Modifying binding molecules to minimize pre-existing interactions

Abstract

To provide binding molecules engineered to minimize or mitigate background reactivity in a sample matrix caused by drug non-specific binding interactions.SOLUTION: The present disclosure is directed towards modifying binding molecules in order to minimize pre-existing binding interactions, including binding molecules engineered to minimize or mitigate background reactivity in a sample matrix caused by drug non-specific binding interactions.SELECTED DRAWING: Figure 4

Description

Technical Field

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

[0002] Sequence Listing This application contains a Sequence Listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on Jul. 8, 2019, is named “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 and includes binding molecules engineered to minimize or mitigate background reactivity in a sample substrate caused by drug-nonspecific binding interactions.

Background Art

[0004] Biological therapies are a valuable means of treating disease by removing, replenishing, or replacing components of a subject's immune system. Substances derived from foreign organisms have the potential to induce an immune response in 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 therapies is the anti-drug antibody (ADA) assay. Using this assay, it is detected whether the immune system of a subject has produced antibodies against the administered biological therapy. For an assay to be effective, the signal-to-noise ratio must be such that meaningful data can be observed and analyzed. In some subjects, the detection of true ADA occurring under treatment is obscured by background signal and the effective ADA assay is diluted, such that high background (noise) already exists even without the administration of any biological therapy.

[0006] The compositions and methods of the present invention provide a way to reduce or eliminate the existing drug-nonspecific background reactivity present in some human serum or plasma samples observed in some ADA assays. It should also be understood that this drug-nonspecific 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 a binding molecule that has been engineered to mitigate existing drug-nonspecific background reactivity in a sample of a subject, either before or after administration of the binding molecule. The present invention is directed to modifying a binding molecule to minimize existing binding interactions and includes a binding molecule engineered to minimize or mitigate background reactivity in a sample matrix caused by drug-nonspecific binding interactions.

[0008] The present disclosure further provides a binding molecule that is specific for one or more specific targets. In one aspect, the present disclosure is directed to a binding molecule that is specific for IL4Rα (interleukin 4 alpha) or IL13R (interleukin 13). In another aspect, the present disclosure provides a binding molecule directed to an IgG4 antibody or a fragment 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 well 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 comprises the C-terminal heavy chain sequence LSPG (SEQ ID NO: 21) or an antigen-binding portion thereof, which molecule has been engineered to minimize or mitigate background reactivity in a sample matrix caused by drug-nonspecific binding interactions.

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

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

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

[0012] In certain embodiments of the non-natural binding molecules of the present disclosure, the binding molecule comprises a V that includes the amino acid sequence of SEQ ID NO: 9 H CDR1 region; a V that includes the amino acid sequence of SEQ ID NO: 10 H CDR2 region; a V that includes the amino acid sequence of SEQ ID NO: 11 H CDR3 region; a V that includes the amino acid sequence of SEQ ID NO: 12 L CDR1 region; a V that includes the amino acid sequence of LGS L CDR2 region; a V that includes 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 comprises a 3 domain.

[0013] In certain embodiments of the present invention, the binding molecule comprises a V that includes the amino acid sequence of SEQ ID NO: 9 H CDR1 region; a V that includes the amino acid sequence of SEQ ID NO: 10 H CDR2 region; a V that includes the amino acid sequence of SEQ ID NO: 11 H CDR3 region; a V that includes the amino acid sequence of SEQ ID NO: 12 L CDR1 region; a V that includes the amino acid sequence of LGS L CDR2 region; a V that includes the amino acid sequence of SEQ ID NO: 14 L CDR3 region, and a C that includes the amino acid sequence of SEQ ID NO: 21 H comprises a 3 domain.

[0014] The present disclosure provides a non-natural binding molecule, the binding molecule comprising a V that includes the amino acid sequence of SEQ ID NO: 9 H CDR1 region; a V that includes the amino acid sequence of SEQ ID NO: 10 H CDR2 region; a V that includes the amino acid sequence of SEQ ID NO: 11 H CDR3 region; a V that includes the amino acid sequence of SEQ ID NO: 12 L CDR1 region; a V that includes the amino acid sequence of LGS L CDR2 region; a V that includes the amino acid sequence of SEQ ID NO: 14 L CDR3 region, and a C that includes 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 comprises a 3 domain.

[0015] In certain embodiments of the present 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 certain embodiments of the present invention, the binding molecule comprises an IgG4 C H 1 region comprising the amino acid sequence of SEQ ID NO: 1.

[0017] In certain embodiments of the present invention, the binding molecule comprises an IgG4 C H 2 region comprising the amino acid sequence of SEQ ID NO: 2.

[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 includes (a) a solid support to which a first component is operably bound, and (b) at least one capture reagent to which a second component is operably bound, the first non-natural binding molecule being a monoclonal antibody or the like, the first non-natural binding molecule being a monoclonal antibody or the like, the V H sequence encoding the CDR1 region, the V H sequence encoding the CDR2 region, the V H sequence encoding the CDR3 region, the V L sequence encoding the CDR1 region, the V L sequence encoding the CDR2 region, the V LAt least one capture reagent comprising an array encoding a CDR3 region and an array encoding a heavy chain constant region, wherein the heavy chain constant region comprises the array 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, wherein the sequence encoding the detection reagent is that of the first non-natural binding molecule of (b), V H An array encoding a CDR1 region, V H An array encoding a CDR2 region, V H An array encoding a CDR3 region, V L An array encoding a CDR1 region, V L An array encoding a CDR2 region, V L At least one detection reagent comprising an array encoding a CDR3 region, and an assay comprising the same, wherein the first component and the second component selectively bind to each other.

