METHOD FOR DETERMINING THE IMMUNOGENICITY LEVEL OF A MONOCLONAL ANTIBODY THERAPY IN A PATIENT

Description

54 METHOD FOR DETERMINING THE IMMUNOGENICITY LEVEL OF A MONOCLONAL ANTIBODY THERAPY IN A PATIENT Separated from BR112020026784-8, filed on 10 / 07 / 2019 RELATED ORDERS

[001] This application claims priority and benefit of the Application U.S. Provisional Order No. 62 / 695,988, filed July 10, 2018, the contents of which are incorporated herein by reference in their entirety. LISTING OF SEQUENCES

[002] This request contains a Sequence Listing that was sent in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The said ASCII copy, created on July 8, 2019, is named “REGE-004_SeqList.txt” and is 32,813 bytes in size. FIELD OF DISSEMINATION

[003] This disclosure is directed toward modifying binding molecules to minimize pre-existing binding interactions, including binding molecules designed to minimize or mitigate background reactivity in a sample matrix caused by non-drug specific binding interactions. BACKGROUND OF THE TECHNIQUE

[004] Biological therapy is a valuable tool for eliminating, supplementing, or replacing elements of a patient's immune system to treat diseases. The foreign biological material has the potential to induce an immune response in the patient being treated. This immune response can be triggered by the administration of a biological therapeutic. However, a patient may have elements of their serum proteins that produce a signal before the introduction of a biological product, thus creating a high level of background or noise when tested in an anti-drug antibody (ADA) assay. Petition 870260045338, dated 05 / 13 / 2026, page 15 / 90 / 54

[005] A standard assay employed during the development and surveillance of a biological therapy is the anti-drug antibody (ADA) assay. This assay is used to detect whether a patient's immune system has produced antibodies against an administered biological agent. For an assay to be effective, the signal-to-noise ratio must be such that meaningful data can be obtained and analyzed. In certain patients, there is already a high background noise, even without the administration of any biological product, so that the detection of true treatment-emergent ADA is obscured due to the background signal and an effective ADA assay is attenuated.

[006] The compositions and methods of the present invention provide a way to reduce or eliminate pre-existing non-specific drug background reactivity present in some human serum or plasma samples that is observed in some ADA assays. It should also be understood that this non-specific drug binding may or may not interfere with the ability of a therapeutic to be active or remain in circulation. BRIEF DESCRIPTION

[007] The present disclosure is directed to binding molecules designed to mitigate pre-existing non-specific drug background reactivity in a patient sample before or after drug administration. The present invention is directed to modifying binding molecules to minimize pre-existing binding interactions, including binding molecules designed to minimize or mitigate background reactivity in a sample matrix caused by non-specific drug binding interactions.

[008] This disclosure also provides specific binding molecules for one or more particular targets. In one aspect, the disclosure is directed to specific binding molecules for IL4Ra (Interleukin 4 alpha) or IL13R (Interleukin 13). In another aspect, the disclosure provides Petition 870260045338, dated 05 / 13 / 2026, p. 16 / 90 / 54 binding molecules targeted to IgG4 antibodies or fragments thereof. A binding molecule as understood herein is a molecule that interacts specifically with a particular 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 IL4Ra. In another aspect of the present invention, a non-naturally occurring binding molecule comprising a C-terminal LSPG heavy chain sequence (SEQ ID NO: 21) or an antigen-binding portion thereof is disclosed, which is designed to minimize or mitigate background reactivity in a sample matrix caused by non-specific drug binding interactions.

[009] The present disclosure provides a non-naturally occurring binding molecule, comprising a C-terminal heavy chain sequence SEQ ID NO: 21, or its antigen-binding portion, wherein said binding molecule mitigates interaction with pre-existing serum proteins and, as such, reduces the high background signal during ADA analysis.

[0010] In certain embodiments of the present invention, the linking 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 linking 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 linking molecule comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 7.

[0011] In certain embodiments of the present invention, the linking molecule comprises a truncated CH domain (REGN-E), wherein the sequence is SEQ ID NO: 22. Petition 870260045338, dated 05 / 13 / 2026, page 17 / 90 / 54

[0012] In certain embodiments of the non-naturally occurring binding molecule of disclosure, the binding molecule comprises a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 VH region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 VH region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 VL region comprising the amino acid sequence LGS; a CDR3 VL region comprising the amino acid sequence SEQ ID NO: 14, and a CH3 domain selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0013] In certain embodiments of the invention, the linking molecule further comprises a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 VH region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 Vh region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 VL region comprising the amino acid sequence LGS; a CDR3 VL region comprising the amino acid sequence SEQ ID NO: 14 and a CH3 domain comprising the amino acid sequence SEQ ID NO: 21.

[0014] The disclosure provides a non-naturally occurring linking molecule, wherein the linking molecule comprises a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 Vh region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 Vh region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 Vl region comprising the amino acid sequence LGS; a CDR3 Vl region comprising the Petition 870260045338, dated 05 / 13 / 2026, page 18 / 90 / 54 amino acid sequence of SEQ ID NO: 14, and a Ch3 domain comprising the amino acid sequence of SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 21, or SEQ ID NO: 22.

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

[0016] In certain embodiments of the present invention, the binding molecule comprises a Ch1 region of IgG4 comprising the amino acid sequence SEQ ID NO: 1.

[0017] In certain embodiments of the invention, the binding molecule comprises a Ch2 region of IgG4 comprising the amino acid sequence SEQ ID NO: 2.

[0018] In certain embodiments of the non-naturally occurring disclosure linking molecule, the linking molecule comprises a hinge region. In certain embodiments, the hinge region comprises the APEFLG sequence (SEQ ID NO: 17).

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

[0020] The disclosure provides an assay comprising (a) a solid support, wherein a first component is operationally linked to the solid support; (b) at least one capture agent, wherein a second component is operationally linked to at least one capture agent, wherein the capture agent comprises a first non-naturally occurring binding molecule, such as a monoclonal antibody, wherein the first non-naturally occurring binding molecule, such as a monoclonal antibody, comprises a sequence encoding a CDR1 Vh region, Petition 870260045338, dated 05 / 13 / 2026, page. 19 / 90 / 54 a sequence encoding a CDR2 Vh region, a sequence encoding a CDR3 Vh region, a sequence encoding a CDR1 Vl region, a sequence encoding a CDR2 Vl region, a sequence encoding a CDR3 Vl region, and a sequence encoding a heavy chain constant region, wherein the heavy chain constant region comprises sequence SEQ ID NO: 21, and (c) at least one detection agent, wherein the detectable marker is operationally linked to the detection agent, wherein the detection agent comprises a second non-naturally occurring binding molecule, such as a monoclonal antibody, wherein the sequence encoding the detection agent comprises the sequence encoding a CDR1 Vh region, the sequence encoding a CDR2 Vh region, the sequence encoding a CDR3 Vh region, the sequence encoding a CDR1 Vl region, the sequence encoding a CDR2 Vl region,the sequence that encodes a CDR3 VL region of the first non-naturally occurring binding molecule of (b), and in which the first component and the second component selectively bind to each other.

[0021] In certain embodiments of the disclosed assays, the first unnaturally occurring linking molecule comprises a sequence encoding a variable heavy chain region and a sequence encoding a variable light chain region, and wherein the second unnaturally occurring linking molecule comprises the sequence encoding a variable heavy chain region and the sequence encoding a variable light chain region of the first unnaturally occurring linking molecule.

[0022] In certain embodiments of the disclosed assays, the first non-naturally occurring linking molecule comprises a sequence encoding a heavy chain constant region comprising SEQ ID NO: 7. Petition 870260045338, dated 05 / 13 / 2026, page 20 / 90 / 54

[0023] In certain embodiments of the disclosed assays, the first unnaturally occurring linking molecule comprises a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 Vh region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 VH region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 VL region comprising the amino acid sequence LGS; a CDR3 VL region comprising the amino acid sequence SEQ ID NO: 14.

[0024] In certain embodiments of the disclosed assays, the first unnaturally occurring linking molecule comprises a variable region of the heavy chain comprising the amino acid sequence SEQ ID NO: 15 and a variable region of the light chain comprising the amino acid sequence SEQ ID NO: 16.

