Antibodies that bind to IL4R and their use

Abstract

The present invention provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human IL4Rα. The present invention also provides nucleic acid molecules encoding the antibody or antigen-binding portion thereof, expression vectors, host cells, and methods for expressing the antibody or antigen-binding portion thereof. The present invention further provides bispecific molecules, oncolytic viruses, and pharmaceutical compositions comprising the antibody or antigen-binding portion thereof, as well as therapeutic methods using the anti-IL4Rα antibody or antigen-binding portion thereof of the present invention.

Description

Detailed Description of the Invention

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 982,521, filed on February 27, 2020.

[0002] All documents cited or examined in the foregoing application and this application (the "documents cited in the application"), all documents cited or referenced in this application (including but not limited to all documents cited in this application, patents, published patent applications) (the "documents cited in this application"), and all documents cited or referenced in the documents cited in this application are hereby incorporated by reference into and can be used in the practice of the present invention together with the instructions, descriptions, product specifications, and product sheets of the manufacturers of the products mentioned in this specification or the products mentioned in the documents incorporated by reference herein. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. All Genbank sequences referred to in the present invention are incorporated by reference to the sequence as of the earliest effective filing date of the present invention.

[0003] Technical Field The present invention relates to isolated monoclonal antibodies, particularly mouse, chimeric or humanized monoclonal antibodies or their antigen - binding sites, that bind to human IL4R, particularly IL4Rα, with high affinity and functionality. The present invention also provides nucleic acid molecules encoding the antibodies or their antigen - binding sites of the present invention, expression vectors, host cells and methods for expressing the antibodies or their antigen - binding sites of the present invention. The present invention further provides bispecific molecules, immune complexes, chimeric antigen receptors, oncolytic viruses and pharmaceutical compositions comprising the antibodies or their antigen - binding sites of the present invention, and methods of treatment using the anti - IL4Rα antibodies or their antigen - binding sites of the present invention.

[0004] Background Art Allergic diseases associated with type 2 inflammation, such as atopic dermatitis, hypersensitivity, allergic rhinitis, and allergic asthma, afflict more than 3 billion people worldwide, and their incidence continues to increase. According to the hygiene hypothesis, the high incidence is due to the decrease in exposure to infectious diseases as living standards improve, enabling the immune system to better handle certain harmless allergens (Stephen J. Galli et al., (2008) Nature 454(7203):445-454). Two key factors central to type 2 immunity are interleukin-4 (IL-4) and IL-13. They are required, for example, to drive most of the major features associated with type 2 inflammation, such as immunoglobulin E production and innate cell recruitment to the site of inflammation. (Gruning G et al., (1998) Science 282:2261-2263; Rankin JA et al., (1996) Proc Natl Acad Sci USA 93:7821-7825; Wills-Karp M et al., (1998) Science 282:2258-2261).

[0005] IL-4 and IL-13 are adjacent to each other on human chromosome 5 and may share regulatory elements. T helper 2 cells (T HIn (2), both cooperative and non - cooperative expressions of these two cytokines are observed (Katherine Bao et al., (2015) Cytokine 75(1):25 - 37). The two cytokines bind to cell - surface receptors to regulate cell functions and activate the transcriptional machinery. Specifically, IL - 4 first binds to the IL - 4Rα chain with picomolar affinity, recruits the IL - 2Rγ γc chain to form a type I receptor complex, or recruits IL - 13Rα1 to form a type II receptor complex. Depending on the levels or availability of IL - 2Rγ γc and IL - 13Rα1, which one to recruit in receptor complex formation is determined. Non - hematopoietic cells do not express or express low levels of IL - 2Rγ γc, but highly express IL - 13Rα1, while in lymphocytes, the opposite has been found. Myeloid cells are in between these two types of cells. The formation of the type II IL - 4 receptor complex is initiated by IL - 13 binding to the IL - 13Rα1 chain (nanomolar affinity binding), and can further recruit the IL - 4Rα chain. In addition to the type II IL - 4 receptor, IL - 13 can bind to IL - 13Rα2 with picomolar affinity, which is considered a decoy receptor (Irina G. Luzina et al., (2012) J Leukoc Biol 92(4):753 - 764). When the IL - 4 receptor complex is assembled, intracellular signaling molecules are activated, and among them, STAT6 and IRS signaling respond to the activation of the type I IL - 4 receptor, but the type II IL - 4 receptor cannot significantly activate IRS (Heller NM et al., (2008) Sci Signal 1(51):ra17 - ra17). STAT6 signaling is important for the differentiation of T H cells and IL - 4 production, and IRS molecules activate signaling pathways such as PI3K and mTOR (Gadani SP et al., (2012) J Immunol 189:4213 - 4219).

[0006] Researchers have suggested that excessive IL-4 / IL-13 signaling may cause allergic diseases, and thus several therapeutic antibodies have been developed to modify signaling via IL-4 and IL-13. For example, Leprikizumab, Anrukinzumab and Tralokinumab that bind to IL-13, and Pascolizumab that targets IL-4. Dupilumab and Pitrakinra are IL-4Rα antagonists, and when Pitrakinra binds to IL-4Rα, it blocks both type I and type II IL-4 receptors (Antoniu SA (2010) Curr Opin Investig Drugs 11:1286-1294). And STAT6 inhibitors have been found to inhibit the growth of prostate cancer cells, suggesting that targeting IL-4 / IL-13 may be useful for cancer treatment (Nappo G et al., (2017) Oncogenesis 2017, 6(5):e342). Therefore, more antibodies targeting IL-4, IL-13 and their receptors (especially IL-4Rα) with more desirable therapeutic properties are desired.

[0007] Summary of the Invention The present invention provides an isolated monoclonal antibody or an antigen-binding site thereof, which is a monoclonal antibody of, for example, mouse, human, chimeric or humanized, that binds to IL4Rα (e.g., human IL4Rα) and has a binding affinity / ability equal to or higher than that of anti-IL4Rα antibodies such as Dupilumab in the prior art, and a blocking activity equal to or higher than that against IL4Rα-IL4 / IL13-IL13Rα1 interaction and corresponding intracellular signaling.

[0008] The antibody or antigen-binding site thereof of the present invention can be used in various applications, including the detection of IL4Rα protein, and the treatment and prevention of IL4, IL13 or IL4R-related diseases such as allergic diseases and cancer.

[0009] Accordingly, on one hand, the present invention provides an isolated monoclonal antibody (e.g., a mouse antibody, a chimeric antibody or a humanized antibody) or an antigen-binding site thereof. The monoclonal antibody or its antigen-binding site binds to IL4Rα and contains a heavy-chain variable region. The heavy-chain variable region contains a CDR1 region, a CDR2 region and a CDR3 region. Here, the CDR1 region, the CDR2 region and the CDR3 region are (1) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 1, 5 and 10 respectively; (2) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 1, 6 and 11 respectively; (3) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 2, 7 and 12 respectively; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 3, 8 and 13 respectively; (5) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 4, 8 and 13 respectively; or (6) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 3, 9 and 14 respectively.

[0010] On one side, the isolated monoclonal antibody or antigen-binding site thereof of the present invention comprises a heavy chain variable region. The heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 32, 33 (X1 = W, X2 = S; X1 = L, X2 = A; X1 = W, X2 = A), 34, 38, 40, 41 (X1 = A, X2 = K, X3 = V, X4 = H; X1 = V, X2 = K, X3 = V, X4 = H; X1 = A, X2 = Q, X3 = V, X4 = H; X1 = A, X2 = K, X3 = M, X4 = H; X1 = A, X2 = K, X3 = V, X4 = Y; X1 = V, X2 = K, X3 = M, X4 = H), 42 (X1 = R, X2 = A, X3 = S, X4 = N; X1 = K, X2 = V, X3 = S, X4 = N; X1 = K, X2 = A, X3 = T, X4 = N; X1 = K, X2 = A, X3 = S, X4 = D; X1 = R, X2 = V, X3 = T, X4 = N), 43, 44, 47, 49, 51 or 53. Here, the antibody or its antigen-binding site binds to IL4Rα. The amino acid sequence shown in SEQ ID NO: 32 can be encoded by the nucleotide sequence shown in SEQ ID NO: 59 or 60. The amino acid sequence shown in SEQ ID NO: 40 can be encoded by the nucleotide sequence shown in SEQ ID NO: 65 or 66. The amino acid sequences shown in SEQ ID NO: 33 (X1 = W, X2 = A) and 41 (X1 = V, X2 = K, X3 = M, X4 = H) can be encoded by the nucleotide sequences shown in SEQ ID NO: 61 or 67, respectively.

[0011] On one side, the isolated monoclonal antibody or its antigen-binding site of the present invention binds to IL4Rα. The monoclonal antibody or its antigen-binding site includes a light chain variable region. The light chain variable region includes a CDR1 region, a CDR2 region, and a CDR3 region. Here, the CDR1 region, the CDR2 region, and the CDR3 region are: (1) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 15, 22, and 26 respectively; (2) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 16, 22, and 27 respectively; (3) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 17, 23, and 28 respectively; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 18, 24, and 29 respectively; (5) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 19, 24, and 30 respectively; (6) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 20, 25, and 31 respectively; or (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 21, 25, and 31 respectively are included.

[0012] On one side, the isolated monoclonal antibody or antigen-binding site thereof of the present invention comprises a light chain variable region. The light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 35, 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), 37, 39, 45, 46, 48, 50, 52 or 54. Here, the antibody or antigen-binding site thereof binds to IL4Rα. The amino acid sequence shown in SEQ ID NO: 35 may be encoded by the nucleotide sequence shown in SEQ ID NO: 62 or 63. The amino acid sequence shown in SEQ ID NO: 45 may be encoded by the nucleotide sequence shown in SEQ ID NO: 68 or 69. The amino acid sequences shown in SEQ ID NO: 36 (X1 = F, X2 = V) and 46 may be encoded by the nucleotide sequences shown in SEQ ID NO: 64 or 70, respectively.

[0013] On one side, the isolated monoclonal antibody or antigen-binding site thereof of the present invention comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region and the light chain variable region each comprise a CDR1 region, a CDR2 region, and a CDR3 region.Here, the heavy chain variable region CDR1, CDR2, and CDR3 and the light chain variable region CDR1, CDR2, and CDR3 are: (1) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 1, 5, 10, 15, 22, and 26, respectively; (2) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 1, 6, 11, 16, 22, and 27, respectively; (3) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 2, 7, 12, 17, 23, and 28, respectively; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 3, 8, 13, 18, 24, and 29, respectively; (5) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 4, 8, 13, 19, 24, and 30, respectively; (6) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 3, 9, 14, 20, 25, and 31, respectively; or (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 3, 9, 14, 21, 25, and 31, respectively. Here, the antibody or its antigen-binding site binds to IL4Rα.