[0021] In certain embodiments of the disclosed assay, the first non-natural binding molecule comprises an array encoding a heavy chain variable region and an array encoding a light chain variable region, and the second non-natural binding molecule comprises an array encoding a heavy chain variable region and an array encoding the 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 an array encoding a heavy chain constant region comprising SEQ ID NO: 7.

[0023] In certain embodiments of the disclosed assay, the first non-natural binding molecule comprises a V comprising the amino acid sequence of SEQ ID NO: 9 H CDR1 region; a V comprising the amino acid sequence of SEQ ID NO: 10 H CDR2 region; a V comprising the amino acid sequence of SEQ ID NO: 11 H CDR3 region; a V comprising the amino acid sequence of SEQ ID NO: 12 L CDR1 region; a V comprising the amino acid sequence of LGS L CDR2 region; a V comprising the amino acid sequence of SEQ ID NO: 14 L Comprising a 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 invention, the C H 3 domain of an IgG4 antibody such as dupilumab is changed to an IgG1 C H 3 domain. In further embodiments, the C H 3 domain of an IgG4 antibody such as dupilumab is cleaved. In one aspect, the cleavage occurs at serine 444.

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

[0027] In certain embodiments of the disclosed assay, the first component comprises streptavidin. In 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 at least one 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 certain embodiments, the first component comprises streptavidin. In certain embodiments, the second component comprises biotin. In certain embodiments, a binding molecule that does not specifically bind to the sequence of the variable region of dupilumab does not bind to at least one capture reagent. In certain embodiments, a binding molecule that specifically binds to the 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 the subject with an assay of the present disclosure under conditions suitable for at least one binding molecule in the biological sample to bind to at least one capture reagent and at least one detection reagent, wherein the subject has been administered a 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, or (d) identifying the level of immunogenicity of the subject as low if the signal from (b) is below the threshold.

[0030] Certain embodiments of the invention are directed to a method for determining the level of immunogenicity of a biological therapy, the biological therapy comprising a binding molecule as described herein. In one aspect, the biological 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 dose of the biological therapy is a therapeutically effective amount, which is an amount sufficient to achieve the intended purpose when the therapeutic agent, which is, for example, a binding molecule of the invention, is administered to the subject.

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

[0034] In other embodiments of the present invention, the subject is a participant in a clinical trial. In one aspect, the subject is a patient undergoing treatment. In another aspect, the treatment has just started and the level of immunogenicity is at the baseline level. In yet another aspect, the treatment is in progress and the level of immunogenicity is at a subsequent level. In still yet another aspect, the treatment is about to end and the level of immunogenicity is at the final level. In another aspect, the subject is a healthy individual.

[0035] In still further embodiments 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 response, or a combination thereof. The subject can be any medical condition or state.

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

[0037] A non-natural monoclonal antibody comprising a C H 3 domain or an antigen-binding portion thereof selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13.

[0038] A non-natural 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-natural monoclonal antibody, wherein the antibody comprises a V H CDR1 region comprising the amino acid sequence of SEQ ID NO: 9; a V H CDR2 region comprising the amino acid sequence of SEQ ID NO: 10; a V H CDR3 region comprising the amino acid sequence of SEQ ID NO: 11; a V L CDR1 region comprising the amino acid sequence of LGS; a V L CDR2 region comprising the amino acid sequence of SEQ ID NO: 14; a V L CDR3 region, and a C H 3 domain 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, or an antigen-binding portion thereof.

[0040] The aforementioned antibody comprises a V H CDR1 region comprising the amino acid sequence of SEQ ID NO: 9; a V H CDR2 region comprising the amino acid sequence of SEQ ID NO: 10; a V H CDR3 region comprising the amino acid sequence of SEQ ID NO: 11; a V L CDR1 region comprising the amino acid sequence of LGS; a V L CDR2 region, a V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region may further be included.

[0041] The present disclosure provides a non-natural monoclonal antibody, wherein the antibody comprises a V H CDR1 region comprising the amino acid sequence of SEQ ID NO: 9; a V H CDR2 region comprising the amino acid sequence of SEQ ID NO: 10; a V H CDR3 region comprising the amino acid sequence of SEQ ID NO: 11; a V L CDR1 region comprising the amino acid sequence of LGS; a V LCDR2 region; V comprising the amino acid sequence of SEQ ID NO: 14 L CDR3 region, and C comprising 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 domain, or an antigen-binding portion 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 1 region. The aforementioned antibody may comprise an IgG4 C H 2 region. IgG4 C H 2 regions may comprise a hinge region. The hinge region may comprise the sequence of APEFLG (SEQ ID NO: 17).