[0025] In one embodiment of the present invention, the CH3 domain of an IgG4 antibody, such as dupilumab, is exchanged for a CH3 domain of IgG1. In yet another embodiment, the CH3 domain of an IgG4 antibody such as dupilumab is truncated. In one aspect, the truncation occurs at Serine 444.

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

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

[0028] The disclosure provides an assay comprising (a) a solid support, in which a first component is operationally connected to the solid support; (b) at least one capture agent, where a second component is operationally connected to at least one capture agent. Petition 870260045338, dated 05 / 13 / 2026, p. 21 / 90 / 54 and wherein the capture agent comprises the non-naturally occurring binding molecule of the disclosure or a composition of the disclosure; and (c) at least one detection agent, wherein a detectable marker is operationally linked to the detection agent and wherein the detection agent comprises 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 a sequence of a variable region of dupilumab does not bind to at least one capture agent. In certain embodiments, a binding molecule that specifically binds to a sequence of a variable region of dupilumab binds to at least one capture agent and at least one detection agent.

[0029] The disclosure provides a method for determining the immunogenicity level of a biological therapy in a patient, comprising (a) placing a biological sample from the patient in contact with the disclosure assay under conditions suitable to allow binding of at least one binding molecule in the biological sample to at least one capture agent and to at least one detection agent, wherein the patient received the binding molecule therapy prior to the contact step, (b) detecting a signal from at least one detection agent, and (c) identifying the patient's immunogenicity level as high when the signal from (b) is above a threshold value or (d) identifying the patient's immunogenicity level as low when the signal from (b) is below the threshold value.

[0030] Certain embodiments of the present invention are directed to methods for determining the immunogenicity level of a biological therapy, wherein the biological therapy comprises a binding molecule described herein. In one aspect, the biological comprises dupilumab.

[0031] In particular embodiments of the present invention, methods Petition 870260045338, dated 13 / 05 / 2026, p. 22 / 90 / 54, to determine an immunogenicity level of a biological therapy, is disclosed, where the limit is a predetermined value. In certain aspects, the limit is a safety limit.

[0032] In certain embodiments of the present invention, the amount of biological therapy is a therapeutically effective dose, wherein a therapeutically effective dose is an amount of therapeutic agent, for example, a binding molecule of the present invention, which when administered to a patient is in sufficient quantity to achieve the intended objective.

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

[0034] In other embodiments of the present invention, the patient is a participant in a clinical trial. In one aspect, the patient is a patient undergoing medical treatment. In another aspect, the medical treatment is beginning and the immunogenicity level is a baseline level. In yet another aspect, the medical treatment is ongoing and the immunogenicity level is a subsequent level. In yet another aspect, the medical treatment is ending and the immunogenicity level is a final level. In another aspect, the patient is a healthy individual.

[0035] In still other embodiments of the present invention, the patient has an inflammatory disease or disorder, an autoimmune disease or disorder, an allergic disease or disorder, an immunological disease or disorder, or a benign proliferative disease or disorder. In one aspect, the patient has 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 patient may have any disease or medical condition. Petition 870260045338, dated 05 / 13 / 2026, page 23 / 90 / 54

[0036] This disclosure provides a non-naturally occurring monoclonal antibody comprising a C-terminal heavy chain sequence comprising a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13, or an antigen-binding portion thereof. A C-terminal heavy chain sequence may be a CH3 domain sequence. A C-terminal heavy chain sequence may comprise SEQ ID NO: 7. A C-terminal heavy chain sequence may comprise SEQ ID NO: 13. A C-terminal heavy chain sequence may comprise a CH domain sequence comprising SEQ ID NO: 8. A C-terminal heavy chain sequence may comprise a CH3 domain sequence comprising SEQ ID NO: 22.

[0037] Non-naturally occurring monoclonal antibody comprising a CH3 domain consisting of a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13, or an antigen-binding portion thereof.

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

[0039] A non-naturally occurring monoclonal antibody, wherein the antibody comprises a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 VH region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 VH region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 VL region comprising the amino acid sequence LGS; a CDR3 VL region comprising the amino acid sequence SEQ ID NO: 14, and a CH3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, Petition 870260045338, dated 05 / 13 / 2026, page 24 / 90 / 54 SEQ ID NO: 7 and SEQ ID NO: 13, or an antigen-binding portion thereof.

[0040] The aforementioned antibodies may further comprise a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 VH region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 VH region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 VL region comprising the amino acid sequence LGS; a CDR3 VL region comprising the amino acid sequence SEQ ID NO: 14.

[0041] The present disclosure provides a non-naturally occurring monoclonal antibody, wherein the antibody comprises a CDR1 VH region comprising the amino acid sequence SEQ ID NO: 9; a CDR2 VH region comprising the amino acid sequence SEQ ID NO: 10; a CDR3 VH region comprising the amino acid sequence SEQ ID NO: 11; a CDR1 VL region comprising the amino acid sequence SEQ ID NO: 12; a CDR2 VL region comprising the amino acid sequence LGS; a CDR3 VL region comprising the amino acid sequence SEQ ID NO: 14, and a CH3 domain comprising the 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.

[0042] The preceding antibodies may comprise a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 16 or an antigen-binding portion thereof. The preceding antibodies may comprise a CH1 region of IgG4 comprising the amino acid sequence of SEQ ID NO: 1. The preceding antibodies may comprise a CH2 region of IgG4 Petition 870260045338, dated 05 / 13 / 2026, page 25 / 90 / 54, comprising the amino acid sequence of SEQ ID NO: 2. A Ch2 region of IgG4 may comprise a hinge region. A hinge region may comprise the APEFLG sequence (SEQ ID NO: 17).

[0043] The antibodies in this disclosure may mitigate high background signal during an immunogenicity analysis.

[0044] This disclosure provides an assay comprising: (a) a solid support, wherein a first component is operationally attached to the solid support; (b) at least one capture agent, wherein a second component is operationally attached to at least one capture agent, wherein the capture agent comprises a first non-naturally occurring monoclonal antibody of the present disclosure; and (c) at least one detection agent, wherein a detectable marker is operationally attached to the detection agent, wherein the detection agent comprises a second non-naturally occurring monoclonal antibody of the present disclosure and wherein the first component and the second component selectively bind to each other.

[0045] A detection agent may comprise dupilumab. A second, unnaturally occurring monoclonal antibody may comprise dupilumab. A first component may comprise streptavidin. A second component may comprise biotin.

[0046] This disclosure provides an assay comprising: (a) a solid support, wherein a first component is operationally linked to the solid support; (b) at least one capture agent, wherein a second component is operationally linked to at least one capture agent and wherein the capture agent comprises the non-naturally occurring monoclonal antibody of this disclosure; and (c) at least one detection agent, wherein a detectable marker is operationally linked to the detection agent and wherein the detection agent comprises dupilumab; wherein the first component and the second component are linked Petition 870260045338, dated 05 / 13 / 2026, p. 26 / 90 / 54 selectively to each other. A first component may comprise streptavidin.

[0047] A second component may comprise biotin.

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

[0049] This disclosure provides a method for determining the immunogenicity level of a monoclonal antibody therapy in a patient, comprising: (a) placing a biological sample from the patient in contact with any assay of this disclosure under conditions suitable to allow binding of at least one antibody in the biological sample to at least one capture agent and to at least one detection agent, wherein the patient received the monoclonal antibody therapy prior to the contact step; (b) detecting a signal from at least one detection agent; and (c) identifying the patient's immunogenicity level as high when the signal from (b) is above a threshold value; or (d) identifying the patient's immunogenicity level as low when the signal from (b) is below the threshold value.