[0014] On one side, the isolated monoclonal antibody or antigen-binding site thereof of the present invention comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region and the light chain variable region are (1) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 32 and 35 respectively; (2) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 33 (X1 = W, X2 = S) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) respectively; (3) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 33 (X1 = W, X2 = S) and 37 respectively; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 34 and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) respectively; (5) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 34 and 37 respectively; (6) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 33 (X1 = L, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) respectively;(7) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 33 (X1 = L, X2 = A) and 37, respectively; (8) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 33 (X1 = W, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (9) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 33 (X1 = W, X2 = A) and 37, respectively; (10) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 38 and 39, respectively; (11) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 40 and 45, respectively; (12) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 41 (X1 = A, X2 = K, X3 = V, X4 = H; X1 = V, X2 = K, X3 = V, X4 = H; X1 = A, X2 = Q, X3 = V, X4 = H; X1 = A, X2 = K, X3 = M, X4 = H; X1 = A, X2 = K, X3 = V, X4 = Y; X1 = V, X2 = K, X3 = M, X4 = H) and 46, respectively; (13) An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 42 (X1 = R, X2 = A, X3 = S, X4 = N; X1 = K, X2 = V, X3 = S, X4 = N; X1 = K, X2 = A, X3 = T, X4 = N; X1 = K, X2 = A, X3 = S, X4 = D;(X1 = R, X2 = V, X3 = T, X4 = N) and an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in 46; (14) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 43 and 46 respectively; (15) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 44 and 46 respectively; (16) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 47 and 48 respectively; (17) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 49 and 50 respectively; (18) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 51 and 52 respectively; or (19) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 53 and 54 respectively is included. Here, the antibody or its antigen-binding site binds to IL4Rα.;

[0015] In some embodiments, the isolated monoclonal antibody or antigen-binding site thereof of the present invention comprises a heavy chain and a light chain. The heavy chain and the light chain are linked by disulfide bonds. The heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region. Here, the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. Here, the heavy chain variable region and the light chain variable region comprise the aforementioned amino acid sequences, and the antibody or its antigen-binding site binds to IL4Rα. The heavy chain constant region can be a human IgG4 constant region having the amino acid sequence shown in SEQ ID NO: 55. The light chain constant region can be a human κ constant region having the amino acid sequence shown in SEQ ID NO: 56. The amino acid sequences shown in SEQ ID NOs: 55 and 56 can be encoded by the nucleotide sequences shown in SEQ ID NOs: 71 and 72, respectively.

[0016] In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains. Here, each heavy chain comprises the above heavy chain constant region, the above heavy chain variable region or CDR sequence, and each light chain comprises the above light chain constant region, the above light chain variable region or CDR sequence. Here, the antibody binds to IL4Rα. The antibody of the present invention can be a full-length antibody, for example, an IgG1, IgG2 or IgG4 isotype full-length antibody, preferably an IgG4 isotype full-length antibody having weak ADCC activity. The light chain constant region can be a κ constant region. In other embodiments, the antibody of the present invention can be a single-chain variable region (scFv) antibody, or an antibody fragment, for example, a Fab or F(ab’)2 fragment.

[0017] For example, compared with prior art anti-IL4Rα antibodies such as Dupilumab, the antibody of the present invention, or its antigen-binding site, has equivalent (not higher) binding affinity / ability for human IL4Rα and / or monkey IL4Rα. And it has equivalent (not higher) blocking activity against the IL4Rα-IL4 / IL13-IL13Rα1 interaction and the corresponding intracellular signal transduction.

[0018] The present invention also provides a bispecific molecule comprising an antibody of the present invention or an antigen-binding site thereof, which is linked to a second functional molecule (e.g., a second antibody) having a binding specificity different from that of the antibody or its antigen-binding site. The present invention further provides an immune complex comprising an antibody of the present invention or an antigen-binding site thereof, such as an antibody-drug conjugate. Here, the antibody or its antigen-binding site is linked to a therapeutic agent (e.g., a cytotoxin). In another aspect, the antibody or its antigen-binding site of the present invention can be part of a chimeric antigen receptor (CAR). The present invention further provides immune cells such as T cells comprising a chimeric antigen receptor. The antibody or its antigen-binding site of the present invention can be encoded by a tumor-lytic virus or used in combination with a tumor-lytic virus.

[0019] The present invention provides a composition comprising an antibody of the present invention or an antigen-binding site thereof, an immune complex, a bispecific molecule, a tumor-lytic virus, a CAR or a CAR-T cell, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may further comprise an anti-allergic agent or an anti-tumor agent.

[0020] The present invention provides a nucleic acid molecule encoding an antibody of the present invention or an antigen-binding site thereof, and an expression vector comprising the nucleic acid molecule and a host cell comprising the expression vector. The present invention provides a method for preparing an anti-IL4Rα antibody or an antigen-binding site thereof by a host cell comprising an expression vector, which comprises (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell or its cell culture.

[0021] In another aspect, a method for reducing IL4 / IL13 signaling is provided. IL4 signals through a receptor comprising IL-4Rα and γC, and IL13 signals through a receptor comprising IL-4Rα and IL13Rα1. Non-limiting examples of IL4 / IL13 signaling include activation and / or proliferation of B cells, eosinophils, macrophages (e.g., activated macrophages), proliferation of fibroblasts, and proliferation of smooth muscle such as airway smooth muscle.

[0022] On the other hand, the present invention provides a method for treating a disease associated with excessive IL4 / IL13 signaling, said method comprising administering to a subject a therapeutically effective amount of an antibody of the present invention or an antigen-binding site thereof.

[0023] The disease may be an allergic disease. The allergic disease may be atopic dermatitis, an allergic reaction, allergic rhinitis or allergic asthma. In some embodiments, the method for treating an allergic disease may comprise administering to the subject a composition of the present invention, a bispecific molecule, or an oncolytic virus encoding or carrying an antibody of the present invention, or a nucleic acid molecule or vector capable of expressing the same in the subject. The method may also comprise administering an anti-allergy agent. The anti-allergy agent may be an antihistamine, a corticosteroid, a beta-adrenergic receptor agonist, a drug targeting cyc-LT, or a drug targeting IgE.

[0024] The disease can be a tumor disease. The tumor can be a solid tumor or a non-solid tumor. In some embodiments, the tumor is prostate cancer. In some embodiments, the method comprises administering to the subject a composition of the present invention, an immune complex such as a bispecific molecule, an antibody-drug conjugate, a CAR-T cell, or a oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule or vector capable of expressing the same in the subject. In some embodiments, at least one additional anti-cancer antibody, for example, an anti-VISTA antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM 3 antibody, an anti-STAT3 antibody, and / or an anti-ROR1 antibody can be administered together with the antibody of the present invention or its antigen-binding site. In another embodiment, the antibody of the present invention or its antigen-binding site can be administered together with a cytokine (e.g., IL-2, IL-21, and / or GM-CSF) or a co-stimulatory antibody (e.g., an anti-CD137 antibody and / or an anti-GITR antibody). The antibody of the present invention can be, for example, a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody.

[0025] In another aspect, the present invention provides a method for reducing a type 2 immune response, which comprises administering to a subject a therapeutically effective amount of the antibody of the present invention or its antigen-binding site. In some embodiments, the method comprises administering to the subject a composition of the present invention, a bispecific molecule, or an oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule or vector capable of expressing the same in the subject.

[0026] In another aspect, the present invention provides methods, compositions, and kits for diagnosis. In some embodiments, the antibody of the present invention is used to determine the presence and expression of IL4Rα in cells or tissues to determine prognosis and appropriate treatment and follow-up.

[0027] Other features and advantages of the present invention will become apparent from the following detailed description and examples, which should not be construed as limiting. The content of all documents cited in this application, Genbank records, patents, and published patent applications are hereby expressly incorporated herein by reference. Brief Description of the Drawings

Brief Description of the Drawings

[0028]

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[0029] The following detailed description and the accompanying drawings, given by way of example, may be best understood, but the invention should not be limited to the specific embodiments described.

[0030] Detailed Description of the Invention To better understand the present invention, some terms are first defined. Other definitions are set forth throughout the detailed description.

[0031] The term "IL4Rα" refers to the α subunit of the interleukin 4 receptor. The term "IL4Rα" includes variants, subtypes, homologs, orthologs, and paralogs. For example, an antibody specific for the human IL4Rα protein may, in some cases, cross-react with an IL4Rα protein from a non-human species (e.g., monkey). In other embodiments, an antibody specific for the human IL4Rα protein is completely specific for the human IL4Rα protein and does not show cross-reactivity with other species or other types, or may cross-react with IL4Rα from a specific other species but not with all others.

[0032] The term "human IL4Rα" refers to an IL4Rα protein having an amino acid sequence derived from a human, such as the amino acid sequence of human IL4Rα having Genbank accession number NP_001244335.1. The terms "cynomolgus monkey IL4Rα" and "marmoset monkey IL4Rα" refer to IL4Rα sequences having the amino acid sequences of Genbank accession numbers EHH60265.1 and NP_001244161.1, respectively.

[0033] The term "antibody" as referred to herein includes full-length antibodies and any antigen-binding fragment (i.e., "antigen-binding site") or single-chain thereof. A full-length antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V H and a heavy chain constant region. The heavy chain constant region is composed of three domains, C H1 , C H2 and C H3 Each light chain is composed of a light chain variable region (abbreviated herein as V L(abbreviated as) and a light chain constant region. The light chain constant region consists of one domain C L composed of. V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) in which more conserved regions called framework regions (FRs) are interspersed. Each V H and V L is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0034] The term "antigen-binding site" (or simply "antibody site") of an antibody, as used herein, refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (e.g., the IL4Rα protein). It has been shown that the antigen-binding function of an antibody can be carried out by fragments of the full-length antibody. Examples of binding fragments included in the term "antigen-binding site" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of V L V H C L and C H1 domains; (ii) an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V H and C H1 ; (iv) an Fv fragment consisting of the V L and V H of a single arm of the antibody; (v) V HdAb fragments consisting of (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains; are included. And the two domains of the Fv fragment, V L and V H are encoded by separate genes but can be joined by a synthetic linker that enables them to be made as single-chain proteins using recombinant methods. Here, V L and V H pair to form a monovalent molecule (referred to 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). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding site" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0035] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to the IL4Rα protein is substantially free of antibodies that specifically bind to antigens other than the IL4Rα protein). However, an isolated antibody that specifically binds to the human IL4Rα protein may have cross-reactivity with other antigens such as the IL4Rα protein from other species. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemical substances.

[0036] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0037] As used herein, the term "mouse antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Further, when the antibody includes a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of the invention can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, the term "mouse antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.

[0038] The term "chimeric antibody" refers to an antibody produced by combining genetic material from a non-human source with genetic material from a human. Or, more generally, a chimeric antibody is an antibody that has genetic material from one species and genetic material from another species.

[0039] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to enhance its similarity to antibodies naturally produced in humans.

[0040] The term "isotype" refers to the antibody class encoded by the heavy chain constant region gene (e.g., IgM or IgG1).