[0043] The antibodies of the present disclosure are capable of mitigating the high background signal 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 each other.

[0045] The detection reagent may comprise dupilumab. The second non-natural monoclonal antibody may comprise dupilumab. The first component may comprise streptavidin. The second component may comprise biotin.

[0046] 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 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 dupilumab, wherein the first component and the second component selectively bind to each other. The first component may comprise streptavidin.

[0047] The second component may comprise biotin.

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

[0049] The present disclosure provides a method for determining the level of immunogenicity of monoclonal antibody therapy in a subject, the method comprising: (a) contacting a biological sample from the subject with any assay of the present disclosure under conditions suitable for at least one antibody in the biological sample to bind to at least one capture reagent and at least one detection reagent, wherein the subject has been administered monoclonal antibody therapy prior to this 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 level of immunogenicity of the subject as low if the signal from (b) is below the threshold.

[0050] The monoclonal antibody therapy may comprise an antibody of the present disclosure.

[0051] Monoclonal antibody therapy may include dupilumab. The threshold may be a predetermined value. The threshold may be a safety threshold. The dosage of the monoclonal antibody therapy may be a therapeutically effective amount. The level of immunogenicity may be a baseline level. The level of immunogenicity may 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 have been initiated and the level of immunogenicity may be a baseline level. Treatment may be ongoing and the level of immunogenicity may be a subsequent level. Treatment may be ending and the level of immunogenicity may be 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 other aspects.

[0054] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification, the singular forms also include the plural forms unless the context clearly dictates otherwise. For example, the terms "a", "an", and "the" are to be understood as either singular or plural, and the term "or" is to be understood as inclusive. For example, "an element" means one or more elements. Throughout this specification, the word "comprising", or variations such as "comprise" or "comprises", are intended to mean the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. The term "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numerical values provided in this specification are modified by the term "about".

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

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

[0057] Unless otherwise defined, 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 hereby incorporated 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) and IL-4Rα (SEQ ID NO: 18). The term "human interleukin-13" (hIL-13) refers to a cytokine that specifically binds to the IL-13 receptor, and the "hIL-13 / hIL-13R1 complex" refers to a complex formed by hIL-13 binding to the hIL-13R1 complex, which complex binds to 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 and binds to a particular target. The target can include biological molecules or small (chemical) molecules. The target molecule can define an antigen or antigenic portion. 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 of skill in the art.

[0060] As used herein, the term "antibody" is an example of a binding molecule and is also referred to as an immunoglobulin and typically includes four polypeptide chains, two heavy (H) chains and two light (L) chains, which are linked to each other by disulfide bonds. Each heavy chain includes a heavy chain variable region (HCVR or V H ) and a heavy chain constant region. The heavy chain constant region includes three domains C H 1, C H 2 and C H 3. Each light chain includes a light chain variable region (LCVR or V L ) and a light chain constant region. The light chain constant region includes one domain (CL1). V H and V L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) and incorporate regions called framework regions (FRs) that are more conserved. V H and V LEach consists of three CDRs and four FRs, and is arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody may include an antibody of the IgG1, IgG2, IgG3 or IgG4 subclass. The antibody may also include a combination of regions from different subclasses. IgG4 antibodies may include, but are not limited to, dupilumab and semiprimab.

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

[0062] As used herein, the term "antigen-binding portion" (or simply "antibody portion" or "antibody fragment") of an antibody 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 included within the term "antigen-binding portion" of an antibody include (i) V L , V H , C L 1 and C H(i) A Fab fragment, which is a monovalent fragment containing 1 domain; (ii) An F(ab’)2 fragment, which is a divalent fragment containing two F(ab’) fragments linked by a disulfide bridge in the hinge region; (iii) An Fc fragment containing 1 domain; (iv) The V H and C H domains of one arm of the antibody; (v) An Fv fragment containing the V L and V H domains; (vi) A dAb fragment containing the V H domain (Ward et al. (1989) Nature 241:544 - 546), and (vi) CDRs. Further, the two domains V L and V H of the Fv fragment are encoded by different genes, but they can be linked by recombinant methods using a synthetic linker such that they form a single contiguous chain where the V L and V H regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, for example, 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 by the term “antigen-binding portion” of an antibody. Other forms of single-chain antibodies, such as bispecific antibodies, are also encompassed (see, for example, Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444 - 6448).

[0063] As used herein, the term "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. Inhibition of this hIL-4 and / or IL-13 biological activity can be evaluated by measuring one or more indicia of hIL-4 and / or hIL-13 biological activity known in the art, such as intracellular activities induced by hIL-4 and / or IL-13 and hIL-4 binding to hIL-4Rα (see the examples below).

[0064] "CDR" or complementarity determining region is a hypervariable region dispersed within a more conserved region called the "framework region" (FR). In various embodiments of the disclosed anti-hIL-4Rα antibodies or fragments, the FR may be identical to the human germline sequence or may be modified, either naturally or artificially.