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

[0051] Monoclonal antibody therapy may include dupilumab. A threshold may be a predetermined value. A threshold may be a safety limit. An amount of monoclonal antibody therapy may be a therapeutically effective dose. An immunogenicity level may be a baseline level. An immunogenicity level may be a subsequent level. Petition 870260045338, dated 05 / 13 / 2026, p. 27 / 90 / 54

[0052] A patient may be a participant in a clinical trial. A patient may be a patient undergoing medical treatment. Medical treatment may be beginning and an immunogenicity level may be a baseline level. Medical treatment may be ongoing and an immunogenicity level may be a subsequent level. Medical treatment may be ending and an immunogenicity level may be a final level. A patient may be a healthy individual. A patient 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. A patient may have atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

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

[0054] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as is generally understood by a person skilled in the art to which this disclosure pertains. In the descriptive report, singular forms also include the plural, unless the context clearly indicates otherwise; for example, the terms a, an, and the are understood as singular or plural, and the term or is considered inclusive. By way of example, “an element” means one or more elements. Throughout the descriptive report, the word comprising or variations such as comprises or including, will be understood as implying the inclusion of a stated element, whole or step, or group of elements, whole or step, but not the exclusion of any other element, whole or step, or group of elements, whole or step."Approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the declared value. Unless otherwise clearly stated. Petition 870260045338, dated 05 / 13 / 2026, page 28 / 90 / 54 context, all numerical values ​​provided in this document are modified by the term approximately. BRIEF DESCRIPTION OF THE FIGURES

[0055] Figure 1 is a graph characterizing the specificity of pre-existing reactivity in baseline patient samples using competitive antibody constructs in an anti-drug antibody (ADA) assay. On the y-axis, the percentage inhibition from 0 to 100 in increments of 25. On the x-axis, the specific competitive antibody construct used in the bridging assay, listed from left to right: dupilumab, REGN-A, wt IgG4, wt IgG1, wt IgG2, wt IgG3, REGN-B, and REGN-C. The six patient samples (S1 to S6) are represented, in order, as a circle, a square, an upward-pointing triangle, an asterisk, a diamond, and a downward-pointing triangle. The antibody reagents mentioned in the graph were used at 200 pg / mL as competitive inhibitors in the ADA confirmatory assay format.The high percentage of inhibition in the assay indicates that the specific competitor was able to inhibit pre-existing signals in these samples, suggesting that the competing molecule contains a region to which pre-existing reactivity binds. Lower percentage inhibitions indicate that the competing molecule does not contain a region to which pre-existing reactivity can bind.

[0056] Figure 2 is a diagram showing a schematic representation of the three main constructs and their structural differences compared to dupilumab. REGN-B, REGN-C, and REGN-E are molecular analogs of dupilumab. All three constructs are IgG4k constructs that have the same specificity and similar affinity for anti-IL4R as dupilumab and also possess the same sequence mutation of the CPPC hinge region present in dupilumab.

[0057] Figure 3 is a drawing that shows a representation Petition 870260045338, dated 05 / 13 / 2026, page 29 / 90 / 54 diagram of the amino acid sequence alignment and comparison of the Ch3 domain of wild-type IgG4, IgG1, and IgG2 subtypes. Leucine at position 445 is the third from the C-terminal end of the CH3 domain sequence, as indicated by the arrow.

[0058] Figure 4 is an embodiment illustrating a diagrammatic representation of a drug-specific bridge (panel A, ADA assay #1), a non-drug-specific bridge due to pre-existing reactivity in the current ADA assay (panel B), and no non-drug-specific bridge due to the use of REGN-C as the capture agent (panel C, ADA assay #2). Streptavidin on the plate is shown as a gray cross, the biotin moiety as a small black square, and the Ruthenium label as a star. The biotinylated REGN-D capture agent (dupilumab) (black bifurcated structure) is shown in panels A and B, while in panel C, biotinylated REGNC is shown (black and white checkered bifurcated structure). Pre-existing reactivity is represented in panels B and C as a smaller dashed outlined bifurcated structure.

[0059] Figure 5A is a graph showing the assay signals from a subset of baseline patient samples using the original ADA assay (assay #1) that is diagrammed in panel A of Figure 4 and uses biotinylated REGN-D as the capture agent. On the y-axis, Signal-to-Noise ratio on a logarithmic scale from 0 to 100. On the x-axis, individual patient samples requested by Signal-to-Noise ratio. The cutoff point is given as a dashed line.

[0060] Figure 5B is a graph showing the assay signals from the same baseline patient samples using a revised ADA assay (assay #2), which is diagrammed in panel C of Figure 4 and uses biotinylated REGNC as the capture agent. On the y-axis, Signal-to-Noise ratio on a logarithmic scale from 0 to 100. On the x-axis, individual patient samples in the same order represented in Figure 5A. The cutoff point is given as Petition 870260045338, dated 05 / 13 / 2026, page 30 / 90 / 54 a dashed line.

[0061] Figure 6A is a graph showing the assay signal of a subset of patient baseline samples using a drug-specific bridging antidrug antibody (ADA) assay, similar to that described in Figure 4, with intact dupliumab used as capture and detection reagents.

[0062] Figure 6B is a graph showing the assay signal of the same subset of patient baseline samples tested in Figure 6A, using a revised bridged ADA assay with REGN-B (Figure 2, a human IgG4 mAb in which the entire Ch3 domain of IgG4 has been swapped for an IgG1 CH3 domain) as the capture and detection reagents.

[0063] Figure 6C is a graph showing the assay signal of the same subset of patient baseline samples tested in Figure 6A, using a revised bridged ADA assay with REGN-E (Figure 2, a human IgG4 mAb in which the CH3 domain is truncated with a stop codon after Serine 444) as capture and detection reagents.

[0064] Figure 7 is a series of graphs characterizing the specificity of pre-existing reactivity in baseline patient samples using competitive antibody constructs in an anti-drug antibody (ADA) assay. On the y-axis, the percentage inhibition from 0 to 100 in increments of 25. On the x-axis, the specific competitive antibody construct used in the bridging assay, listed from left to right: dupilumab, REGN-F, and REGN-F (L445P) in the upper graph or dupilumab, REGN-G, and REGN-G (L445P) in the lower graph. The six patient samples (S1 to S6) are represented, in order, as a circle, a square, an upward-pointing triangle, an asterisk, a diamond, and a downward-pointing triangle. The antibody reagents mentioned in the graph were used at 200 pg / mL as competitive inhibitors in the ADA confirmatory assay format. The high percentage of inhibition in the assay Petition 870260045338, dated 05 / 13 / 2026, page 31 / 90 / 54 indicates that the determined competitor was able to inhibit the pre-existing signals in these samples, suggesting that the competing molecule contains a region to which the pre-existing reactivity binds. Lower percentage inhibitions indicate that the competing molecule does not contain a region to which the pre-existing reactivity can bind.

[0065] Figure 8 is a series of graphs showing the assay signal of a subset of patient baseline samples using a drug-specific bridge antidrug antibody (ADA) assay, similar to that described in Figure 4, with REGN-F used as capture and detection reagents (top) or with REGN-F (L445P) used as capture reagent in combination with REGN-F as detection reagent.

[0066] Figure 9 is a series of graphs showing the assay signal of a subset of patient baseline samples using a drug-specific antidrug antibody (ADA) bridging assay, similar to that described in Figure 4, with REGN-G used as capture and detection reagents (top) or with REGN-G used as capture reagent in combination with REGN-G (L445P) as detection reagent. DETAILED DESCRIPTION

[0067] It should be understood that this disclosure is not limited to the compositions and methods described in this document, as well as the experimental conditions described, as these may vary. It should also be understood that the terminology used in this document is intended to describe certain embodiments only and is not intended to be limiting, since the scope of this disclosure will be limited only by the appended claims.

[0068] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is generally understood by a person skilled in the art to which this disclosure pertains. Although any similar compositions, methods and materials Petition 870260045338, dated 05 / 13 / 2026, p. 32 / 90 / 54 or equivalents to those described in this document may be used in the practice or testing of the present invention. All postings mentioned are incorporated into this document by reference in their entirety.

[0069] The term human IL4R (hIL-4R), as used herein, is intended to refer to a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4Ra (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 the complex formed by the binding of hIL-13 to the hIL-13R1 complex, which complex binds to the hIL-4 receptor to initiate biological activity.

[0070] The term binding molecule, as used herein, is intended to refer to molecules that specifically interact with and bind to a specific target. The target may comprise a biological or small (chemical) molecule. The target molecule may define an antigen or antigenic portion. Examples of a binding molecule include, but are not limited to, antibodies (including monoclonal antibodies, bispecific antibodies, as well as antibody fragments), fusion proteins, and other antigen-binding molecules known to those skilled in the art.