[0041] The terms "antibody that recognizes an antigen", "antibody specific for an antigen", and "antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

[0042] As used herein, an antibody that "specifically binds to human IL4Rα" refers to an antibody that binds to human IL4Rα protein (and possibly IL4Rα proteins from one or more non-human species), but does not substantially bind to non-human IL4Rα proteins. Preferably, the antibody binds to human IL4Rα protein with "high affinity". That is, 5.0× -8 M or less, preferably 1.0×10 -8 M or less, more preferably 7.0×10 -9 M or less of K D to bind to human IL4Rα protein.

[0043] As used herein, the term "does not substantially bind to" a protein or cell means, as used herein, does not bind to or binds with low affinity to the protein or cell, i.e., 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, more preferably 1.0×10 -3 M or higher, even more preferably 1.0×10 -2 M or higher of K D to bind to the protein or cell.

[0044] The term "high affinity" for an IgG antibody refers to an antibody having 1.0×10 -6 M or less, more preferably 5.0×10 -8M or less, even more preferably 1.0×10 -8 M or less, even more preferably 7.0×10 -9 M or less, even more preferably 1.0×10 -9 M or less of K D for the target antigen. However, "high affinity" binding may be different for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having 1.0×10 -6 M or less, preferably 1.0×10 -7 M or less, more preferably 1.0×10 -8 M or less of K D for the target antigen.

[0045] As used herein, the term "K assoc " or "K a " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K dis " or "K d " as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "K D " as used herein refers to the dissociation constant obtained from the ratio of K d to K a (i.e., K d / K a ) and is intended to be expressed as a molar concentration (M). The K D value of an antibody can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably by using a biosensor system such as a Biacore TM system.

[0046] The term "EC 50 ", also known as the median effective dose, refers to the concentration of an antibody that elicits a response midway between the baseline and the maximum value after a specific exposure time.

[0047] The term "IC 50 ", also known as the half-maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a particular biological or biochemical function by 50% compared to the absence of the antibody.

[0048] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates that are mammals and non-mammals, for example, non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, but mammals such as non-human primates, sheep, dogs, cats, cows, horses, etc. are preferred.

[0049] The term "therapeutically effective amount" means an amount of an antibody of the present invention sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and / or to reduce the severity of the disease or condition. A therapeutically effective amount is understood to be related to the condition being treated and the actual effective amount can be readily identified by one of ordinary skill in the art.

[0050] As used herein, the term "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. Sequence comparisons and percent identity determinations between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithms available at the National Center For Biotechnology Institute Web site.

[0051] Aspects of the present invention are described in more detail below.

[0052] Anti-IL4Rα antibody with improved binding affinity for human IL4Rα and improved IL4 / IL13 signal transduction blocking activity The antibody of the present invention, or antigen-binding portion thereof, specifically binds to human IL4Rα with (if not higher) equivalent binding affinity / ability as compared to previously described anti-IL4Rα antibodies (such as Dupilumab).

[0053] The antibody of the present invention or antigen-binding portion thereof can block the binding of IL4 or IL13-IL13Rα1 to IL4Rα, thereby blocking the corresponding intracellular signaling, which has equivalent or higher blocking activity as compared to reported anti-IL4Rα antibodies (such as Dupilumab).

[0054] Preferably, the antibody of the present invention is a humanized monoclonal antibody. Additionally, or alternatively, the antibody of the present invention may be, for example, a chimeric monoclonal antibody.

[0055] Anti-IL4Rα monoclonal antibody The antibody of the present invention is a monoclonal antibody structurally and chemically characterized as described below and in the examples. The amino acid sequence IDs of the heavy chain / light chain variable regions of the antibody are summarized in Table 1 below. Some antibodies have the same V H or V L . The heavy chain constant region of the antibody can be a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 55. The light chain constant region of the antibody can also be a human κ constant region having the amino acid sequence shown in SEQ ID NO: 56.

[0056]

Table 1-1

Table 1-2

Table 1-3

[0057] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system. However, as is well known in the art, the CDR regions can also be determined by other numbering systems such as the Chothia, IMGT, AbM or Contact numbering systems / methods based on the heavy chain / light chain variable region sequences.

[0058] The V H and V L sequences (or CDR sequences) of other anti-IL4Rα antibodies that bind to human IL4Rα can be "mixed and paired" with the V H and V L sequences (or CDR sequences) of the anti-IL4Rα antibody of the present invention. Preferably, when the V H and V L chains (or CDRs of these chains) are mixed to form pairs, the V H / V L sequences from a particular V H pair are structurally similar to the V His replaced by an array. Similarly, preferably, a specific V H / V L from the pair of V L arrays are replaced by structurally similar V L arrays.

[0059] Thus, in some embodiments, the antibody or antigen-binding site thereof of the present invention comprises: (a) a heavy chain variable region comprising the amino acid sequences listed in Table 1; and (b) a light chain variable region comprising the amino acid sequences listed in Table 1, or the V L of another anti-IL4Rα antibody that specifically binds to human IL4Rα. In another embodiment, the antibody or antigen-binding site thereof of the present invention comprises: (a) CDR1, CDR2, and CDR3 of the heavy chain variable region listed in Table 1; and (b) CDR1, CDR2, and CDR3 of the light chain variable region listed in Table 1, or the CDRs of another anti-IL4Rα antibody that specifically binds to human IL4Rα.

[0060] In another embodiment, the antibody, or antigen-binding site thereof, of the present invention comprises CDR2 of the heavy chain variable region of an anti-IL4Rα antibody and the CDRs of other antibodies that bind to human IL4Rα, such as CDR1 and / or CDR3 of the heavy chain variable region from another anti-IL4Rα antibody, and / or CDR1, CDR2, and / or CDR3 of the light chain variable region.

[0061] Furthermore, the CDR3 domain can, independently of the CDR1 and / or CDR2 domains, alone determine the binding specificity of an antibody to a homologous antigen, and can predictably generate multiple antibodies having the same binding specificity based on a common CDR3 sequence. For example, see Klimka et al., British J. of Cancer 83(2):252-260(2000); Beiboer et al., J. Mol. Biol. 296:833-849(2000); Rader et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915(1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162(1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533(1995); Ditzel et al., J. Immunol. 157:739-749(1996); Berezov et al., BIAjournal 8: Scientific Review 8(2001); Igarashi et al., J. Biochem(Tokyo)117:452-7(1995); Bourgeois et al., J. Virol 72:807-10(1998); Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8(1993); Polymenis and Stoller, J. Immunol. 152:5218-5329(1994) and Xu and Davis, Immunity 13:37-45(2000). Also see U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of these references is hereby incorporated by reference in its entirety.

[0062] Accordingly, in another embodiment, the antibody of the present invention comprises the CDR2 of the heavy chain variable region of an anti-IL4Rα antibody and at least the CDR3 of the heavy chain variable region and / or the light chain variable region of an anti-IL4Rα antibody or the CDR3 of the heavy chain variable region and / or the light chain variable region of another anti-IL4Rα antibody, and the antibody specifically binds to human IL4Rα. These antibodies preferably (a) compete with the anti-IL4Rα antibody of the present invention for binding to IL4Rα; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinities. In another embodiment, the antibody of the present invention may also comprise the CDR2 of the light chain variable region of an anti-IL4Rα antibody or the CDR2 of the light chain variable region of another anti-IL4Rα antibody, and the antibody specifically binds to human IL4Rα. In another embodiment, the antibody of the present invention may further comprise the CDR1 of the heavy chain and / or the light chain variable region of an anti-IL4Rα antibody or the CDR1 of the heavy chain and / or the light chain variable region of another anti-IL4Rα antibody, and the antibody specifically binds to human IL4Rα.

[0063] Conservative modification In another embodiment, the antibody of the present invention comprises a heavy chain variable region and / or a light chain variable region each containing CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 sequences are different from the CDR1, CDR2, and CDR3 sequences of the anti-IL4Rα antibody of the present invention. The "different" is due to one or more conservative modifications. It is understood in the art that certain conservative sequence modifications do not abrogate antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0064] Thus, in some embodiments, the antibody of the present invention comprises a heavy chain variable region and / or a light chain variable region each containing CDR1, CDR2, and CDR3. Among them, (a) CDR1 of the heavy chain variable region comprises the sequences listed in Table 1 and / or conservative modifications thereof; and / or (b) CDR2 of the heavy chain variable region comprises the sequences listed in Table 1 and / or conservative modifications thereof; and / or (c) CDR3 of the heavy chain variable region comprises the sequences listed in Table 1 and / or conservative modifications thereof; and / or (d) CDR1, and / or CDR2, and / or CDR3 of the light chain variable region comprises the sequences listed in Table 1 and / or conservative modifications thereof; and (e) This antibody specifically binds to human IL4Rα.

[0065] The antibodies of the present invention have one or more of the functional properties such as high affinity binding to human IL4Rα and blocking activity against IL4Rα-IL4 binding or IL4Rα-IL13-IL13Rα1 binding.

[0066] In some embodiments, the antibody can be, for example, a murine antibody, a human antibody, a humanized antibody or a chimeric antibody.

[0067] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. The modifications can be introduced into the antibodies of the present invention by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine, etc.), acidic side chains (e.g., aspartic acid, glutamic acid, etc.), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan, etc.), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of the antibodies of the present invention can be replaced with other amino acid residues from the same side chain family, and the modified antibody can be tested for retained function (i.e., the functions described above) using the functional assays described herein.

[0068] Engineered and modified antibody The antibodies of the present invention are the V of the anti-IL4Rα antibodies of the present invention H / V LAn engineered antibody can be prepared using as a starting material an antibody having one or more arrays. The antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V H and / or V L ), for example, within one or more CDR regions and / or within one or more framework regions).

[0069] In certain embodiments, CDR grafting can be used to alter the variable regions of an antibody. Antibodies interact with target antigens primarily through amino acid residues in the six complementarity-determining regions (CDRs) of the heavy and light chains. Thus, the amino acid sequences within the CDRs are more diverse among different antibodies than the amino acid sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular natural antibody by constructing an expression vector capable of grafting the CDR sequences of a particular natural antibody into the framework sequences of an antibody with different properties (see, for example, Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. U.S.A. 86:10029-10033; see also U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0070] Accordingly, another embodiment of the invention relates to an isolated monoclonal antibody or antigen-binding site thereof comprising a heavy chain variable region and / or a light chain variable region. The heavy chain variable region comprises CDR1, CDR2 and CDR3 of the present invention, and the light chain variable region comprises CDR1, CDR2 and CDR3 of the present invention. These antibodies comprise the V H and V L CDR sequences of the monoclonal antibodies of the present invention, but may comprise different framework sequences.