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

[0066] The term "immunogenicity" refers to the ability of an antigen or immunogen to induce an immune response in a human or animal body. Protein therapeutics have the ability to elicit harmful immune reactions 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] When referring to a nucleic acid or a fragment thereof, the terms "substantially identical" or "substantially the same" mean that, when optimally aligned with another nucleic acid (or its complementary strand), including appropriate nucleotide insertions or deletions, at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases have nucleotide sequence identity, as measured by any well-known sequence identity algorithm such as, for example, FASTA, BLAST or Gap, as described below.

[0068] When used in reference to polypeptides, the terms "substantially similar" or "substantially identical" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned using, for example, the programs GAP or BESTFIT with the default gap weights. Typically, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are substitutions 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, conservative amino acid substitutions will not substantially change the functionality of the protein. If 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 upward 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) aliphatic-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 substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. "Moderately conservative" substitutions are any change having a non-negative value in the PAM250 log-likelihood matrix.

[0069] Sequence similarity to a polypeptide, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences by measuring similarities assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that 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 a wild-type protein and its 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 an alignment of the most overlapping regions between the query sequence and the search sequence and the percentage of sequence identity (Pearson (2000) supra). Another algorithm for comparing the disclosed sequences to databases containing numerous sequences from various organisms is the computer program BLAST, specifically BLASTP or TBLASTN, using default parameters. See, for example, 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] Examples of methods for producing human antibodies include those described in U.S. Patent No. 6,596,541, Green et al. (1994) Nature Genetics 7:13-21), U.S. Patent Nos. 5,545,807, 6,787,637.

[0071] Any method known in the art can be used to immunize rodents (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). The 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 are replaced with the corresponding human variable regions are inoculated with the antigen of interest, and lymphocytes (e.g., B cells) are recovered from the mice that express the antibody. These lymphocytes can be fused with a myeloma cell line to prepare immortalized hybridoma cell lines, and such hybridoma cell lines can be screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to the constant regions of the desired isotypes of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding the variable regions of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0072] DNA encoding the variable regions of the heavy and light chains of the antibody can be isolated and operably ligated to DNA encoding the constant regions of the human heavy and light chains. And this DNA can be expressed in cells capable of expressing fully human antibodies. In certain embodiments, the cells are CHO cells.

[0073] Antibodies can be therapeutically useful in blocking ligand-receptor interactions or inhibiting receptor component interactions rather than by cell death mediated by complement fixation (complement-dependent cytotoxicity) (CDC) and cell death involving antibody-dependent cellular cytotoxicity (ADCC). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of the antibody can be selected based on whether it is desirable for an antibody that mediates 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 approximately 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 molecules of approximately 75 - 80 kDa are formed, which are composed of covalently linked light and heavy chains (half-antibodies). These forms were extremely difficult to separate even after affinity purification. The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences related to the isotype of the hinge region of the antibody. In fact, one amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form relative to the levels typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure encompasses antibodies having one or more mutations within the hinge region of C H 2 or C H 3 that may be desired, for example, in production to improve the yield of the desired form of the antibody.

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

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

[0077] The present disclosure provides an immunogenicity assay, which includes an anti-IL-4Rα binding molecule that is an antibody of the present disclosure or its antigen-binding fragment, etc. 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 the ADA bridging assay, a biotinylated form of the binding molecule of interest binds to streptavidin on the plate. And a binding molecule similar to an antibody, when present in the sample, binds to both the biotinylated binding molecule and a labeled form of the same binding molecule to form 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 fluorophore. The ADA bridging assay can include titration of a sample that performs a bridging reaction to generate a standard curve and the use of an unlabeled cold-competition antibody that inhibits the reaction.

[0078] Figure 4 shows the drug-specific bridging ADA assay in Panel A, the non-drug-specific bridging assay due to the existing reactivity in the current ADA assay in Panel B, and the ADA assay without non-drug-specific bridging due to the use of REGN-C as a capture reagent in Panel C. The existing reactivity is seen in Panels B and C as the smaller cruciform structures with dashed outlines. The molecular analogs REGN-A, B, C, D, and E of the assay reagents of the disclosed embodiments are interchangeable with each other, whereby one skilled in the art should understand that various permutations of the described assay are permitted. 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 an arrangement such that the sequence order is "capture reagent" + "detection reagent" (reversed such as D + B, D + C, D + E, or B + D, C + D, or E + D) to form new combinations. In another aspect, one skilled in the art may make the following pairs: B + B, C + C, or E + E to reduce the background signal.

[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 antigen-binding portion thereof). In certain embodiments, the level of immunogenicity is evaluated for dupilumab using an IgG4 antibody. One problem typically requiring addressing in these immunogenicity tests is the high background signal, which is often associated with the IgG4 antibody used. To mitigate this high background, the present disclosure provides an IgG4 antibody comprising 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 present disclosure, a binding molecule, such as an antibody or antigen-binding portion thereof, comprises one or more V selected from the group consisting of (1) SEQ ID NO: 12, LGS, SEQ ID NO: 14, and combinations thereof LOne or more Vs selected from the group consisting of a heavy chain sequence, (2) SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H A heavy chain sequence, and (3) a C selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 7, and SEQ ID NO: 8 H Those containing a 3 or CH sequence, wherein the binding molecule is (1) one or more Vs selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14, and combinations thereof L A heavy chain sequence, (2) one or more Vs selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H A heavy chain sequence, and (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 Compared to a binding molecule containing a 3 or CH sequence, it exhibits reduced background reactivity in an immunogenicity (ADA) assay. In certain embodiments, 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, for example, the C H 3 domain of an IgG4 antibody such as dupilumab is H Changed to the IgG1 C H 3 domain, resulting in a lower background signal (Figure 6B). In a further embodiment, the C

[0082] In some aspects, the present disclosure provides (1) one or more Vs selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14, and combinations thereof L A heavy chain sequence, (2) one or more Vs selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof H A heavy chain sequence, and (3) a C 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 HProvide a binding molecule comprising 3 or a CH sequence.