[0071] The term antibody, as used herein, is an example of a binding molecule and refers to an immunoglobulin that typically comprises four polypeptide chains, two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain comprises a variable region of the heavy chain (HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain comprises three domains, Ch1, Ch2, and Ch3. Each light chain comprises a variable region of the light chain (LCVR or VL) and a constant region of the light chain. The constant region of the light chain comprises one domain (CL1). The Vh and Vl regions can be further subdivided into regions of hypervariability, called complementarity-determining regions. Petition 870260045338, dated 05 / 13 / 2026, p. 33 / 90 / 54 (CDR), interspersed with more conserved regions called structural regions (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies may include antibodies in the IgG1, IgG2, IgG3, or IgG4 subclasses. Antibodies may also comprise a combination of regions from different subclasses. IgG4 antibodies may include, but are not limited to, dupilumab and cemiplimab.

[0072] Other examples of binding molecules include, but are not limited to, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and pentaspecific antibodies. In some respects, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and pentaspecific antibodies may comprise an Fc portion of an antibody. In some respects, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and pentaspecific antibodies may comprise an IgG4 structure. Another example of a binding molecule is an antibody-drug conjugate (ADC). In some respects, an ADC may comprise an IgG4 structure. Another example of a binding molecule is a bispecific T-cell engager (BiTE). In some respects, BiTE may comprise an IgG4 structure. Another example of a binding molecule is a TRAP fusion protein.In some respects, the TRAP fusion protein has an IgG4 structure. Another example of a binding molecule is a finomer.

[0073] The term antigen-binding portion of an antibody (or simply antibody portion or antibody fragment), as used herein, refers to one or more fragments of an antibody that retain the ability to bind specifically to an antigen (e.g., hIL4Ra). The antigen-binding function of an antibody has been shown to... Petition 870260045338, dated 05 / 13 / 2026, page 34 / 90 / 54, can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment comprising the V1, Vh, CL1 and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge in the hinge region; (iii) an Fc fragment comprising the VH and CH1 domains; (iv) an Fv fragment comprising the V1 and Vh domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241: 544-546), which comprises a Vh domain; and (vi) a CDR.Furthermore, although the two domains of the Fv fragment, Vl and Vh, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made as a single contiguous chain in which the Vl and Vh regions pair up to form monovalent molecules (known as 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. These 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 diabodies, are also included (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448).

[0074] A neutralizing or blocking antibody, as used in this document, is intended to refer to an antibody whose binding to hIL-4Ra results in the inhibition of the biological activity of hIL-4 and / or hIL-13. This inhibition of the biological activity of hIL-4 and / or IL-13 can be assessed by measuring one or more indicators of the biological activity of hIL-4 and / or hIL-13 known in the art, such as hIL-4- and / or IL13-induced cell activation and hIL-4 binding to hIL-4Ra (see examples below).

[0075] A CDR or complementarity-determining region is Petition 870260045338, dated 05 / 13 / 2026, p. 35 / 90 / 54 a region of hypervariability interspersed within regions that are more conserved, called structure regions (FRs). In different modalities of the anti-hIL-4Ra antibody or fragment of disclosure, FRs can be identical to human germline sequences or can be naturally or artificially modified.

[0076] The term epitope is an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, an epitope may include saccharide fragments, phosphoryl groups, or sulfonyl groups in the antigen.

[0077] The term immunogenicity refers to the ability of an antigen or immunogen to induce an immune response in the body of a human or animal. Therapeutic proteins have the ability to provoke adverse immune responses that can interfere with the pharmacokinetics and efficacy of the drug. This immune response can take the form of the production of antidrug antibodies (ADAs).

[0078] The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when ideally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST or Gap, as discussed below. Petition 870260045338, dated 05 / 13 / 2026, p. 36 / 90 / 54

[0079] As applied to polypeptides, the term substantial similarity or substantially similar means that two peptide sequences, when optimally aligned, such as by GAP or BESTFIT programs using standard gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Typically, the positions of residues that are not identical differ by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein.In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. The means of making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307–331, herein incorporated by reference. Examples of groups of amino acids that have 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: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine ​​and methionine.In certain embodiments, the conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change with... Petition 870260045338, dated 05 / 13 / 2026, p. 37 / 90 / 54 a positive value in the PAM250 logarithmic likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, incorporated here by reference. A “moderately conservative” replacement is any change with a non-negative value in the PAM250 log likelihood matrix.

[0080] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures attributed to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as Gap and Bestfit that can be used with standard 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 a mutant thereof. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using standard or recommended parameters, a program in GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between query and search sequences (Pearson (2000) supra). Another algorithm when comparing a disclosure sequence to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using standard parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403 to 410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389 to 402, each of which is incorporated here by reference.

[0081] Methods for generating human antibodies include those described in, for example, US Patent No. 6,596,541, Green et al. (1994) Petition 870260045338, dated 05 / 13 / 2026, page 38 / 90 / 54 Nature Genetics 7: 13 to 21), US Patent Nos. 5,545,807, 6,787,637.

[0082] Rodents can be immunized by any method known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, 1988 Cold Spring Harbor Laboratory; Malik and Lillehoj (1994) Antibody Techniques, Academic Press, CA). Disclosure antibodies are typically prepared using VELOCIMMUNE® technology (US Patent No. 6,596,541). A transgenic mouse in which the variable regions of the endogenous immunoglobulin light and heavy chains are replaced by the corresponding human variable regions is challenged with the antigen of interest, and lymphatic cells (such as B cells) are retrieved from the antibody-expressing mice. The lymphatic cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest.The DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to desirable isotypic constant regions of the heavy and light chains. This antibody protein can be produced in a cell, such as a CHO cell. Alternatively, the DNA encoding the antigen-specific chimeric antibodies or the variable regions of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0083] The DNA encoding the variable regions of the antibody heavy and light chains can be isolated and operationally ligated to the DNA encoding the constant regions of the human heavy and light chains. The DNA can then be expressed in a cell capable of expressing the fully human antibody. In one specific embodiment, the cell is a CHO cell.

[0084] Antibodies may be therapeutically useful in blocking Petition 870260045338, dated 05 / 13 / 2026, pp. 39 / 90 / 54, refers to a ligand-receptor interaction or the inhibition of the receptor component interaction, rather than killing cells through complement fixation (complement-dependent cytotoxicity) (CDC) and participation in antibody-dependent cell-mediated cytotoxicity (ADCC). The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0085] Human immunoglobulins can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four-chain construct of approximately 150 to 160 kDa in which the dimers are held together by an interchain heavy-chain disulfide bond. In a second form, the dimers are not linked by interchain disulfide bonds, and a molecule of about 75 to 80 kDa is formed, composed of a covalently coupled light and heavy chain (semiantibody). These forms have been 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, isotype-associated structural differences in the antibody hinge region.In fact, a single amino acid substitution in the hinge region of human IgG4 can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels normally observed using a human IgG1 hinge. The present disclosure covers antibodies with one or more mutations in the hinge, CH2 or CH3 region that may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0086] Initially, high-affinity chimeric antibodies are isolated with a human variable region and a constant region of Petition 870260045338, dated 05 / 13 / 2026, page 40 / 90 / 54 mouse. As described below, the antibodies are characterized and selected for desirable characteristics, including binding affinity to hIL-4Ra, ability to block hIL-4 binding to hIL-4Ra, and / or selectivity for the human protein. The mouse constant regions are replaced with desired human constant regions to generate fully human disclosure antibodies, for example, wild-type or modified IgG4 or IgG1 (e.g., SEQ ID NO: 4, 19, 20, and 23). Although the selected constant region may vary according to the specific use, the characteristics of high antigen-binding affinity and target specificity reside in the variable region. Immunogenicity assays

[0087] This disclosure provides a non-naturally occurring binding molecule, such as a monoclonal antibody, or antigen-binding portion thereof, comprising a C-terminal LSPG heavy chain sequence (SEQ ID NO: 21). In certain embodiments, the C-terminal heavy chain is a human CH3 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.