[0071] Such framework arrays can be obtained from publicly available DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes are available from the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase); Kabat et al., (1991) as described above; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836, which are hereby expressly incorporated by reference. In another embodiment, the germline DNA sequences of human heavy chain variable region genes and human light chain variable region genes are available from the Genbank database. For example, the following heavy chain germline sequences from the HCo7 HuMAb mouse have Genbank accession numbers 1-69 (NG--0010109, NT--024637 & BC070333), 3-33 (NG--0010109 & NT--024637)) and 3-7 (NG--0010109 & NT--024637). In another embodiment, the following heavy chain germline sequences from the HCo12 HuMAb mouse have Genbank accession numbers 1-69 (NG--0010109, NT--024637 & BC070333), 5-51 (NG--0010109 & NT--024637), 4-34 (NG--0010109 & NT--024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

[0072] Antibody protein sequences are compared to compiled protein sequence databases using a sequence similarity search method known to those of skill in the art called Gapped BLAST (Altschul et al., (1997), as described above).

[0073] The antibody framework sequences used in the present invention are preferably those that are structurally similar to the antibody framework sequences of the present invention. V HThe CDR1, CDR2, and CDR3 sequences can be grafted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence is derived. Alternatively, the CDR sequences can be grafted into a framework region that contains one or more mutations compared to the germline sequence. For example, it may be beneficial to mutate residues in the framework region in order to maintain or enhance the antigen-binding ability of the antibody. (See, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0074] Another type of variable region modification is to H and / or L mutate the amino acid residues within the CDR1, CDR2, and / or CDR3 regions of V

[0075] thereby improving one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in vitro or in vivo assays as is known in the art. Preferably, conservative modifications (known in the art) are introduced. The mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Further, typically, one, two, three, four, or five or fewer residues within the CDR region are altered. H CDR1 sequence of the invention, or a V H CDR1 region that contains an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (b) the V H CDR2 sequence of the invention, or a V H CDR2 region that contains an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (c) the V HA V containing a CDR3 sequence, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions H CDR3 region; (d) the V of the present invention L A V containing a CDR1 sequence, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions L CDR1 region; (e) the V of the present invention L A V containing a CDR2 sequence, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions L CDR2 region; and (f) the V of the present invention L A V containing a CDR3 sequence, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions L CDR3 region.

[0076] The engineered antibodies of the present invention include, for example, those in which the framework region residues of V H and / or V L are modified to improve antibody properties. Generally, such framework region modifications can be used to reduce the immunogenicity of the antibody. For example, one or more framework region residues are "reverted to wild type" to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutations may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived.

[0077] Another type of framework modification is to mutate one or more residues within the framework region or within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in more detail in US Patent Publication No. 20030153043.

[0078] Alternatively, or as an alternative to the modifications made within the framework or CDR regions, the antibodies of the present invention can typically be engineered to include modifications within the Fc region in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Furthermore, the antibodies of the present invention can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody), or modified to alter their glycosylation, again altering one or more functional properties of the antibody.

[0079] In some embodiments, the H1 C-hinge region is modified such that the number of cysteine residues within the hinge region is altered (e.g., increased or decreased). This method is further described in U.S. Patent Nos. 5,677,425. H1 The number of cysteine residues in the C-hinge region is altered, for example, to facilitate light and heavy chain assembly or to increase or decrease the stability of the antibody.

[0080] In another embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 domain interface region to impair binding to staphylococcal protein A (SpA) compared to the native Fc hinge domain. This method is described in further detail in U.S. Patent Nos. 6,165,745.

[0081] In another embodiment, the glycosylation of the antibody is modified. For example, a deglycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to enhance the affinity of the antibody for the antigen. Such glycosylation modifications can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made to effect the removal of glycosylation sites in one or more variable region frameworks, thereby removing glycosylation at those sites. Such aglycosylation can potentially enhance the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0082] Furthermore, it is possible to produce antibodies with altered glycosylation types, such as afucosylated antibodies with a reduced amount of fucosyl residues or antibodies with an increased bisecting GlcNac structure. Such altered glycosylation patterns have been demonstrated to increase or decrease the ADCC ability of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell having a modified glycosylation mechanism. Cells having a modified glycosylation mechanism are described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies with modified glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), so the antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their sugar chains. The Ms704, Ms705, and Ms709 FUT8− / − cell lines were created by disrupting the target FUT8 gene in CHO / DG44 cells using two replacement vectors (see US Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87: 614-22). As another example, EP 1,176,195 describes a cell line having a functionally disrupted FUT8 gene encoding fucosyltransferase, and antibodies expressed in such cell lines exhibit afucosylation by reducing or removing α-1,6 linkage-related enzymes. EP 1,176,195 also describes cell lines with reduced or eliminated enzyme activity for adding fucose to N-acetylglucosamine that binds to the Fc region of the antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662).PCT Publication WO 03 / 035835 describes the Lec13 cell line, a mutant CHO cell line that has a reduced ability to attach fucose to the Asn(297)-linked sugar chain and also results in hypofucosylation of antibodies expressed in its host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with an altered glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with an altered glycosylation profile can be produced in plant cells such as moss. Methods for producing antibodies in a plant system are disclosed in a U.S. patent application corresponding to Attorney Docket No. 040989 / 314911 of Alston & Bird LLP, filed on August 11, 2006. The fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14:5516-23).

[0083] Another modification of the antibodies of the present invention is pegylation. The antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, typically the antibody or a fragment thereof is reacted with polyethylene glycol (PEG), such as an active ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups bind to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation or alkylation reaction with an active PEG molecule (or similar active water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP0154316 and EP0401384.

[0084] Physical properties of the antibody The antibodies of the present invention can be characterized by their various physical properties in order to detect and / or distinguish classes.

[0085] For example, an antibody can contain one or more glycosylation sites in either the light chain or the heavy chain variable region. Such glycosylation sites can lead to an increase in the immunogenicity of the antibody or a change in the pK of the antibody due to a change in antigen binding. (Marshall et al (1972) Annu Rev Biochem 41:673 - 702; Gala and Morrison (2004) J Immunol 172:5489 - 94; Wallick et al (1988) J Exp Med 168:1099 - 109; Spiro (2002) Glycobiology 12:43R - 56R; Parekh et al (1985) Nature 316:452 - 7; Mimura et al., (2000) Mol Immunol 37:697 - 706). Glycosylation is known to occur at motifs containing the N - X - S / T sequence. In some cases, an anti - IL4Rα antibody that does not contain variable region glycosylation is preferred. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylation region.

[0086] In a preferred embodiment, the antibody does not contain an asparagine isomerization site. Deamidation of asparagine occurs at N - G or D - G sequences and results in the formation of isoaspartic acid residues that introduce a cross - link into the polypeptide chain and reduce its stability (the isoaspartic acid effect).

[0087] Each antibody has a unique isoelectric point (pI), which is generally in the pH range of 6 - 9.5. The pI of IgG1 antibodies is usually in the pH range of 7 - 9.5, and the pI of IgG4 antibodies is usually in the pH range of 6 - 8. There is a speculation that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferred to have an anti - IL4Rα antibody with a pI value within the normal range. This can be achieved by selecting antibodies with a pI within the normal range or by mutating charged surface residues.

[0088] Nucleic acid molecule encoding the antibody of the present invention On the other hand, the present invention provides a nucleic acid molecule encoding the heavy chain and / or light chain variable region, or CDR, of an antibody of the present invention. The nucleic acid can be present in whole cells, cell lysates; or can be present in a partially purified or substantially pure form. When the nucleic acid is purified from other cellular components or other contaminants, such as nucleic acids or proteins of other cells, by standard techniques, it is "isolated" or "substantially purified". The nucleic acid of the present invention can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0089] The nucleic acid of the present invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, further described below), the cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene libraries.

[0090] Preferably, the nucleic acid molecule of the present invention includes those encoding the V H and V L sequences or CDRs of the IL4Rα monoclonal antibody. When DNA fragments encoding the V H and V L segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, V H or V LThe DNA fragment encoding it is operably linked to another DNA fragment encoding another protein by an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0091] V H The DNA encoding the region is operably linked to another DNA molecule encoding the heavy chain constant region (C H1 , C H2 and C H3 ) to convert the isolated DNA encoding V H into a full-length heavy chain gene. The sequences of the human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably an IgG1 or IgG4 constant region. In the case of the Fab fragment heavy chain gene, the DNA encoding V H can be operably linked to another DNA molecule encoding only the heavy chain C H1 constant region.

[0092] V L The DNA encoding the region is operably linked to another DNA molecule encoding the light chain constant region C L to convert the isolated DNA encoding V L into a full-length light chain gene (and Fab light chain gene). The sequences of the human light chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a κ or λ constant region.

[0093] To prepare the scFv gene, V H and V LThe DNA fragment encoding it is operably linked to another fragment encoding a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3, such that the V H and V L sequences can be expressed as a continuous single-chain protein. The V L region and the V H region are linked by this flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0094] Production of the monoclonal antibody of the present invention The monoclonal antibodies (mAbs) of the present invention can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256: 495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. For example, they are described in U.S. Patent Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are hereby specifically incorporated by reference in their entirety.

[0095] Generation of transfectomas producing the monoclonal antibody of the present invention The antibodies of the present invention can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In some embodiments, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the gene is operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked to the vector such that the transcriptional and translational control sequences within the vector perform their defined function of regulating the transcription and translation of the antibody gene.

[0096] The term "regulatory sequences" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include, for example, viral elements that direct high-level protein expression in mammalian cells, such as those derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyomavirus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or β-globin promoter can be used. Additionally, regulatory elements can be composed of sequences of various origins, such as the SRα promoter system that includes sequences from the SV40 early promoter and the long terminal repeat sequence of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

[0097] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In a preferred embodiment, the V H segment is operably linked to the C H segment within the vector, and the variable region is inserted into an expression vector encoding the heavy chain constant region and the light chain constant region of the desired isotype such that the V L segment is operably linked to the C L segment within the vector, thereby creating a full-length antibody gene of any antibody isotype. Alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0098] For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the diverse techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies of the present invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably in mammalian host cells, is most preferred. This is because eukaryotic cells, particularly mammalian cells, are more likely to construct and secrete immunocompetent antibodies that are properly folded than prokaryotic cells.

[0099] Preferred mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells for use with the DHFR selection marker, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, and the DHFR selection marker is described, for example, in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, when used in NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841. After a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the host cell is cultured for a time sufficient to express the antibody within the host cell or, preferably, for a time sufficient to secrete the antibody into the medium in which the host cell grows, and the antibody is prepared. The antibody can be recovered from the medium using standard protein purification methods.

[0100] Bispecific molecule On the other hand, provided are bispecific molecules comprising one or more antibodies of the present invention linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or a ligand for a receptor), to generate binding sites to at least two different binding sites. Thus, the "bispecific molecule" used in the present invention includes molecules having three or more specificities.

[0101] In some embodiments, the multispecific molecule has a third specificity in addition to the binding specificity for Fc and the binding specificity for IL4Rα.

[0102] Bispecific molecules can be of many different forms and sizes. At one end of the size spectrum, bispecific molecules retain the conventional antibody format, the difference being that the two binding arms have different specificities rather than the same specificity. At the other end are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, so-called Bs(scFv)2 constructs. Intermediate-sized bispecific molecules include two different F(ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, e.g., Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644(1998); van Spriel et al., Immunology Today, 21(8), 391-397(2000), and references cited therein.