[0083] In some embodiments, the present disclosure provides one or more V L chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14, and combinations thereof, (2) one or more V H chain sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof, and (3) C H 3 or C H sequences, a C H sequence 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, and (3) C H 3 or C

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

[0085] In some embodiments, the present disclosure provides one or more V L chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14, and combinations thereof, (2) one or more V H chain sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and combinations thereof, and (3) C H sequences, a C H sequence comprising one or more sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 21, and SEQ ID NO: 22, and (3) C

[0086] In some embodiments, the present disclosure provides CH An array C comprising one or more arrays selected from the group consisting of array number 8, array number 21, and array number 22 H Provided is a binding molecule comprising the array.

[0087] In some embodiments, the present disclosure provides a C H A C3 array comprising one or more arrays selected from the group consisting of array number 7, array number 13, or array number 21 H Provided is a binding molecule comprising the C3 array.

[0088] In some embodiments, the present disclosure provides a binding molecule comprising an IgG4 C domain with an amino acid substitution from proline to leucine at position 445 H .

[0089] In some embodiments, the present disclosure provides a binding molecule comprising array number 1, array number 2, and array number 5

[0090] In some embodiments, the present disclosure provides a binding molecule comprising array number 1, array number 2, and array number 6

[0091] In some embodiments, the present disclosure provides a binding molecule comprising: (1) one or more V - chain sequences selected from the group consisting of array number 12, LGS, array number 14, and combinations thereof; (2) one or more V - chain sequences selected from the group consisting of array number 9, array number 10, array number 11, and combinations thereof; and (3) a C array comprising array number 1, array number 2, and array number 5 L . H . H .

[0092] In some embodiments, the present disclosure provides a binding molecule comprising: (1) one or more V - chain sequences selected from the group consisting of array number 12, LGS, array number 14, and combinations thereof; (2) one or more V - chain sequences selected from the group consisting of array number 9, array number 10, array number 11, and combinations thereof; and (3) a C array comprising array number 1, array number 2, and array number 6 L . H .H Provided is a binding molecule comprising an array.

[0093] In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having 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 having 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 having 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 having 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 having 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 having 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 having at least about 93% sequence identity to SEQ ID NO: 7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having at least about 92% sequence identity to SEQ ID NO: 7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having at least about 91% sequence identity to SEQ ID NO: 7. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having 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 having 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 having 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 having 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 having 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 having 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 having 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 having at least about 93% sequence identity to SEQ ID NO: 8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having at least about 92% sequence identity to SEQ ID NO: 8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having at least about 91% sequence identity to SEQ ID NO: 8. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having 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 having 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 having 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 having 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 having 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 having 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 having 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 having at least about 93% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having at least about 92% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having at least about 91% sequence identity to SEQ ID NO: 13. In some embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 embodiments, the present disclosure provides a binding molecule comprising at least one polypeptide sequence having 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 can exhibit reduced background reactivity in an immunogenicity (ADA) assay as compared to a binding molecule comprising the C sequence of SEQ ID NO: 3. H In some embodiments, any binding molecule of the present disclosure can exhibit reduced background reactivity in an immunogenicity (ADA) assay as compared to a binding molecule comprising the C sequence of SEQ ID NO: 4. H In some embodiments, any binding molecule of the present disclosure can exhibit reduced background reactivity in an immunogenicity (ADA) assay as compared to a binding molecule comprising the C sequence of SEQ ID NO: 4.

[0099] Therapeutic Administration and Formulations The present disclosure provides a therapeutic composition comprising an anti-IL-4Rα binding molecule of the present disclosure. Administration of a therapeutic composition according to the present disclosure will be administered with suitable carriers, excipients, and other reagents that are included in the formulation to provide improved transport, delivery, resistance, etc. A number of suitable formulations can be found in the following formularies known to all pharmacists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (e.g., LIPOFECTIN™, etc.), DNA complexes, anhydrous absorbent pastes, oil-in-water emulsions 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 can vary depending on the age and physical build of the subject to be administered, the target disease, symptoms, route of administration, etc. When the binding molecule of the present disclosure is used to treat various symptoms and diseases related to IL-4Rα in adult patients, the binding molecule of the present disclosure is usually administered intravenously 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 molecule of the present disclosure is used to treat various symptoms and diseases related to IL-4Rα, the dosing schedule can be 300 mg once every two weeks (Q2W) and can be extended up to once every four weeks (Q4W). The frequency and duration of treatment can be adjusted according to the severity of the symptoms.