[0088] The disclosure provides immunogenicity assays comprising an anti-IL-4Ra binding molecule, such as an antibody or antigen-binding fragments of the present disclosure. The disclosure's immunogenicity assays may take the form of anti-drug antibody (ADA) assays. The disclosure's ADA assays may be ADA bridge assays or direct enzyme-linked immunosorbent assays (ELISA). In an ADA bridge assay, a biotinylated form of the binding molecule in question is ligated to streptavidin in a plate. Binding molecules, such as antibodies present in the sample, bind to the biotinylated binding molecule and to a labeled form of the same binding molecule. Petition 870260045338, dated 05 / 13 / 2026, pp. 41 / 90 / 54 forming a bridging interaction with a detectable marker signal. Suitable markers will be known to a person skilled in the art. Exemplary markers include ruthenium, horseradish peroxidase, alkaline phosphatase, and fluorophores. The ADA bridging assay may include titrating the sample by performing the bridging reaction to generate a standard curve and using a cold, unlabeled competing antibody to inhibit the reaction.

[0089] Figure 4 illustrates a drug-specific ADA bridging assay in Panel A; in Panel B, a non-drug-specific bridging assay due to pre-existing reactivity in the current ADA assay; and no non-drug-specific ADA bridging assay due to the use of REGN-C as the capture agent seen in Panel C. Pre-existing reactivity is plotted in Panels B and C as a smaller dashed bifurcated structure. It should be understood that, for the disclosed embodiment, the molecular analogs of the assay reagent REGN-A, B, C, D, and E are interchangeable, thus allowing a person skilled in the art to design various permutations of the assay as described.In one aspect, in order to reduce the background signal, a person skilled in the art could combine REGN-D with any of the following reagents to form new combinations: REGNB, REGN-C, or REGN-E (D+B, D+C, D+E, or reversed so that B+D, C+D, or E+D) so that the alignment order is capture agent + detection agent. In another aspect, a person skilled in the art could make the following pairs in order to reduce the background signal: B+B, C+C, or E+E.

[0090] In certain embodiments of the present disclosure, immunogenicity levels (or ADA) are determined using binding molecules of such antibodies (or antigen-binding portions thereof). In certain embodiments, immunogenicity levels are assessed for dupilumab using an IgG4 antibody. One issue that typically needs to be addressed in these immunogenicity studies is the high background signal that is frequently associated with the IgG4 antibody used. In order to mitigate this. Petition 870260045338, dated 05 / 13 / 2026, p. 42 / 90 / 54 high background, the present disclosure provides IgG4 antibodies comprising a heavy chain sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 7 or SEQ ID NO: 8 resulting in a lower background signal.

[0091] In certain embodiments of the present disclosure, a binding molecule, such as an antibody, or its antigen-binding moiety, comprises (1) one or more VL chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more VH 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) a Ch3 or CH sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8, wherein the binding molecule exhibits reduced background reactivity in an immunogenicity assay (ADA) compared with a binding molecule comprising (1) one or more VL chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and their combinations, (2) one or more VH string sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and their combinations,and (3) a Ch3 or CH sequence not selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8. In certain embodiments, the CH domain sequence of (3) may include SEQ ID NO: 5 and SEQ ID NO: 6.,

[0092] In one embodiment of the present invention, the Ch3 domain of an IgG4 antibody, such as dupliumab, is exchanged for a Ch3 domain of IgG1, resulting in a lower background signal (Figure 6B). In yet another embodiment, the Ch3 domain of an IgG4 antibody such as dupliumab is truncated. In one aspect, the truncation occurs at Serine 444, resulting in a lower background signal (Figure 6C).

[0093] In some respects, the present disclosure provides a Petition 870260045338, dated 05 / 13 / 2026, p. 43 / 90 / 54 linking molecule comprising (1) one or more Vl chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more Vh 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) a CH3 or CH sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 13, SEQ ID NO: 7 and SEQ ID NO: 8.

[0094] In some respects, the present disclosure provides a linking molecule comprising (1) one or more Vl chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more Vh 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) a Ch 3 or Ch sequence, wherein the Ch 3 or Ch sequence comprises 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.

[0095] In some respects, the present disclosure provides a linking molecule comprising (1) one or more Vl chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more Vh 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) a Ch3 sequence, wherein the Ch3 sequence comprises one or more sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 21.

[0096] In some respects, the present disclosure provides a linking molecule comprising (1) one or more Vl chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more Vh chain sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ Petition 870260045338, dated 05 / 13 / 2026, pp. 44 / 90 / 54 ID NO: 11 and its combinations, and (3) a sequence Ch, wherein the sequence Ch comprises one or more sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 21 and SEQ ID NO: 22.

[0097] In some respects, the present disclosure provides a linking molecule comprising a Ch sequence, wherein the Ch sequence comprises one or more sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 21 and SEQ ID NO: 22.

[0098] In some respects, the present disclosure provides a linking molecule comprising a Ch3 sequence, wherein the Ch3 sequence comprises one or more sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 21.

[0099] In some respects, the present disclosure provides a linking molecule comprising an IgG4 Ch domain comprising a proline amino acid substitution for leucine at position 445.

[00100] In some respects, the present disclosure provides a linking molecule comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5.

[00101] In some respects, the present disclosure provides a linking molecule comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6.

[00102] In some respects, the present disclosure provides a linking molecule comprising (1) one or more Vl chain sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more Vh 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) a Ch sequence comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5.

[00103] In some respects, the present disclosure provides a linking molecule comprising (1) one or more chain sequences Petition 870260045338, dated 05 / 13 / 2026, pp. 45 / 90 / 54 (1) one or more Vl sequences selected from the group consisting of SEQ ID NO: 12, LGS, SEQ ID NO: 14 and combinations thereof, (2) one or more Vh string sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and combinations thereof, and (3) a CH sequence comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6.

[00104] In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO: 7. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO: 7. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO: 7. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO: 7.In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO: 7. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO: 7. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO: 7. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 7. In some respects. Petition 870260045338, dated 05 / 13 / 2026, p. 46 / 90 / 54 aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 91% sequence identity with SEQ ID NO: 7. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 90% sequence identity with SEQ ID NO: 7.

[00105] In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO: 8. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO: 8. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO: 8. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO: 8.In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO: 8. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO: 8. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO: 8. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least... Petition 870260045338, dated 05 / 13 / 2026, p. 47 / 90 / 54 approximately 92% sequence identity with SEQ ID NO: 8. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least approximately 91% sequence identity with SEQ ID NO: 8. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least approximately 90% sequence identity with SEQ ID NO: 8.

[00106] In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO: 13. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO: 13. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 913% sequence identity to SEQ ID NO: 13. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO: 13.In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO: 13. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO: 13. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO: 13. In some respects, the present disclosure provides a linking molecule. Petition 870260045338, dated 05 / 13 / 2026, pp. 48 / 90 / 54 comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 13. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO: 13. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO: 13.

[00107] In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 97% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21.In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 96% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 95% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule. Petition 870260045338, dated 05 / 13 / 2026, pp. 49 / 90 / 54 comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21.In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO: 7, wherein at least one polypeptide sequence comprises SEQ ID NO: 21.

[00108] In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 99% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 98% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least Petition 870260045338, dated 05 / 13 / 2026, page 50 / 90 / 54 approximately 97% sequence identity with SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least approximately 96% sequence identity with SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least approximately 95% sequence identity with SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21.In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 94% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 93% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some aspects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 92% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21.In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 91% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. In some respects, the present disclosure provides a linking molecule comprising at least one polypeptide sequence with at least about 90% sequence identity to SEQ ID NO: 8, wherein at least one polypeptide sequence comprises SEQ ID NO: 21. Petition 870260045338, dated 05 / 13 / 2026, page 51 / 90 / 54 less a polypeptide sequence comprising SEQ ID NO: 21.

[00109] In some respects, any binding molecule of the present disclosure may exhibit reduced background reactivity in an immunogenicity assay (ADA) compared to a binding molecule comprising a CH sequence comprising SEQ ID NO: 3. In some respects, any binding molecule of the present disclosure may exhibit reduced background reactivity in an immunogenicity assay (ADA) compared to a binding molecule comprising a CH sequence comprising SEQ ID NO: 4. Therapeutic Administration and Formulations

[00110] This disclosure provides therapeutic compositions comprising the anti-IL-4Ra binding molecules of this disclosure. The therapeutic compositions according to this disclosure will be administered with suitable vehicles, excipients, and other agents that are incorporated into formulations to provide improved transfer, release, tolerance, and the like. A multitude of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52: 238-311.