[0103] Immune complex The antibodies of the present invention can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear transport inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and mitotic inhibitors. In an ADC, the antibody and the therapeutic agent are preferably linked via a cleavable linker such as a peptidyl, disulfide, or hydrazone linker. More preferred linkers are peptide linkers such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. The ADCs can be prepared according to U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02 / 096910; WO07 / 038,658; WO07 / 051,081; WO07 / 059,404; WO08 / 083,312; and WO08 / 103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295, the disclosures of which are incorporated herein by reference.

[0104] Oncolytic virus encoding or carrying an antibody Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the present invention can be used with oncolytic viruses. Alternatively, an oncolytic virus encoding the antibody of the present invention can be introduced into a human.

[0105] Chimeric antigen receptor The present invention also provides a chimeric antigen receptor (CAR) comprising an anti-IL4Rα scFv comprising the CDRs and heavy / light chain variable regions of the present invention.

[0106] The anti-IL4Rα CAR may comprise: (a) an extracellular antigen-binding domain comprising an anti-IL4Rα scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0107] The CAR may comprise, at the N-terminus of the extracellular antigen-binding domain, a signal peptide that directs the nascent receptor to the endoplasmic reticulum, and at the N-terminus of the extracellular antigen-binding domain, a hinge peptide that makes the receptor more binding-friendly. Preferably, the CAR comprises, in the intracellular signaling domain, a primary intracellular signaling domain and one or more co-stimulatory signaling domains in the intracellular signaling domain. The commonly used and most potent primary intracellular signaling domain is the CD3-zeta cytoplasmic domain containing ITAM, and the phosphorylation of its ITAM leads to the activation of T cells. The co-stimulatory signaling domains are derived from co-stimulatory proteins such as CD28, CD137, and OX40.

[0108] The CAR may further be supplemented with factors that enhance T cell proliferation, persistence, and anti-tumor activity, such as cytokines and co-stimulatory ligands.

[0109] Also provided are engineered immune effector cells comprising the CAR provided herein. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cells are T cells.

[0110] Pharmaceutical composition In another aspect, the invention provides a pharmaceutical composition comprising one or more antibodies of the invention (or antigen-binding sites thereof, or bispecific molecules, CAR-T cells, oncolytic viruses or immune complexes) formulated together with a pharmaceutically acceptable carrier. Where the composition comprises a plurality of antibodies (or antigen-binding sites thereof, or bispecific molecules, CAR-T cells, oncolytic viruses, immune complexes), the antibodies (or antigen-binding sites thereof, or bispecific molecules, CAR-T cells, oncolytic viruses, immune complexes) can be administered separately. The composition can optionally comprise one or more additional pharmaceutically active ingredients, such as another antibody, or a drug such as an anti-tumor agent or anti-allergy agent.

[0111] The pharmaceutical composition can contain any number of excipients. Excipients that can be used include vectors, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizing agents, colorants, flavorants, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of appropriate excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0112] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient can be coated with a material to protect it from the action of acids and other natural conditions that may inactivate the active ingredient. As used herein, the term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, intrathecal, intraspinal, epidural and intrasternal injections and infusions. Alternatively, the antibodies of the present invention can be administered via non-parenteral routes such as topical, epidermal or mucosal administration routes such as intranasal, oral, intravaginal, rectal, sublingual or topical.

[0113] The pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0114] The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the subject to be treated and the particular mode of administration, and will generally be the amount of the composition that produces a therapeutic effect. For such amount, an active ingredient in the range of about 0.01% to about 99%, preferably about 0.1% to about 70%, most preferably about 1% to about 30% on a percentage basis is combined with a pharmaceutically acceptable carrier.

[0115] The dosing regimen is adjusted to provide an optimal desired response (e.g., a therapeutic response). For example, a single bolus dose may be administered, or multiple divided doses may be administered over time, or the dose can be proportionally decreased or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to formulate a composition for parenteral administration in unit dosage form to facilitate administration and to make the dosage uniform. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for the subject to be treated, each unit containing a predetermined amount calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. Alternatively, the antibody may be administered as a sustained release formulation, in which case the required dosing frequency is reduced.

[0116] When administering the composition, the dosage can range from about 0.0001 to 100 mg per kg of the host's body weight, more usually from 0.01 to 5 mg. For example, the dosage can be in the range of 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or 1 - 10 mg / kg body weight. Exemplary treatment schedules involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferred dosing schedules for the anti-IL4Rα antibody of the present invention include intravenous administration at 1 mg / kg body weight or 3 mg / kg body weight, and the antibody administration is carried out according to one of the following dosing regimens: (i) six times every four weeks, then every three months; (ii) every three weeks; (iii) once at 3 mg / kg body weight, then at 1 mg / kg body weight every three weeks. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1 - 1000 μg / ml, and in some methods, a plasma antibody concentration of about 25 - 300 μg / ml is achieved.

[0117] A "therapeutically effective amount" of the anti-IL4Rα antibody or antigen-binding portion thereof, bispecific molecule, CAR-T cell, oncolytic virus or immune complex of the present invention can preferably cause a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic period of the disease, or prevention of damage or disorder caused by the pain of the disease. For example, in the treatment of a tumor subject, a "therapeutically effective amount" is an amount that preferably inhibits tumor growth by at least about 20%, preferably at least about 40%, more preferably at least about 60%, more preferably at least about 88% compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can typically reduce tumor size or otherwise improve symptoms in a subject who is human or can be another mammal.

[0118] The pharmaceutical composition can be a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0119] The pharmaceutical composition can be administered by medical devices such as, for example, (1) needleless subcutaneous injection devices (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Patent No. 4,487,603); (3) transdermal drug delivery devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patent Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0120] In certain embodiments, the monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the invention cross the blood-brain barrier, they can be formulated into liposomes and can further include targeting moieties to enhance selective transport to specific cells or organs. See, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0121] Uses and methods of the present invention Compositions comprising the antibodies or antigen-binding sites thereof, or bispecificities, CAR-T cells, oncolytic viruses, immune complexes of the invention have numerous in vitro and in vivo utilities, such as being involved in the treatment of allergic diseases associated with excessive IL4 and / or IL13 signaling.

[0122] Since the anti-IL4Rα antibody of the present invention has the ability to block the binding of IL4Rα to IL4 or IL13-IL13Rα1 and reduce type 2 immunity, the present invention provides a method for treating allergic diseases related to type 2 immunity, including administering the composition of the present invention to a subject. The allergic disease can be atopic dermatitis, allergic reaction, allergic rhinitis or allergic asthma.

[0123] On the other hand, since IL4 or IL13 signaling can activate STAT6 and STAT6 inhibitors have been found to inhibit cancer cell proliferation, the present invention provides a method for inhibiting tumor cell proliferation in a subject. The method includes administering the composition of the present invention to a subject, thereby inhibiting tumor growth in the subject. Non-limiting examples of tumors that can be treated with the antibody of the present invention include melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, and urothelial cancer, but are not limited thereto.

[0124] On the other hand, the present invention provides a method for reducing or inhibiting the activation of cells responsive to IL-4 or IL-13. In some embodiments, inhibiting activation includes inhibiting the production or secretion of cytokines. In some embodiments, inhibiting activation includes inhibiting proliferation. Cells responsive to IL-4 via stimulation of the hybrid IL-4Rα / γC receptor include, but are not limited to, B cells, eosinophils, and macrophages. Cells responsive to IL-13 via stimulation of the hybrid IL-4Rα / IL-3Rα1 receptor include, but are not limited to, fibroblasts and smooth muscle cells. Thus, in some embodiments, the present invention provides a method for inhibiting the proliferation of smooth muscle cells. In another embodiment, the present invention provides a method for inhibiting fibroblast proliferation.

[0125] In another aspect, the present invention provides methods, compositions, and kits for diagnosis. In some embodiments, the antibodies of the present invention are used to determine the presence and expression of IL4Rα in cells or tissues. In some embodiments, said diagnosis indicates prognosis and / or guides treatment and / or follow-up. For example, overexpression of IL4Rα in human bladder cancer has been found to correlate with the pathological grade and stage of the disease. In some embodiments, the antibodies of the present invention are used to diagnose the grade and stage of bladder cancer. High expression of IL-4Rα has been found to correlate with the occurrence and multiple recurrences of oral cancer. In some embodiments, the antibodies of the present invention are used in an oral cancer diagnostic kit or method for determining prognosis and appropriate treatment and follow-up. IL-4Ra expression in tumors is inversely correlated with the survival rate of patients undergoing surgical resection of epithelial malignant pleural mesothelioma (MPM). In some embodiments, the antibodies of the present invention are used in a diagnostic kit or method for determining the prognosis and appropriate treatment and / or follow-up of MPM.

[0126] Combination therapy In one aspect, the present invention provides a combination therapy comprising combining an anti-IL4Rα antibody or antigen-binding site, bispecific molecule, or oncolytic virus of the present invention with one or more other drugs effective to ameliorate type 2 immune-related allergic diseases. Said drugs can be antihistamines (targeting the H1 histamine receptor) clinically used for the treatment of allergic rhinitis, or corticosteroids, beta-adrenergic receptor agonists, and agents targeting cyc-LT clinically used in the treatment of asthma. Omalizumab, an anti-IgE antibody, can also be used in the treatment of allergic diseases by the antibodies or antigen-binding sites, bispecific molecules, or oncolytic viruses of the present invention. In certain embodiments, the subject is human.

[0127] In another aspect, the present invention provides a combination therapy by combining the anti-IL4Rα antibody or antigen-binding site thereof, bispecific molecule, CAR-T cell, oncolytic virus or immune complex of the present invention with one or more other agents effective to inhibit tumor growth in a subject. In some embodiments, the present invention provides a method of inhibiting tumor growth in a subject comprising administering to the subject an anti-IL4Rα antibody (or antigen-binding site thereof, bispecific molecule, oncolytic virus, CAR-T cell, or immune complex) and one or more other antibodies such as anti-OX40 antibody, anti-TIM-3 antibody, anti-CD137 antibody, anti-GITR antibody, anti-LAG-3 antibody, anti-PD-L1 antibody and anti-PD-1 antibody. In certain embodiments, the subject is human. The IL4Rα pathway inhibitor can be further combined with the standard treatment for cancer. For example, the IL4Rα pathway inhibitor can be combined with LAG-3 and / or PD-1 inhibitors and chemotherapy regimens. The anti-IL4Rα antibody can be administered together with a chemotherapeutic agent which can be a cytotoxic drug. For example, epirubicin, oxaliplatin, and 5-fluorouracil can be administered to patients receiving anti-IL4Rα therapy. Optionally, the combination of anti-IL4Rα and one or more other antibodies (e.g., anti-LAG-3 and / or anti-PD-1 antibodies) can be further combined with an immunogenic substance. The immunogenic substances are, for example, cancer cells, purified tumor antigens (including recombinant proteins, peptides, and sugar molecules), and cells transfected with genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides such as gp100, MAGE antigen, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. Other therapies that can be combined with the anti-IL4Rα antibody include, but are not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone blockade.