[0101] Various delivery systems are known and it is possible to administer the pharmaceutical composition of the present disclosure using them. For example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-dependent endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Routes of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and intraoral routes. The composition can be administered by any conventional route, for example, by infusion or bolus injection, by absorption through the epithelial or mucocutaneous layer (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

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

[0103] In certain embodiments, the pharmaceutical composition can be delivered by a sustained release system. In one embodiment, a pump may be used (see Langer supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). 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 sustained release system is provided near the target of the composition, whereby only a fraction of the minimal systemic dose will be required (see, for example, Goodson in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984). Other sustained release systems are discussed in the review by Langer (1990) Science 249:1527-1533.

[0104] Injectable preparations may include dosage forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, and intravenous infusions. These injectable preparations can be prepared by known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in, for example, a sterile aqueous medium or an oily medium conventionally used for injection. Examples of the aqueous medium for injection include physiological saline and isotonic solutions containing glucose and other adjuvants, and these can be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, and these can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus prepared is preferably filled in a suitable ampoule.

[0105] It is advantageous to prepare the above-described oral or parenteral pharmaceutical compositions in dosage forms of unit doses suitable to be commensurate with the dose of the active ingredient. Examples of such dosage forms of unit doses include tablets, pills, capsules, injectable preparations (ampoules), suppositories, etc. The amount of the aforementioned binding molecule contained is generally about 5 to 500 mg per dosage form of unit dose, and in certain embodiments, particularly in the form of an injectable preparation, the aforementioned binding molecule is contained at about 5 to 100 mg, and in other dosage forms at about 10 to 250 mg.

[0106] Monotherapy and combination therapy The binding molecules of the present disclosure are useful for treating diseases and disorders that can be ameliorated, inhibited, or remitted by reducing IL-4 activity. These diseases are diseases characterized by abnormal or overexpression of IL-4, or abnormal host responses to IL-4 production.

[0107] The present disclosure encompasses combination therapies in which an anti-IL-4Rα binding molecule (e.g., an antibody or antibody fragment) is administered in combination with a second therapeutic agent. Concurrent administration and combination therapies are not limited to simultaneous administration and include a treatment regimen in which the anti-IL-4Rα binding molecule is administered at least once during a series of treatments involving administration of at least one other therapeutic agent to a patient. The second therapeutic agent can be, for example, another IL-4 antagonist such as another binding molecule, or a soluble cytokine receptor, an IgE antagonist, an anti-asthmatic drug (corticosteroid, non-steroidal drug, 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 can be administered in combination with an IL-1 antagonist or an IL-13 antagonist such as rilonacept. In some aspects, an anti-IL-4Rα binding molecule such as a binding molecule of the present disclosure can be administered in combination with a binding molecule that targets cytokines and / or receptors in a type 1 or type 2 inflammatory response. The second agent can include one or more leukotriene receptor antagonists and can treat diseases such as allergic inflammatory diseases such as asthma and allergy. Examples of leukotriene receptor antagonists include, but are not limited to, montelukast, pranlukast, and zafirlukast. Examples of the second agent can include cytokine inhibitors such as one or more of an antagonist 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 present 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 present disclosure.

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

[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 molecule of the present invention is administered systemically. In one aspect, the binding molecule is administered intravenously or subcutaneously. In another aspect, the binding molecule is administered by injection or infusion. In yet another aspect, the binding molecule is administered by subcutaneous injection.

[0117] 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, a therapeutically effective amount comprises a subcutaneous injection of about 75 mg, 150 mg, 300 mg, or 600 mg. In yet another aspect, a 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 still yet another aspect, a therapeutically effective amount comprises a subcutaneous injection of about 75 mg, 150 mg, 300 mg, or 600 mg administered once a week, once every two weeks, once every four weeks, or long term as a maintenance amount 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, a 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 still yet another aspect, including aspects where 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 methods of the present disclosure, 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 immunosuppressive agent. 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-1β 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 immunosuppressive agent, a corticosteroid, a glucocorticoid, a proton pump inhibitor, a non-steroidal 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 condition and / or medical state. The compositions, formulations, kits, and methods of the present disclosure are applicable to any disease condition and / or medical state in which a patient exhibits an existing reactivity to antibody medicaments.

[0123]

Table 1-1

[0124]

Table 1-2

Examples

[0125] Example 1: IgG4 C H Substitution of the 3 constant regions reduces existing immunoreactivity in some patient samples. A competition inhibition test was conducted to characterize the high existing reactivity signals observed in several samples. In these tests, commercially available antibody agents or monoclonal antibodies specially constructed for this purpose were used. A list of antibody constructs or antibody agents useful for elucidating the specificity of this 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) confirmation assay format. This assay format is shown in Figure 4, and the results of this assay are shown in Figure 1 (Figure 1 and Figure 4). A high inhibition rate in this assay indicates that a given competitor was able to inhibit the existing reactivity signals in these samples, suggesting that the competing molecule contains the region to which the existing reactivity binds. A lower inhibition rate indicates that the competing molecule does not contain the region to which the existing reactivity can bind. The drugs listed in Table 1 above can be broadly classified into three categories: IgG4 backbone / Fc hinge region constructs, commercially available isotype antibodies, and dupilumab genetically engineered constructs. In the first experiment, it was investigated whether the high reactivity of this assay was directed towards the CDR of dupilumab or the IgG4 backbone. REGN-A is a human monoclonal antibody that has the same IgG4 backbone as dupilumab but a different CDR sequence and does not bind to IL-4Rα.