[00111] The dose may vary depending on the age and size of the patient being administered to, the target disease, conditions, route of administration, and similar factors. When the binding molecule of this disclosure is used for Petition 870260045338, dated 05 / 13 / 2026, p. 52 / 90 / 54 To treat various conditions and diseases associated with IL-4Ra in an adult patient, it is advantageous to administer the binding molecule of this disclosure intravenously, typically in a single dose of about 0.01 to about 20 mg / kg of body weight, more commonly about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg of body weight. In some respects, when the binding molecule of this disclosure is used to treat various conditions and diseases associated with IL-4Ra, the dosage regimen may be 300 mg once every two weeks (Q2W) and may be extended to every four weeks (Q4W). Depending on the severity of the condition, the frequency and duration of treatment may be adjusted.

[00112] Several delivery systems are known and can be used to administer the pharmaceutical composition of the release, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262: 4429 to 4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local.

[00113] The pharmaceutical composition can also be distributed in a vesicle, 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. Petition 870260045338, dated 05 / 13 / 2026, pp. 53 / 90 / 54

[00114] In certain embodiments, the pharmaceutical composition can be released in a controlled-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, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled-release system may be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115–138, 1984). Other controlled-release systems are discussed in Langer's (1990) review of Science 249: 1527–1533.

[00115] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared, for instance, by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As aqueous media for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent, such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a non-ionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)], etc.As an oily medium, sesame oil, soybean oil, etc., are used, which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably placed in an appropriate ampoule. Petition 870260045338, dated 05 / 13 / 2026, page 54 / 90 / 54

[00116] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a unit dose suitable to match a dose of the active ingredients. These dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned linking molecule contained is generally about 5 to 500 mg per dosage form in a unit dose; especially in the injection form, in certain embodiments, the aforementioned linking molecule is contained in about 5 to 100 mg and in about 10 to 250 mg for the other dosage forms.

[00117] Simple and combined therapies. Disclosure binding molecules are useful for treating diseases and disorders that are improved, inhibited, or enhanced by reducing IL-4 activity. These disorders include those characterized by abnormal or excessive IL-4 expression, or by an abnormal host response to IL-4 production.

[00118] The disclosure covers combination therapies in which the anti-IL-4Ra binding molecule (e.g., antibody or antibody fragment) is administered in combination with a second therapeutic agent. Co-administration and combination therapy are not limited to simultaneous administration but include treatment regimens in which an anti-IL-4Ra binding molecule is administered at least once during a course of treatment involving the administration of at least one other therapeutic agent to the patient. A second therapeutic agent may be another IL-4 antagonist, such as another binding molecule, or a soluble cytokine receptor, an IgE antagonist, an anti-asthma medication (corticosteroids, non-steroidal agents, β-agonists, leukotriene antagonists, xanthines, fluticasone, salmeterol, albuterol) that may be administered by inhalation or other appropriate means. In one modality Petition 870260045338, dated 13 / 05 / 2026, p. 55 / 90 / 54, specifically, the anti-IL-4Ra binding molecule, as a binding molecule of the disclosure, can be administered with an IL-1 antagonist, such as rilonacept, or an IL-13 antagonist. In some respects, an anti-IL-4Ra binding molecule, as a binding molecule of the present disclosure, can be administered in combination with a binding molecule that targets cytokines and / or receptors in the Type 1 or Type 2 inflammatory response. The second agent may include one or more leukotriene receptor antagonists to treat disorders such as allergic inflammatory diseases, for example, asthma and allergies. Examples of leukotriene receptor antagonists include, but are not limited to, montelukast, pranlukast, and zafirlukast. The second agent may include a cytokine inhibitor, such as one or more TNF (etanercept, ENBREL™), IL-9, IL-5, or IL-17 antagonists. Therapeutic Uses

[00119] The disclosure provides compositions and methods for the treatment of a disease or disorder in a patient in need, comprising administering to the patient a therapeutically effective amount of a binding molecule of the present disclosure.

[00120] The disclosure provides compositions and methods for the treatment of a disease or disorder in a patient in need, comprising administering to the patient a therapeutically effective amount of a composition from the disclosure.

[00121] In certain embodiments of this disclosure, the disease or disorder is a Type 1 or Type 2 inflammatory disease or disorder.

[00122] In certain embodiments of this disclosure, the disease or disorder is an autoimmune disease or disorder.

[00123] In certain embodiments of this disclosure, the disease or disorder is an allergic disease or disorder.

[00124] In certain forms of disclosure, the disease or disorder is Petition 870260045338, dated 05 / 13 / 2026, p. 56 / 90 / 54 an immune disease or disorder.

[00125] In certain embodiments of this disclosure, the disease or disorder is a benign proliferative disease or disorder. In certain embodiments of this disclosure, the disease or disorder is a malignant proliferative disease or disorder.

[00126] In certain embodiments of this disclosure, the disease or disorder is atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

[00127] In certain embodiments of the disclosure, a 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.

[00128] 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 dose comprises a subcutaneous injection of approximately 75 mg, 150 mg, 300 mg, or 600 mg. In yet another aspect, the therapeutically effective dose comprises at least one subcutaneous injection, at least two subcutaneous injections, at least three subcutaneous injections, or at least four subcutaneous injections of approximately 75 mg, 150 mg, 300 mg, or 600 mg. In yet another aspect, the therapeutically effective dose comprises a subcutaneous injection of approximately 75 mg, 150 mg, 300 mg, or 600 mg once weekly, once every two weeks, once every four weeks, or administered chronically as a maintenance dose to control disease symptoms.

[00129] In certain forms of dissemination, the binding molecule is administered systemically. In one aspect, the binding molecule is Petition 870260045338, dated 13 / 05 / 2026, p. 57 / 90 / 54 administered by subcutaneous injection. In another aspect, the therapeutically effective dose comprises an initial dose of approximately 600 mg. In yet another aspect, the initial dose comprises a pair of injections of 300 mg each, administered at two distinct injection sites. In yet another aspect, including those in which the therapeutically effective dose comprises an initial dose, the therapeutically effective dose further comprises a maintenance dose of approximately 300 mg. In another aspect, the maintenance dose is administered every two weeks.

[00130] In certain embodiments of the dissemination methods, the binding molecule is administered systemically. In one aspect, the binding molecule is administered intravenously at a dose of approximately 1.0 mg / kg, 3.0 mg / kg, 8.0 mg / kg, or 12.0 mg / kg.

[00131] In certain embodiments of the disclosure, the binding molecule is administered in combination with a second therapeutic agent. In one aspect, the second therapeutic agent comprises an immunosuppressant. In one aspect, the second therapeutic agent comprises an agonist antibody. In one aspect, the second therapeutic agent comprises an immunoactivator. In another aspect, the second therapeutic agent comprises an IL-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 eotoxin-3 inhibitor, an IgE inhibitor, a prostaglandin D2 inhibitor, an immunosuppressant, a corticosteroid, a glucocorticoid, a proton pump inhibitor, a nonsteroidal anti-inflammatory drug (NSAID), or a combination thereof.

[00132] In certain embodiments of the 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.