[0128] The combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier or as separate compositions of each agent in a pharmaceutically acceptable carrier. In another embodiment, the combinations of therapeutic agents can be administered sequentially.

[0129] Furthermore, when multiple combination therapies are administered sequentially, the order of sequential administration can be reversed or maintained the same at each administration time point, sequential administration can be combined with simultaneous administration, or any combination thereof can be performed.

[0130] The present invention is further illustrated by the following examples, which should not be construed as further limitations. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are hereby expressly incorporated herein by reference.

Examples

[0131] Examples Example 1: Preparation of mouse anti-IL4Rα monoclonal antibody using hybridoma technology Immunization Mice were immunized according to the method described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998. Recombinant human IL4Rα-his protein (Sino biological inc., catalog number 10402-H08H) was used as an immunogen, and a self-made human IL4Rα-his protein (amino acid sequence shown in SEQ ID NO: 57) was used for the determination of the titer of the antiserum and the screening of hybridomas secreting antigen-specific antibodies. The immunization doses for the primary immunization and booster immunization were 20 μg of human IL4Rα-his protein per injection per mouse. To enhance the immune response, complete Freund's adjuvant and incomplete Freund's adjuvant (Sigma, St. Louis, Mo., USA) were used for the first immunization and booster immunization, respectively. Briefly, the mixture of adjuvant and immunogen was prepared as follows. First, the adjuvant was gently mixed by vortexing in a vial. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL microcentrifuge tube. The antigen was prepared in PBS or physiological saline to a concentration in the range of 0.2 - 0.3 mg / ml. Next, the calculated amount of antigen was added to the adjuvant in the microcentrifuge tube, and the resulting mixture was gently vortexed for 2 minutes to mix and form an oil-in-water emulsion. Next, the adjuvant-antigen emulsion was aspirated into an appropriate syringe for animal injection. A total of 20 μg of antigen was injected in a volume of 150 - 200 μl. Each animal was immunized and boosted 2 - 3 times according to the antiserum titer. Animals with better titers were given a final booster injection by intraperitoneal injection before cell fusion.

[0132] Fusion and Screening of Hybridomas Immediately prior to fusion, cells of the mouse myeloma cell line (SP2 / 0-Ag14, ATCC#CRL-1581) were cultured until they reached the logarithmic phase. Spleen cells from immunized mice were aseptically prepared and fused with the myeloma cells according to the method described by Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256: 495-497(1975). Subsequently, the fused "hybrid cells" were dispensed into 96-well plates containing DMEM / 20% FCS / HAT medium. Colonies of surviving hybridomas were observed under a microscope 7 to 10 days after fusion. Two weeks later, the supernatant of each well was subjected to ELISA-based screening using recombinant human IL4Rα-his protein. Briefly, human IL4Rα-his protein (2.0 μg / mL) in PBS was coated onto ELISA plates at 60 μL / well and incubated overnight at 4°C. After washing the plates 4 times with PBST, they were blocked with 200 μl of blocking buffer (in PBST containing 5% w / v skim milk). Diluted hybridoma supernatant (60 μl) was added to each well and incubated at 37°C for 40 minutes. The plates were then washed 4 times, and HRP goat anti-mouse IgG (Jackson Immuno research, catalog number 115-036-071) was used for detection, and the bound OD value was observed at 450 nm. Next, positive hybridomas secreting antibodies capable of binding to human IL4Rα-his protein were selected and transferred to 24-well plates. Hybridoma clones producing antibodies showing high specific human IL4Rα binding and IL4RαIL4 or IL4Rα-13Rα1-IL13 blocking activity were subcloned by the limiting dilution method, and after ensuring the clonality of the cell line, monoclonal antibodies were purified. Briefly, a protein A Sepharose column (bestchrom(Shanghai) Biosciences, catalog number AA0273) was washed using PBS buffer at 5 to 10 times the column volume.The cell supernatant was passed through a column, and then the column was washed with PBS buffer until the absorbance of the protein reached the baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and the eluate was immediately collected in a 1.5 mL tube and neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0). Fractions containing immunoglobulins were pooled and dialyzed against PBS at 4°C overnight. Subsequently, the in vitro functional activity of the purified monoclonal antibody was characterized as follows.

[0133] Example 2: Affinity measurement of mouse anti-IL4Rα monoclonal antibody using BIACORE surface plasmon resonance The purified anti-IL4Rα mouse monoclonal antibody (mAb) generated in Example 1 was characterized for binding affinity and binding kinetics by a Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

[0134] Briefly, goat anti-mouse IgG (GE healthcare, Cat. No. BR100838, Mouse Antibody Capture Kit) was covalently coupled to a CM5 chip (carboxymethylated dextran coated chip) via primary amine groups using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA). Unreacted sites on the biosensor surface were blocked with ethanolamine. Then, purified anti-IL4Rα antibody of the present invention at a concentration of 66.67 nM and anti-IL4Rα benchmark (also called Dupilumab®, BM) at 10 μg / ml were flowed over the chip at a flow rate of 10 μL / min. Then, serial dilutions of recombinant human IL4Rα-his (produced in-house, amino acid sequence shown in SEQ ID NO: 57), cynomolgus monkey IL4Rα-his protein (Sino biological inc., catalog number 90897-C08H), or marmoset IL4Rα-his protein (outsourced from Sinobiological Inc., also called cal-IL4Rα-his, amino acid sequence shown in SEQ ID NO: 58) in HBS EP buffer (provided by Biacore) were flowed over the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were followed for 2 min, and dissociation kinetics were followed for 10 min. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using the BIA evaluation software. K D , K a and K d The values were determined and are summarized in Table 2 below.

[0135] [Table 2]

[0136] All murine antibodies of the invention specifically bound to human IL4Rα, and most of them showed comparable or higher binding affinity compared to the benchmark.

[0137] Example 3: IL4Rα binding activity of mouse anti-IL4Rα antibody The binding activity of the mouse anti-IL4Rα antibody of the present invention to IL4Rα was determined by Capture ELISA, flow cytometry (FACS), and indirect ELISA.

[0138] 3.1 Capture ELISA Briefly, 2 μg / ml goat anti-mouse IgG Fcγ fragment specific (Jackson Immuno Research, catalog number 115-005-008) in PBS was coated on a 96-well plate at 100 μl / well and incubated overnight at 4°C. After washing the plate once with wash buffer (PBS + 0.05% w / v Tween-20, PBST), 200 μl / well of blocking buffer (PBST containing 5% w / v skim milk) was added and blocked at 37°C for 2 hours. The plate was washed again, and incubated with 100 μl / well of serially diluted (5-fold dilution with PBST containing 2.5% w / v non-fat milk, starting from 66.7 nM) anti-IL4Rα antibody of the present invention, benchmark or negative control hIgG (for intravenous injection of human immunoglobulin (pH4), Hualan Biological Engineering Inc.) at 37°C for 40 minutes, and the plate was washed 4 times again. 100 μL / well of biotin-labeled human IL4Rα-his protein (self-made with SEQ ID NO: 57, in PBST containing 2.5% w / v non-fat milk, 0.14 nM) was added to the 96-well plate containing the captured anti-IL4Rα antibody. Incubated at 37°C for 40 minutes, the plate was washed 4 times, and HRP-labeled streptavidin (diluted 1:10000 with PBST, Jackson Immuno Research, catalog number 016-030-084) was added at 100 μL / well and incubated at 37°C for 40 minutes. After the last wash, 100 μL / well of ELISA substrate TMB (Innoreagents, catalog number MB-S-002) was added and incubated. After 10 minutes, 50 μL / well of 1M H2SO4 was added at 25°C to stop the reaction, and the absorbance was read at 450 nm. The data was analyzed using Graphpad Prism software to obtain the EC 50 value.

[0139] 3.2 Cell-based binding FACS The binding activity of mouse anti-IL4Rα antibody to IL4Rα expressed on the surface of 293F-IL4Rα cells was tested by flow cytometry (FACS). 293F cells (Thermofisher Inc., catalog number 11625019) were transfected with a pCMV-T-P plasmid construct having nucleotides encoding human IL4Rα (amino acid residues 1-825 of uniprot #P24394-1) between EcoRI and XbaI, and a stable cell pool (named 293F-IL4Rα) was selected for subsequent cell-based binding FACS and cell-based ligand blockade FACS analysis. 293F-IL4Rα cells were recovered from cell culture flasks, washed twice, and resuspended in FACS buffer (phosphate buffered saline (PBS) containing 2% v / v fetal bovine serum). Next, anti-IL4Rα antibody or control substance serially diluted (starting from 80 nM, 4-fold serial dilution) with FACS buffer was added at 100 μL / well to a 96-well plate containing 2×10 5 cells / well, and incubated in an ice bath for 40 minutes. After washing the cells twice with FACS buffer, 100 μL / well of R-phycoerythrin-labeled affinity-purified F(ab')2 fragment goat anti-mouse IgG (H+L) (diluted 1:1000 with FACS buffer, Jackson Immunoresearch, catalog number 115-116-146.) was added. After incubation in the dark at 4 °C for 40 minutes, the cells were washed three times and resuspended in FACS buffer. Fluorescence values were measured using a Becton Dickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software to obtain EC 50 values.

[0140] 3.3 Indirect ELISA The cross-reactivity of the anti-IL4Rα antibody with cynomolgus IL4Rα protein or cal-IL4Rα-his protein was measured. Briefly, 2 μg / mL of cynomolgus IL4Rα-his protein (Sino biological inc., catalog number 90897-C08H) in carbonate / bicarbonate buffer (pH 9.6) or 0.2 μg / mL of cal-IL4Rα-his protein (outsourced from Sinobiological inc., product catalog BAX2) in carbonate / bicarbonate buffer (pH 9.6) was coated onto a 96-well plate at 100 μL / well and incubated at 37 °C for 2 hours. The plate was washed once with wash buffer (PBS + 0.05% w / v Tween-20, PBST), and 200 μL / well of blocking buffer (PBST containing 5% w / v skim milk) was added and blocked at 37 °C for 2 hours. The plate was washed again, and the anti-IL4Rα antibody of the present invention or a control substance serially diluted (0.004 - 66.7 nM, starting from 66.7 nM, diluted 5-fold with PBST containing 2.5% w / v skim milk) was added to each well at 100 μL and incubated at 37 °C for 40 minutes. After the plate was washed 4 times, 100 μL / well of peroxidase-labeled affinity-purified goat anti-mouse IgG (Fcγ fragment specific) (diluted 1:5000 with PBST buffer, Jackson Immunoresearch, catalog number: 115-036-071) was incubated at 37 °C for 40 minutes. After the final wash, 100 μL / well of TMB (Innoreagents) was added and incubated. After 3 - 10 minutes, the reaction was stopped by adding 50 μL / well of 1 M H2SO4 at 25 °C, and the absorbance was read at 450 nm. The data was analyzed using Graphpad Prism software to obtain the EC 50 value.