[0128] As shown in Figure 1, in the competitive inhibition experiments using both dupilumab and REGN-A, significant inhibition of the high assay signal was shown in six baseline samples from the patients examined. Since the CDR region sequences of these two antibodies are different, this inhibition result suggests that the high signal reactivity targets not the CDR portion of dupilumab, but rather some common antibody framework sequences. Both dupilumab and REGN-A contain the "CPPC" hinge region sequence of IgG1, which stabilizes the antibody hinge region within these IgG4 molecules. To determine whether existing immunoreactivity targets this CPPC mutation, a commercially available IgG4κ antibody with the wild-type (wt) CPSC sequence in the hinge region was examined in the competitive inhibition experiment. As can be seen in Figure 1, the wild-type IgG4 antibody also showed significant inhibition similar to that of dupilumab and REGN-A. This result suggests that the high baseline assay signal is not directed against the CPPC hinge region mutation in dupilumab, but rather is likely directed against the wild-type constant region of the IgG4 molecule. These results indicate that the high signal observed at baseline is not specific to dupilumab.

[0129] Further experiments were conducted to determine whether the high existing reactivity of this assay is directed against constant region sequences that would 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 existing reaction was examined. None of these antibodies showed a high baseline signal in these samples (see Figure 1). This suggests that the existing reactivity is likely associated with a region that is specific to the IgG4 constant region sequence and not common to any of the IgG subtypes tested.

[0130] A human monoclonal antibody (REGN-B) was constructed, which has an IgG4 C H The 3 domains are IgG1 C HIt is similar to dupilumab except that it is replaced by 3 domains (see Figure 2). This antibody was examined in a competition assay to determine whether the existing reactivity targeted the C H 3 domains. REGN-B did not significantly inhibit the high signal in the sample (see Figure 1), which suggested that the existing reactivity probably targeted some regions within the C H 3 domains.

[0131] To further identify the regions within the C H 3 domains of dupilumab that would be associated with these high signals, an alignment of the amino acid sequences of the C H 3 domains of IgG4, IgG1, and IgG2 antibodies was performed (see Figure 3). Six distinct amino acid positions of difference between the C H 3 domain sequences of IgG4 and IgG1, and five distinct amino acid positions of difference between the C H 3 domain sequences of IgG4 and IgG2 were recorded (see Figure 3).

[0132] An n IgG4 construct with leucine (L) at position 445 replaced by proline (P) was available and was examined in a competitive inhibition assay. This construct did not show significant inhibition of these high assay signals. This indicated that the existing reactivity was probably specific for the L445 region of dupilumab. For additional samples containing high assay signals, examination in a competitive inhibition assay using this construct with L replaced by P at position 445 showed similar low levels of inhibition. Thus, it appeared that the existing reactivity was confirmed to specifically target the region around L445. Therefore, by changing the leucine (present in wild-type IgG4) in dupilumab to proline (present at the same position in wild-type IgG1, IgG2, and IgG3), this existing reaction that leads to high signals in the ADA assay is suppressed.

[0133] An IgG4 construct with leucine (L) substituted by proline (P) at position 445 was available and examined in a competitive inhibition assay. This construct did not show significant inhibition of these high assay signals. This indicated that the existing reactivity was probably specific to the L445 region of dupilumab. For additional samples containing high assay signals, when examined in a competitive inhibition assay using this construct with L substituted by P at position 445, similar low levels of inhibition were observed. This thus confirmed that the existing reactivity appeared to specifically target the region around L445. Therefore, by changing the leucine (present in wild-type IgG4) in dupilumab to proline (present at the same position in wild-type IgG1, IgG2, and IgG3), this existing reaction that leads to high signals in the ADA assay is suppressed.

[0134] A second dupilumab-based human monoclonal antibody construct (REGN-C) was generated. This construct was identical to dupilumab except that it had a point mutation inserted that changed leucine to proline (abbreviated as L>P) at residue 445 within the antibody sequence. REGN-C was unable to significantly inhibit the high signal in the baseline samples tested (see Figures 1 and 2). This confirmed that the existing reactivity specifically targeted the region around L445 in dupilumab and also suggested that using this genetically engineered dupilumab in the ADA assay removed most, but not all, of the high levels of background signal observed in current ADA assays.