[00133] It should be appreciated that compositions, formulations, kits, Petition 870260045338, dated 05 / 13 / 2026, p. 58 / 90 / 54. The methods of this disclosure are not limited to any disease and / or medical condition. The compositions, formulations, kits, and methods of this disclosure may be applied to any disease and / or medical condition in which a patient exhibits pre-existing reactivity to therapeutic antibodies. TABLE 1: EXEMPLARY SEQUENCES OF THE DISCLOSURE. Name Sequence SEQ ID NO: Ch1 ​​IgG4 astkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsga ltsgvhtfpavlqssglyslssvvtvpsslgtktytcnvdhkpsn TKVDKRV l Ch2 IgG4 apeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfn WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY kckvsnkglpssiektiskak 2 Ch3 IgG4 gqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesng qpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmh EALHNHYTQKSLSLSLGK 3 Ch IgG4 astkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsga ltsgvhtfpavlqssglyslssvvtvpsslgtktytcnvdhkpsn tkvdkrveskygppcpscpapeflggpsvflfppkpkdtlmisrtp evtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnsty rvvsvltvlhqdwlngkeykckvsnkglpssiektskakgqpre pqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpenn ykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhn HYTQKSLSLSLGK 4 Ch3 IgGl gqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesng qpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmh EALHNHYTQKSLSLSPGK 5 Ch3 IgG2 gqprepqvytlppsreemtknqvsltclvkgfypsdisvewesng qpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmh EALHNHYTQKSLSLSPGK 6 Ch3 IgG4 L>Pgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesng qpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmh EALHNHYTQKSLSLSPGK 7 Ch IgG4 L> P astkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsga ltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsn tkvdkrveskygppcpscpapeflggpsvflfppkpkdtlmisrtp evtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnsty rvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqpre pqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpenn ykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhn HYTQKSLSLSPGK 8 Heavy Chain CDR1 GFTFRDYA 9 Heavy Chain CDR2 ISGSGGNT 10 Heavy Chain CDR3 AKDRLSITIRPRYYGL 11 Light Chain CDR1 QSLLYSIGYNY 12 Petition 870260045338, dated 05 / 13 / 2026, pages 59 / 90 / 54 Light chain CDR2 LGS NA Light chain CDR3 MQALQTPYT 14 Variable heavy chain region EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKG LEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVS 15 Variable light chain region DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSG QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYY CMQALQTPYTFGQGTKLEIK 16 CH2 hinge region IgG4 APEFLG 17 IL4Ra MKVLQEPTCVSDYMSISTCEWKMNGPTNCSTELRLLYQLVFLLSE AHTCIPENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQLLWK GSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHL TYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRA WAQCYNTTWSEWSPSTKWHNSYREPFEQH 18 Ch IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 19 Ch IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPAPEFLGPSVFLFPPKPKDTLMISRTP EVTCVVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK 20 IgG4 region of CH3 LSPG 21 CH IgG4 truncated ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPAPEFLGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLS 22 Ch IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSSGAWN LTGSVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS NTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPKPKPKDTLMISRTP EVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 23 CH3 IgG4 truncated GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLS 13 Petition 870260045338, dated 05 / 13 / 2026, pages 60 / 90 / 54 EXAMPLES Example 1: Replacing the constant regions of Ch3 IgG4 reduces pre-existing immunoreactivity in some patient samples.

[00134] Competitive inhibition studies were conducted to characterize the pre-existing high reactivity signals observed in some samples. Commercially obtained antibody reagents or monoclonal antibodies specifically constructed for this purpose were used in these studies. A list of antibody constructs or reagents that helped elucidate the specificity of this pre-existing reactivity is provided in Table 2. TABLE 2 Antibody / Reagent Name Isotype Properties Fc Hinge Region Construct Dupilumab IgG4k CPPC (Fc hinge sequence) REGN-A IgG4k CPPC (Fc hinge sequence) non-specific for IL-4R REGN-B, C and E IgG4k CPPC (Fc hinge sequence) Commercially available wild-type IgG4 (WT) IgG4k CPSC (Fc hinge sequence) wt IgG4 Fc Commercially available isotype antibodies Human IgG1 IgG1K Human IgG2 IgG2λ Human IgG3 IgG3K Dupilumab and Bioengineered Antibodies REGN-D (Dupilumab) IgG4K CPPC (Fc hinge sequence) REGN-B IgG4K CH2-IgG4-CH3-IgG1 regn-c IgG4K CH2-IgG4-C H3 L445> P REGN-E IgG4K CH2-IgG4-CH3 truncated @ 444

[00135] These antibody constructs were used at 200 pg / mL as competitive inhibitors in the anti-drug antibody confirmation assay (ADA) format. The assay format is shown in Figure 4 and the assay results are shown in Figure 1 (Figure 1 and Figure 4). A Petition 870260045338, dated 05 / 13 / 2026, page 61 / 90 / 54. A high percentage of inhibition in the assay indicates that the given competitor was able to inhibit pre-existing reactivity signals in these samples, suggesting that the competitor molecule contains a region to which pre-existing reactivity binds. Lower percentage inhibitions indicate that the competitor molecule does not contain a region to which pre-existing reactivity can bind. The reagents listed in Table 1 above can be broadly grouped into three categories: IgG4 structure / Fc Hinge region constructs, commercially available isotype antibodies, and dupilumab bioengineered constructs. Initial experiments examined whether this high assay reactivity was directed at the CDR or the IgG4 structure of dupilumab. REGN-A is a human monoclonal antibody that has the same IgG4 structure as dupilumab, but a different CDR sequence and does not bind to IL-4Ra.

[00136] As shown in Figure 1, competitive inhibition experiments using dupilumab and REGN-A demonstrated significant inhibition of elevated assay signals in the six baseline samples from the examined patients. Since the CDR region sequences of these two antibodies are different, this inhibition result suggests that the high signal reactivity is not directed at the CDR portion of dupilumab, but rather at some common sequences in the antibody structure. Both dupilumab and REGN-A contain an IgG1 hinge region sequence, “CPPC,” that stabilizes the antibody hinge region in these IgG4 molecules. To determine if pre-existing immunoreactivity targets this CPPC mutation, a commercial IgG4K antibody that has 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 dupilumab and REGN-A. This result suggests that the high baseline assay signal is not directed towards the mutation in the region of. Petition 870260045338, dated 05 / 13 / 2026, p. 62 / 90 / 54 hinge CPPC in dupilumab, but is more likely directed against the wild-type constant regions of the IgG4 molecule. These results indicate that the elevated signals observed at baseline are not specific to dupilumab.

[00137] Further experiments were conducted to determine whether this pre-existing high assay reactivity was directed toward constant region sequences that would be common among the different IgG subtypes. Using the same competitive inhibition approach, the impact of three commercially obtained human IgG1K, IgG2K, and IgG3K antibodies on this pre-existing response was examined. None of these antibodies inhibited the high baseline signals in these samples (see Figure 1). This suggested that the pre-existing reactivity was likely associated with a region that is unique to the IgG4 constant region sequences and that was not shared by any of the IgG subtypes tested.

[00138] A human monoclonal antibody (REGN-B) was constructed that was similar to dupilumab, except that the CH3 domain of IgG4 was replaced by a CH3 domain of IgG1 (see Figure 2). This antibody was examined in competition studies to determine if pre-existing reactivity was directed to the CH3 domain of dupilumab. REGN-B did not significantly inhibit high signals in the samples (see Figure 1), suggesting that pre-existing reactivity is likely directed to some region within the CH3 domain of the IgG4 molecule.

[00139] To further identify the area that may be associated with these elevated signals within the CH3 domain of dupilumab, an amino acid sequence alignment of the CH3 domain of IgG4, IgG1, and IgG2 antibodies was performed (see Figure 3). Differences were observed at six individual amino acid positions between the CH3 domain sequences of IgG4 and IgG1 and at five individual amino acid positions between the CH3 domain sequences of IgG4 and IgG2 (see Figure 3). Petition 870260045338, dated 05 / 13 / 2026, pp. 63 / 90 / 54

[00140] The IgG4 construct with a leucine (L) to proline (P) substitution at position 445 was available and examined in the competitive inhibition assay. This construct did not show significant inhibition of these elevated assay signals. This indicated that the pre-existing reactivity was likely specific to the L445 region of dupilumab. Additional samples with high assay signals were examined in the competitive inhibition assay using this construct with L to P substitution at position 445, and similar low levels of inhibition were observed. This appeared to confirm that the pre-existing reactivity specifically targets the region around L445. Therefore, a change from Leucine (in wt IgG4) to Proline (present at the same position in wt IgG1, IgG2, and IgG3) in dupilumab abolishes this pre-existing response leading to elevated signals in the ADA assay.

[00141] An IgG4 construct with a leucine (L) to proline (P) substitution at position 445 was available and examined in the competitive inhibition assay. This construct did not show significant inhibition of these elevated assay signals. This indicated that the pre-existing reactivity was likely specific to the L445 region of dupilumab. Additional samples with high assay signals were examined in the competitive inhibition assay using this construct with L to P substitution at position 445, and similar low levels of inhibition were observed. This appeared to confirm that the pre-existing reactivity specifically targets the region around L445. Therefore, a change from Leucine (in wt IgG4) to Proline (present at the same position in wt IgG1, IgG2, and IgG3) in dupilumab abolishes this pre-existing response leading to elevated signals in the ADA assay.