[0141] The results of three measurements are shown in Table 3 and Figures 1A - 1C, 2A - 2D, and 3.

[0142] From the results, it was found that the mouse anti-IL4Rα antibodies of the present invention specifically bind to human IL4Rα with high binding ability, and some of them bind to the monkey IL4Rα protein with a binding activity higher than that of the benchmark.

[0143]

Table 3

[0144] Example 4: Blocking activity of mouse anti-IL4Rα antibody against IL4Rα-benchmark or IL4Rα-IL4 interaction 4.1 Ligand-blocking ELISA The ability of the anti-IL4Rα antibodies of the present invention to block the IL4-IL4Rα interaction was determined by competitive ELISA. Briefly, a 96-well plate was coated with 100 μL / well of human IL4Rα-his protein (SEQ ID NO: 57, self-prepared) at a concentration of 2 μg / mL in PBS and incubated overnight at 4°C. The next day, the plate was washed with wash buffer (PBS + 0.05% w / v Tween-20, PBST) and blocked with 5% w / v skim milk in PBST at 37°C for 2 hours. Then, the plate was washed again using the wash buffer.

[0145] Anti-IL4Rα antibody or a control substance was serially diluted (starting from 80 nM, 4-fold serial dilution) with PBST buffer containing 2.5% w / v skim milk, and the serially diluted anti-IL4Rα antibody or control substance was added to the IL4Rα-coated plate at 100 μL / well and incubated with human IL4Rα-his protein at 37 °C for 40 minutes. The plate was washed 4 times with wash buffer, and then 100 μL of biotinylated human IL4 protein (Sinobiological Inc., catalog number 11846-HNAE) at a concentration of 0.56 nM was added to each well and incubated at 37 °C for 40 minutes. The plate was washed again with wash buffer. Next, 100 μL / well of HRP-labeled streptavidin (diluted 1:10,000 in PBST buffer, Jackson Immunoresearch, catalog 016-030-084) was added and incubated at 37 °C for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added, the reaction was stopped with 1 M H2SO4, and the absorbance was read at 450 nm. The data was analyzed using Graphpad Prism software, and the IC 50 value was obtained.

[0146] 4.2 Benchmark Blocking ELISA The ability of the anti-IL4Rα antibody of the present invention to block benchmark-human IL4Rα binding was measured by a competitive ELISA assay. Briefly, a 96-well microplate was coated with 100 μl / well of benchmark at a concentration of 2 μg / mL in PBS and incubated overnight at 4°C. The next day, the plate was washed with wash buffer (PBS + 0.05% w / v Tween-20, PBST) and blocked with 5% w / v non-fat milk in PBST at 37°C for 2 hours. During plate blocking, the anti-IL4Rα antibody of the present invention or a control was serially diluted 4-fold starting from 100 nM with biotinylated human IL4Rα-his protein (SEQ ID NO: 57, in-house preparation, prepared at 0.55 nM with 2.5% w / v non-fat milk in PBST) and incubated at 25°C for 40 minutes. After washing the plate, 100 μl of the antibody / IL4Rα--his mixture was added per well to the benchmark-coated plate. After incubation at 37°C for 40 minutes, the plate was washed using the wash buffer. Then, 100 μl / well of HRP-labeled streptavidin was added to the plate and incubated at 37°C for 40 minutes to detect biotinylated human IL4Rα-his bound to the plate. The plate was washed again with the wash buffer. Finally, TMB was added and the reaction was stopped with 1 M H2SO4, and the absorbance was read at 450 nm. The data was analyzed using Graphpad Prism software to obtain the IC 50 value.

[0147] 4.3 Cell-based Ligand Blocking FACS Using the 293F-IL4Rα cells prepared above, the activity of the anti-IL4Rα antibody that blocks the binding of IL4 protein to cell surface IL4Rα was evaluated by flow cytometry (FACS).

[0148] Briefly, the 293F-IL4Rα cells were recovered from the cell culture flask, washed twice, and resuspended in FACS buffer (PBS containing 2% v / v fetal bovine serum). Next, the anti-IL4Rα antibody or control substance serially diluted (starting from 80 nM, 4-fold serial dilution) with FACS buffer was added at 100 μL / well to 1×105 It was added to a 96-well plate containing cells / wells and ice-bathed for 40 minutes. The plate was washed twice with FACS buffer, 100 μl / well of 1.67 nM biotin-labeled human IL4 protein (Sino biological inc., catalog number 11846-HNAE) was added, and it was incubated at 4 °C in the dark for 40 minutes. After the plate was washed twice with FACS buffer, 100 μl / well of R-phycoerythrin streptavidin (diluted 1:500 with FACS buffer, Jackson Immunoresearch, catalog number 016-110-084) was added, and it was incubated at 4 °C in the dark for 40 minutes. The cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS device. The data was analyzed using Graphpad Prism software, and IC 50 values were obtained.

[0149] The results of the three measurements are shown in Table 4 and Figures 4A - 4B, 5A - 5B, and 6A - 6C below.

[0150] From Table 4 and Figures 4A - 4B, it was found that all anti-IL4Rα antibodies of the present invention were able to block the human IL4-human IL4Rα interaction with a blocking activity comparable to the benchmark.

[0151] Figures 5A and 5B show that some of the antibodies of the present invention were able to block human IL4Rα-benchmark binding, suggesting that they may bind to the same or similar epitopes as the benchmark.

[0152] Furthermore, as shown in Table 4 and Figures 6A - 6C, all anti-IL4Rα antibodies were able to block the binding of IL4 to cell surface IL4Rα, and their blocking ability was very close to that of the reference product (however, the IC 50 values were slightly higher than those of the benchmark).

[0153]

Table 4

[0154] Example 5: Cell-based functional measurement of mouse anti-IL4Rα antibody IL4 and IL13 can bind to IL4Rα and induce phosphorylation of STAT6 in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. The phosphorylation step is important in the IL4 / IL13 signaling pathway.

[0155] Briefly, using the pcDNA3.1-Puro (YouBio biological inc., catalog number VT9222) plasmid construct (containing nucleotides encoding human IL4Rα between BamHI and XhoI), the STAT6 plasmid (Sino biological inc., catalog number HG13190-NH) (containing nucleotides encoding human STAT6 between KpnI and XbaI), and the STAT6 luciferase reporter gene plasmid STAT6-Luc (Yeasen biological inc., catalog number 11588ES03), HEK293T cells (ATCC CRL-11268) that natively express IL13Rα1 were stably transfected to prepare HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells in-house. Next, the single cell clone LB2 was selected for all subsequent functional assays.

[0156] The anti-IL4Rα antibody of the present invention was tested for its inhibitory effect on IL4- and IL13-induced STAT6 phosphorylation.

[0157] Briefly, HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells in the logarithmic phase were resuspended in medium (RPMI1640 + 10% FBS), seeded in 96-well plates at 100 μL / well, and each well contained 5×10 5Cells were included. Next, 50 μL of serially diluted anti-IL4Rα antibody or control (including self-prepared anti-CD22 antibody) (starting from 100 nM, 5-fold serial dilution) was added to each well and incubated at 37 °C for 30 minutes. Next, 50 μL of IL4 protein (600 pg / mL, Sino biological inc., catalog number 11846-HNAE) or IL13 protein (50 ng / mL, Sino biological inc., catalog number 10369-HNAC) was added to each well and incubated at 37 °C for 20 minutes. The plate was centrifuged and washed twice with staining buffer (prepared in-house, DPBS + 0.5% w / v BSA + 2 mM EDTA), then 50 μL of fixation buffer (BD biosciences inc., catalog 5545655) was added to each well and incubated at 4 °C for 30 minutes. The cells were washed twice, 200 μL of permeabilization buffer (BD biosciences inc., catalog number 558050) was added to each well, and incubated in an ice bath for 30 minutes. The plate was washed three times with staining buffer. Then, anti-pSTAT6 antibody (20-fold dilution of pSTAT6 stock solution, BD biosciences inc., catalog number 562079) was added and left standing on ice for 60 minutes. Finally, the plate was washed twice and resuspended in staining buffer. Fluorescence values were measured using a Becton Dickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software and IC 50 values were obtained.

[0158] The results are shown in Table 5 and Figures 7 and 8 below.

[0159] The results indicate that all anti-IL4Rα antibodies can block IL4- or IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells with a blocking activity equal to or greater than that of the reference substance.

[0160]

Table 5

[0161] Example 6: Generation and characterization of chimeric antibodies The variable regions of the heavy and light chains of the anti-IL4Rα mouse monoclonal antibody were sequenced, and the sequence IDs were summarized in Table 1.

[0162] The variable domains of the heavy and light chains of the anti-IL4Rα mouse mAb monoclonal antibodies C2C1A1A1 and B8G11F2B7G5E8 were cloned into a vector containing the human IgG4 heavy chain constant region (SEQ ID NO: 55) and a vector containing the human κ light chain constant region (SEQ ID NO: 56), respectively, where the C-terminus of the variable region is ligated to the N-terminus of the corresponding constant region.

[0163] A vector containing nucleotides encoding the heavy chain variable region linked to the human IgG4 heavy chain constant region and a vector containing nucleotides encoding the light chain variable region linked to the human κ light chain constant region were transiently transfected into 50 mL of 293F suspension cells at 1 mg / mL PEI at a ratio of light chain construct:heavy chain construct of 60%:40%.

[0164] After culturing in a shaking flask for 6 days, the cell supernatant was collected, spun down to pellet the cells, and filtered through a 0.22 μm filter for immunoglobulin separation. The chimeric antibody was purified by protein A affinity chromatography. Briefly, a protein A Sepharose column (from bestchrom (Shanghai) Biosciences, catalog number AA0273) was washed with PBS buffer at 5 to 10 column volumes. The cell supernatant was passed through the protein A Sepharose column, and the column was washed with PBS buffer until the absorbance of the protein reached the baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collected into a 1.5 ml tube containing neutralization buffer (1 M Tris-HCl, pH 9.0) and neutralized. The fractions containing immunoglobulin were pooled and dialyzed against PBS at 4°C overnight.

[0165] The purified antibodies were tested in Capture ELISA, competitive ELISA, BIAcore affinity assays, cell-based binding FACS, and cell-based functional measurements according to the protocols of the above examples (with minor modifications described below).

[0166] In Capture ELISA, instead of Goat anti-Mouse IgG (Fcγ fragment specific), 2 μg / ml of Goat anti-Human IgG (Affinity purified Goat anti-Human IgG, Fcγ fragment specific, Jackson Immunoresearch, catalog number 109-005-098) was used at 100 μL / well.

[0167] In indirect ELISA, instead of peroxidase-labeled affinity purified Goat anti-Mouse IgG (Fcγ fragment specific), peroxidase-labeled affinity purified F(ab’)2 fragmented Goat anti-Human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-036-098) was used at 100 μL / well.