[0135] To demonstrate that the leucine-to-proline modification at residue 445 within the antibody sequence suppresses existing reactivity, two additional antibodies, REGN-F and REGN-G, were used for further experiments. As shown in Figure 7, competitive inhibition experiments using dupilumab, REGN-F, and REGN-G demonstrated significant inhibition of the high assay signals in six baseline samples, indicating that dupilumab, REGN-F, and REGN-G exhibit a high level of existing reactivity. However, when REGN-F and REGN-G were unable to inhibit the high assay signals, the existing reactivity was suppressed by substituting leucine with proline at position 445 of REGN-F and REGN-G, designated 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 agents in a drug-specific bridging anti-drug antibody assay similar to that shown in Figure 4. As shown in Figures 8 and 9, when both REGN-F and REGN-G were used as capture and detection reagents, they showed high assay signals, indicating signs of high existing reactivity. In contrast, when REGN-F(L445P) was used as the capture reagent in combination with REGN-F as the detection reagent (Figure 8), or when REGN-G was used as the capture reagent in combination with REGN-G(L445P) as the detection reagent (Figure 9), the assay signals were significantly reduced, demonstrating that the L445P substitution suppresses existing reactivity. REGN-F and REGN-G contain distinct 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 existing reactivity is independent of the variable domain and CDR, but instead is specific to the region around L445. Furthermore, this demonstrates that the L445P substitution can generally be applied to IgG4 antibodies regardless of the CDR identity of the IgG4 antibody to reduce existing reactivity.

[0136] Regions within the dupilumab array that target at least a majority of the existing reactivity were identified. The high signal in the ADA assay was due to a substrate composition in some of these serum samples that was able to cross-link labeled dupilumab molecules in the assay by binding at or near L445 within the C H 3 domain. This appears to have resulted from the fact that this existing reactivity is not drug-specific to dupilumab but can bind to any IgG4 molecule. Furthermore, this result suggests that using this modified form of dupilumab with the L445P mutation in the ADA assay removes most, but not all, of the high level of background signal.

[0137] Example 2: Development of a modified anti-drug antibody (ADA) assay that reduces background immunoreactivity in patient samples A modified ADA assay was developed that uses biotinylated REGN-C (with the L445P mutation) as a capture reagent. Figure 4 illustrates the differences in assay design 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 signals obtained by the current ADA assay #1 versus the 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 exact same samples in the ADA assay #2. The assay format of the ADA assay #2 significantly reduced the high signals observed in the current ADA screening assay. Although there were still samples showing reactivity in the ADA assay #2, the number of screening positives was more in harmony with the predicted false positive rate for the screening assay, and the signal response levels for these positive samples were generally much lower than the levels observed using the current ADA assay. The reduction of these high assay signals to near baseline values in most cases of the ADA assay #2 as observed should enable improved detection of ADA expressed under treatment and drug-specific ADA in the patient population.

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

[0139] Other embodiments Although specific embodiments of the present disclosure have been described and set forth, various other changes and modifications can be made without departing from the spirit and scope of the present disclosure. The appended claims cover all such changes and modifications that are within the scope of the present disclosure.

Claims

1. Heavy chain variable domain (V) containing the amino acid sequence of SEQ ID NO: 9 H ), complementarity determining region (CDR) CDR1 region, V containing the amino acid sequence of SEQ ID NO: 10 H CDR2 region, and V containing the amino acid sequence of SEQ ID NO: 11 H CDR3 region; Light chain variable domain (V) containing the amino acid sequence of SEQ ID NO: 12 L ), V containing the amino acid sequence of LGS L CDR2 region, and V containing the amino acid sequence of SEQ ID NO: 14 L CDR3 region; and A C-terminal heavy chain sequence represented by SEQ ID NO: 8: An anti-human interleukin-4 alpha (hIL4Rα) monoclonal antibody comprising the same.

2. The anti-hIL4Rα monoclonal antibody according to 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 bound, and (b) At least one capture reagent to which a second component is operably bound, the at least one capture reagent comprising the first monoclonal antibody according to claim 1 or 2, and (c) At least one detection reagent to which a detectable label is operably bound, the at least one detection reagent comprising the second monoclonal antibody according to 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 each other.

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

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

6. (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 the monoclonal antibody according to claim 1 or 2; and (c) At least one detection reagent to which a detectable label is operably bound, 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 each other.

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

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

9. The kit according to any one of claims 6 to 8, wherein the at least one capture reagent does not bind an antibody that specifically binds to the sequence of the variable region of dupilumab.

10. The kit according to 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 the sequence of the variable region of dupilumab.

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

12. The monoclonal antibody therapy 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 , 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 comprises an anti-hIL4Rα antibody comprising the C-terminal heavy chain sequence of SEQ ID NO: 8, The method according to claim 11.

13. The monoclonal antibody therapy comprises dupilumab, the method according to claim 12.

14. The method according to any one of claims 11 to 13, wherein the threshold value is a predetermined value.

15. The method according to claim 14, wherein the threshold value is a safety threshold value.

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

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

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

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

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

21. The method according to claim 20, wherein the treatment has just started and the level of immunogenicity is a baseline level.

22. The method according to claim 20, wherein the treatment is in progress and the level of immunogenicity is a subsequent level.

23. The method according to claim 20, wherein the treatment is about to end and the level of immunogenicity is at a final level. **Claim 24** The method according to any one of claims 11 to 23, wherein the subject is a healthy individual. **Claim 25** The method according to any one of claims 11 to 23, wherein 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. **Claim 26** The method according to claim 25, wherein the subject is atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

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