[00142] A second human monoclonal antibody construct based on dupilumab (REGN-C) was generated. This construct is identical to dupilumab except for the insertion of a point mutation at residue 445 in Petition 870260045338, dated 05 / 13 / 2026, page 64 / 90 / 54 antibody sequence, where a Leucine is altered to Proline (abbreviated as L>P). REGN-C cannot significantly inhibit the high signals in the baseline samples that were tested (see Figure 1 and Figure 2). This confirmed that the pre-existing reactivity specifically targets the region around L445 in dupilumab and suggested that the use of this bioengineered dupilumab in the ADA assay eliminates most, if not all, of the high-level background signal observed in the current ADA assay.

[00143] Further experiments were performed using two additional antibodies, REGN-F and REGN-G, to demonstrate that modifying residue 445 in the antibody sequence from Leucine to Proline abolishes pre-existing reactivity. As shown in Figure 7, competitive inhibition experiments using Dupilumab, REGN-F, and REGN-G demonstrated significant inhibition of elevated assay signals in the six baseline samples, thus showing that Dupilumab, REGN-F, and REGN-G exhibit a high level of pre-existing reactivity. However, a substitution of Leucine for Proline at position 445 of REGN-F and REGN-G, here referred to as REGN-F (L445P) and REGN-G (L445P), respectively, abolished this pre-existing reactivity, as REGN-F and REGN-G were unable to inhibit the high assay signals. REGN-F, REGN-F (L445P), REGN-G, and REGN-G (L445P) were also tested as reagents in a drug-specific bridging antidrug antibody assay, similar to that described in Figure 4.As shown in Figures 8 and 9, when REGN-F and REGN-G were used as the capture and detection reagents, a high assay signal was demonstrated, indicative of pre-existing high 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 signal was significantly reduced, demonstrating that the L445P substitutions... Petition 870260045338, dated 05 / 13 / 2026, pp. 65 / 90 / 54, cancels out pre-existing reactivity. REGN-F and REGN-G comprise distinct variable domains that are also different from the variable domains of the antibodies tested in the results shown in Figure 1. Thus, the results shown in Figures 7 to 9 demonstrate that pre-existing reactivity is independent of variable domains and CDRs and, instead, is specific to the region around L445. Furthermore, it demonstrates that L445P substitution can generally be applied to IgG4 antibodies, regardless of the identity of their CDRs, to reduce pre-existing reactivity.

[00144] A region in the dupilumab sequence to which at least most of the pre-existing reactivity is directed has been identified. The elevated signals in the ADA assay appear to have been generated due to some matrix constituent in these serum samples that can bridge the gap between the labeled dupilumab molecules in the assay by binding to or near L445 in the CH3 domain. This indicates that this pre-existing reactivity is not specific to dupilumab but can bind to any IgG4 molecule. Furthermore, the results suggest that the use of this modified version of dupilumab with the L445P mutation in the ADA assay eliminates most, if not all, of the high-level background signal. Example 2: Development of a modified anti-drug antibody (ADA) assay that reduces background immunoreactivity in patient samples

[00145] A modified ADA assay was developed that uses a biotinylated REGN-C (with the L445P mutation) as the capture agent. Figure 4 illustrates the difference in assay design between the current ADA assay and the modified ADA assay using REGN-C. For clarity, the current ADA assay will be considered as ADA assay number 1 and the modified ADA assay will be referred to as ADA assay number 2. Figure 5 shows a comparative analysis of the ADA screen signals obtained using the current ADA assay number 1 versus ADA assay number 2, from all the Petition 870260045338, dated 05 / 13 / 2026, page 66 / 90 / 54 patient baseline samples. Panel 5A shows a graph of the signal-to-noise ratio generated by these high-signal baseline ADA samples in the current ADA screening assay (Assay number 1), while panel 5B shows a graph of the signal-to-noise ratio generated by the exact same samples in the ADA assay number 2. The assay format of the ADA assay number 2 significantly reduces the high signals observed in the current ADA screening assay. Some samples still demonstrate reactivity in the ADA assay number 2, but the number of screening positives falls more in line with the expected false positive rate for a screening assay, and the signal response level for these positive samples is generally much lower than that observed using the current ADA assay.The observed drop in these high assay signals to near baseline values ​​in most cases in the ADA #2 trial should allow for improved detection of treatment-emergent and drug-specific ADAs in a patient population. Incorporation by Reference

[00146] Each document cited herein, including any cross-reference or related patent or application, is incorporated herein by reference in its entirety, unless expressly excluded or otherwise limited. Citation of any document is not an admission that it is prior art with respect to any disclosure disclosed or claimed in this document or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any disclosure. Furthermore, 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 ascribed to that term in this document shall prevail. OTHER MODALITIES

[00147] Although certain forms of disclosure have been Petition 870260045338, dated 05 / 13 / 2026, p. 67 / 90 / 54 illustrated and described, various other changes and modifications may be made without departing from the spirit and scope of the disclosure. The scope of the attached claims includes all changes and modifications that are within the scope of this disclosure. Petition 870260045338, dated 05 / 13 / 2026, pp. 68 / 90

Claims

1 / 3 CLAIMS 1. A method for determining the immunogenicity level of a monoclonal antibody therapy in a patient, characterized in that it comprises (a) placing a biological sample from the individual in contact with the assay comprising: (i) a solid support comprising streptavidin operably linked to the solid support; (ii) at least one capture agent comprising biotin operably linked to an isolated monoclonal antibody comprising a C-terminal heavy chain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13; and (iii) at least one detection agent comprising dupilumab operably linked to a detectable label;wherein streptavidin and biotin selectively bind to each other, thereby binding at least one capture agent to the support, and at least one antibody in the biological sample binds to at least one capture agent and at least one detection agent, and wherein the individual was administered monoclonal antibody therapy prior to the contact step, (b) detect a detectable signal from at least one detection agent, and (c) identify the immunogenicity level of the monoclonal antibody therapy in the individual as high when the signal from (b) is above a threshold value, or (d) identify the immunogenicity level of the monoclonal antibody therapy in the individual as low when the signal from (b) is below the threshold value. Petition 870260045338, dated 05 / 13 / 2026, p. 69 / 90 2 / 3; 2. Method according to claim 1, characterized in that the monoclonal antibody therapy comprises an antibody comprising a C-terminal heavy chain sequence comprising a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO:

13.

3. Method according to claim 1, characterized in that the limit value is a predetermined value or a safety limit.

4. Method according to claim 1, characterized in that the monoclonal antibody therapy is: (i) beginning and the immunogenicity level is a baseline level; (ii) ongoing and the immunogenicity level is a subsequent level; or (iii) ending and the immunogenicity level is a final level.

5. Method according to claim 1, characterized in that the individual has an inflammatory disease or disorder, an autoimmune disease or disorder, an allergic disease or disorder, an immune disease or disorder, or a benign proliferative disease or disorder.

6. The method of claim 1, characterized by the fact that the individual is a participant in a clinical trial.

7. The method of claim 1, characterized by the fact that the individual is a patient undergoing medical treatment.

8. Method of claim 1, characterized in that the monoclonal antibody therapy comprises dupilumab.

9. The method of claim 1, characterized in that the individual was administered a therapeutically effective dose of monoclonal antibody therapy.

10. Method of claim 1, characterized by the fact that the detectable marker is selected from the group consisting of ruthenium, horseradish peroxidase, alkaline phosphatase and a fluorophore.

11. Method of claim 1, characterized in that the individual has atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

12. Method of claim 1, characterized in that at least one antibody in the biological sample is an anti-drug antibody.

13. Invention, characterized by any of its forms or categories of claims covered by the subject matter initially disclosed in the patent application or in its examples presented herein, such as (but not limited to) monoclonal antibody, assay, compositions, formulations, kits and methods, among others. Petition 870260045338, dated 05 / 13 / 2026, pp. 71 / 90

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