[0168] In BIAcore, instead of Goat anti-Mouse IgG, Goat anti-Human IgG (GE healthcare, catalog number BR100839, Human Antibody Capture Kit) was used and covalently bound to the CM5 chip.

[0169] In cell-based binding FACS, instead of R-phycoerythrin-labeled affinity purified F(ab')2 fragmented Goat anti-Mouse IgG (H+L), R-phycoerythrin-labeled affinity purified Goat anti-Human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog number 109-115-098) was diluted 1:1000 in FACS buffer and used at 100 μL / well.

[0170] The results are shown in Table 6 and Figures 9 - 13. The data show that the chimeric antibodies have similar binding affinity / ability and blocking activity as their parental mouse antibodies.

[0171]

Table 6

[0172] Example 7: Humanization of anti-IL4Rα monoclonal antibodies B8G11F2B7G5E8 and C2C1A1A1 The mouse anti-IL4Rα antibodies B8G11F2B7G5E8 and C2C1A1A1 were humanized and further characterized. The humanization of the mouse antibodies was performed as described below using the established CDR grafting method.

[0173] To select acceptor frameworks for the humanization of mouse antibodies B8G11F2B7G5E8 and C2C1A1A1, the light and heavy chain variable region sequences of each mouse antibody were blasted against the human immunoglobulin gene database. The human germline with the highest homology was selected as the acceptor framework for humanization. The CDRs of the heavy / light chain variable regions of the mouse antibodies were inserted into the selected frameworks, and the framework residues were further subjected to back mutations to obtain more candidate heavy / light chain variable regions. A total of 13 exemplary humanized B8G11F2B7G5E8 antibodies, namely, from huB8G11F2B7G5E8-V1 to huB8G11F2B7G5E8-V11, huB8G11F2B7G5E8-V13 and huB8G11F2B7G5E8-V14, and 16 exemplary humanized C2C1A1A1 antibodies, namely, from huC2C1A1A1-V1 to huC2C1A1A1-V16, were obtained, and the sequence IDs of their heavy / light chain variable regions are shown in Table 1.

[0174] Vectors containing nucleotides encoding humanized heavy chain variable regions linked to the human IgG4 heavy chain constant region (SEQ ID NO: 55) and vectors containing nucleotides encoding humanized light chain variable regions linked to the human κ light chain constant region (SEQ ID NO: 56) were transiently transfected into 50 mL of 293F suspension cells at 1 mg / mL PEI at a ratio of light chain construct:heavy chain construct of 60%:40%.

[0175] After culturing for 6 days in a shaking flask, the cell supernatant was collected. The cells in the supernatant were pelleted by centrifugation and filtered through a 0.22 μm filter to separate immunoglobulins. The antibody was purified by protein A affinity chromatography. Briefly, a protein A Sepharose column (from bestchrom(Shanghai) Biosciences, catalog number AA0273) was washed with PBS buffer at 5 to 10 column volumes. The cell supernatant was passed through the protein A Sepharose column, and the column was washed with PBS buffer until the absorbance of the protein reached the baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collected in a 1.5 ml tube containing neutralization buffer (1 M Tris-HCl, pH 9.0) for neutralization. The fractions containing immunoglobulins were pooled and dialyzed against PBS at 4°C overnight.

[0176] Example 8: Characterization of humanized antibodies

[0177]

Table 7

[0178] The binding affinity of the humanized antibody against human IL4Rα was evaluated by BIAcore technology according to the protocol of the previous example. The measured K a , K d and K D values were measured and summarized in Table 7 and Table 8.

[0179]

Table 8

[0180] The results showed that the humanized antibody had a binding affinity similar to that of the chimeric antibody against human IL4Rα, and all humanized huC2C1A1A1 antibodies showed higher binding affinity against human IL4Rα compared to the reference.

[0181] The humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, huB8G11F2B7G5E8-V14, huC2C1A1A1-V14 and huC2C1A1A1-V15 were tested in Biacore, capture ELISA, indirect ELISA, cell-based binding FACS, competitive ELISA and cell-based functional assays according to the protocols of the above examples (with minor modifications described below).

[0182] In capture ELISA, instead of goat anti-mouse IgG (Fcγ fragment specific), 2 μg / mL goat anti-human IgG (affinity purified goat anti-human IgG, Fcγ fragment specific, Jackson Immunoresearch, catalog number 109-005-098) was used at 100 μL / well.

[0183] In indirect ELISA, instead of peroxidase-labeled affinity purified goat anti-mouse IgG (Fcγ fragment specific), peroxidase-labeled affinity purified F(ab’)2 fragmented goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-036-098) was used at 100 μl / well.

[0184] In Biacore, instead of goat anti-mouse IgG, goat anti-human IgG (GE healthcare, catalog number BR100838, Human Antibody Capture Kit) was used and covalently coupled to the CM5 chip.

[0185] In cell-based binding FACS, instead of R-phycoerythrin-labeled affinity purified F(ab')2 fragmented goat anti-mouse IgG (H+L), R-phycoerythrin-labeled affinity purified goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog number 109-115-098) was diluted 1:1000 with FACS buffer and used at 100 μL / well.

[0186] The thermal stabilities of the humanized antibodies huB8G11F2B7G5E8-V14 and huC2C1A1A1-V15 were also tested. Briefly, GloMelt TM Using the Thermal Shift Protein Stability Kit (Biotium, catalog number 33022-T, catalog number 181214), the melting temperature (Tm) was measured by a protein thermal shift assay. Briefly, GloMelt TM The dye was thawed at room temperature. The vial containing the dye was vortexed and centrifuged. Next, 5 μL of 200× dye was added to 95 μL of PBS to prepare 10× dye. 2 μL of 10× dye and 10 μg of the humanized antibody were added to the reaction system and made up to 20 μL with PBS. The microcentrifuge tube containing the dye and the antibody was briefly centrifuged and placed in a real-time PCR thermal cycler (Roche, LightCycler 480 II) with the melting curve program set using the parameters in Table 9.

[0187] [Table 9]

[0188] The results are shown in Tables 10-1 to 10-3 and Figures 14A to 14B to 22.

[0189] [Table 10-1]

[0190] [Table 10-2]

[0191] [Table 10-3]

[0192] The data shows that the humanized C2C1A1A1 antibody exhibits equivalent (if not better) binding affinity / activity for human IL4Rα and blocking ability for IL4Rα-IL4 / IL13, while the humanized B8G11F2B7G5E8 antibody showed significantly superior blocking ability for the IL4 / IL13-IL13Rα1-IL4Rα interaction.

[0193] Although the present invention has been described above in connection with one or more embodiments, it should be understood that the invention is not limited to those embodiments, but is intended to cover all alternatives, modifications, and equivalents included within the spirit and scope of the appended claims. All documents cited herein are incorporated by reference in their entirety.

[0194] The sequence information for this application is summarized in the following table.

[0195]

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Table 11-5

Table 11-6

Table 11-7

Table 11-8

Table 11-9

Table 11-10

Table 11-11

Table 11-12

Table 11-13

Table 11-14

Table 11-15

Table 11-16

Claims

**Claim 1** An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the interleukin-4 receptor alpha subunit (IL4Rα), wherein the heavy-chain variable region CDR1, CDR2, and CDR3 and the light-chain variable region CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs: 1, 5, 10, 15, 22, and 26, the isolated monoclonal antibody or antigen-binding fragment thereof. **Claim 2** The heavy-chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO: 32, 33 (X1 = W, X2 = S; X1 = L, X2 = A; X1 = W, X2 = A), or 34, the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1. **Claim 3** The light-chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO: 35, 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), or 37, the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1. **Claim 4** The heavy-chain variable region and the light-chain variable region are: (1) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 32 and 35, respectively; (2) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = S) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (3) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = S) and 37, respectively; (4) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 34 and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (5) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 34 and 37, respectively; (6) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 33 (X1 = L, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (7) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 33 (X1 = L, X2 = A) and 37, respectively; (8) amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively;Or (9) an isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences shown in SEQ ID NO: 33 (X1 = W, X2 = A) and 37, respectively.; **Claim 5**: The heavy chain variable region and the light chain variable region are: (1) the amino acid sequences shown in SEQ ID NOs: 32 and 35, respectively; (2) the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = S) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (3) the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = S) and 37, respectively; (4) the amino acid sequences shown in SEQ ID NOs: 34 and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (5) the amino acid sequences shown in SEQ ID NOs: 34 and 37, respectively; (6) the amino acid sequences shown in SEQ ID NOs: 33 (X1 = L, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; (7) the amino acid sequences shown in SEQ ID NOs: 33 (X1 = L, X2 = A) and 37, respectively; (8) the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively; or (9) the amino acid sequences shown in SEQ ID NOs: 33 (X1 = W, X2 = A) and 37, respectively, and the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1. **Claim 6**: The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region linked to the heavy chain variable region and a light chain constant region linked to the light chain variable region, wherein the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 55, and the light chain constant region has the amino acid sequence shown in SEQ ID NO:

56. **Claim 7** The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 5, comprising a heavy chain constant region linked to the heavy chain variable region and a light chain constant region linked to the light chain variable region, wherein the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 55, and the light chain constant region has the amino acid sequence shown in SEQ ID NO:

56. **Claim 8** The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof: (a) binds to human IL4Rα; (b) binds to simian IL4Rα; (c) blocks the IL4Rα-IL4 interaction; and (d) blocks the IL4Rα-IL13-IL13Rα1 interaction. **Claim 9** The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is a mouse antibody, a chimeric antibody or a humanized antibody.

10. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is of the IgG1, IgG2 or IgG4 isotype.

11. The antigen-binding fragment of the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment comprises a scFv, a Fab fragment, or an F(ab’)2 fragment.

12. A nucleotide encoding the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

13. An expression vector comprising the nucleotide according to claim 12.

14. A host cell comprising the nucleotide according to claim 12 or the expression vector according to claim 13.

15. A bispecific molecule comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

16. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleotide according to claim 12, the expression vector according to claim 13 or the host cell according to claim 14, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, further comprising an anti-allergic agent or an anti-tumor agent.

18. The pharmaceutical composition according to claim 17, wherein the anti-allergic agent is an antihistamine, a corticosteroid, a β-adrenergic receptor agonist, a drug targeting cyc-LTs or a drug targeting IgE.

19. The pharmaceutical composition according to claim 16 for treating an allergic disease.

20. The pharmaceutical composition according to claim 19, wherein the allergic disease is atopic dermatitis, allergic rhinitis or allergic asthma.

21. The pharmaceutical composition according to claim 19 or 20, wherein the treatment comprises administering an anti-allergic agent.

22.

22. The pharmaceutical composition according to claim 21, wherein the anti-allergic agent is an antihistamine, a corticosteroid, a β-adrenergic receptor agonist, a drug targeting cyc-LTs or a drug targeting IgE.

23. The pharmaceutical composition according to claim 16 for treating a tumor associated with an increase in STAT6 activation in a subject.

24. The pharmaceutical composition according to claim 23, wherein the tumor is a solid tumor.

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