current ADA assay will be referred to as "ADA assay #1" and the modified ADA assay will be referred to as "ADA assay #2". Figure 5 shows a comparative analysis of the ADA screening signal obtained by the current ADA assay #1 versus ADA assay #2 from all patient baseline samples. Panel 5A shows a plot of the signal to noise ratio generated by these high ADA signal baseline samples in the current ADA screening assay (assay #1), while panel 5B shows a plot of the signal to noise ratio generated by the very same samples in ADA assay #2. The assay format of ADA assay #2 significantly reduced the high signal observed in the current ADA screening assay. Although some samples were still reactive in ADA Assay #2, the number of screen positives was more in line with the expected false positive rate for the screening assay, and the signal response levels for these positive samples were generally much lower than those observed with the current ADA assay. This observed reduction of these high assay signals to near baseline values ​​in most cases for ADA Assay #2 should allow for improved detection of treatment-emergent and drug-specific ADA in patient populations.

[0138] Incorporation by Reference All documents cited herein, including any cross-referenced or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art to any disclosure disclosed or claimed herein, or that it alone or in any combination with other references teaches, suggests or discloses such disclosure. Furthermore, to the extent that any meaning or definition of a word in this document conflicts with any meaning or definition of the same word in a document incorporated by reference, the meaning or definition assigned to that word in this document shall control.

[0139] Other embodiments While particular embodiments of the present disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the present disclosure, and the appended claims are intended to cover all such changes and modifications that are within the scope of the present disclosure.

Claims

1. A heavy chain variable domain (V H Complementarity determining region (CDR) CDR1 region, V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region, and a V H CDR3 region; A light chain variable domain (V L ) CDR1 region, V containing the amino acid sequence of LGS L CDR2 region, and a V L A CDR3 region; and C-terminal heavy chain sequence represented by SEQ ID NO:8: An anti-human interleukin 4 alpha (hIL4Rα) monoclonal antibody comprising:

2. The anti-hIL4Rα monoclonal antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

16.

3. (a) a solid phase to which a first component is operably attached; and (b) at least one capture reagent to which a second component is operably linked, the capture reagent comprising a first monoclonal antibody according to claim 1 or 2; and (c) at least one detection reagent to which a detectable label is operably linked, the at least one detection reagent comprising the second monoclonal antibody of claim 1 or 2; A kit for use in an anti-drug antibody (ADA) assay comprising: The kit, wherein the first component and the second component selectively bind to one another.

4. The kit of claim 3 , wherein the first component comprises streptavidin.

5. The kit of claim 3 or 4, wherein the second component comprises biotin.

6. (a) a solid phase to which the first component is operably attached; (b) at least one capture reagent to which a second component is operably linked, the capture reagent comprising the monoclonal antibody of claim 1 or 2; and (c) at least one detection reagent operably linked to a detectable label, the at least one detection reagent comprising dupilumab; A kit for use in an anti-drug antibody (ADA) assay comprising: The kit, wherein the first component and the second component selectively bind to one another.

7. The kit of claim 6 , wherein the first component comprises streptavidin.

8. The kit of claim 6 or 7, wherein the second component comprises biotin.

9. 9. The kit of any one of claims 6-8, wherein the at least one capture reagent does not bind antibodies that do not specifically bind to a variable region sequence of dupilumab.

10. 9. The kit of any one of claims 6 to 8, wherein the at least one capture reagent and the at least one detection reagent bind to an antibody that specifically binds to a sequence of the variable region of dupilumab.

11. A method for determining the level of immunogenicity of a monoclonal antibody therapy in a subject with a kit according to any one of claims 3 to 10, the method comprising the steps of: (a) contacting a biological sample from a subject with the at least one capture reagent and the at least one detection reagent of the kit under conditions suitable to allow binding of at least one antibody in the biological sample to the at least one capture reagent and the at least one detection reagent of the kit, the subject having been administered the monoclonal antibody therapy prior to the contacting step; (b) detecting a signal from the at least one detection reagent; and (c) identifying the level of immunogenicity of the subject as high if the signal from (b) exceeds a threshold; or (d) identifying the subject's level of immunogenicity as low if the signal from (b) is below the threshold.

12. The monoclonal antibody therapy comprises: A V comprising the CDR1 amino acid sequence of SEQ ID NO:9, the CDR2 amino acid sequence of SEQ ID NO:10, and the CDR3 amino acid sequence of SEQ ID NO:

11. Ｈ , A V comprising the CDR1 amino acid sequence of SEQ ID NO:12, the CDR2 amino acid sequence of LGS, and the CDR3 amino acid sequence of SEQ ID NO:

14. Ｌ , and C-terminal heavy chain sequence of SEQ ID NO:8; The method of claim 11, comprising an anti-hIL4Rα antibody comprising:

13. 13. The method of claim 12, wherein the monoclonal antibody therapy comprises dupilumab.

14. 14. The method of claim 11, wherein the threshold is a predetermined value.

15. The method of claim 14 , wherein the threshold is a safety threshold.

16. 16. The method of any one of claims 11 to 15, wherein the subject has been administered a therapeutically effective dose of the monoclonal antibody therapy.

17. 17. The method of any one of claims 11 to 16, wherein the level of immunogenicity is a baseline level.

18. 17. The method of any one of claims 11 to 16, wherein the level of immunogenicity is a subsequent level.

19. 17. The method of any one of claims 11 to 16, wherein the subject is a participant in a clinical trial.

20. 20. The method of any one of claims 11 to 19, wherein the subject is a patient undergoing treatment.

21. 21. The method of claim 20, wherein the treatment has begun and the level of immunogenicity is at a baseline level.

22. 21. The method of claim 20, wherein the treatment is ongoing and the level of immunogenicity is a subsequent level.

23. 21. The method of claim 20, wherein the treatment is terminating and the level of immunogenicity is a final level.

24. 24. The method of any one of claims 11 to 23, wherein the subject is a healthy individual.

25. 24. The method of any one of claims 11 to 23, wherein the subject has an inflammatory disease or disorder, an autoimmune disease or disorder, an allergic disease or disorder, an immune disease or disorder, or a benign proliferative disease or disorder.

26. 26. The method of claim 25, wherein the condition is atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

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