Antibodies binding to il4r and uses thereof

Abstract

To provide an isolated monoclonal antibody that specifically binds to human IL4Rα, or an antigen-binding portion thereof.SOLUTION: The present invention provides an isolated monoclonal antibody that binds to IL4Rα or an antigen-binding portion thereof. the monoclonal antibody or the antigen-binding portion thereof comprises a heavy chain variable region. The heavy chain variable region having an amino acid sequence having a specific sequence. The present invention also provides nucleic acid molecules encoding the antibody or the antigen-binding portion thereof, expression vectors, host cells, and methods for expressing the antibody or the antigen-binding portion thereof. The present invention also provides bispecific molecules, oncolytic viruses, and pharmaceutical compositions comprising the antibody or the antigen-binding portion thereof, as well as therapeutic methods using an anti-IL4Rα antibody or an antigen-binding portion thereof of the present invention.SELECTED DRAWING: Figure 7

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 982,521, filed February 27, 2020.

[0002] All documents cited or reviewed in the aforementioned applications and in this application ("Documents Cited in the Application"), all documents cited or referenced in this application (including, but not limited to, all documents, patents, and published patent applications cited in this application) ("Documents Cited in the Application"), and all documents cited or referenced in the documents cited in this application, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products referred to herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of this invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference. All Genbank sequences referred to in this application are incorporated by reference as being the sequences of the earliest effective filing date of the present invention.

[0003] Technical area The present invention relates to isolated monoclonal antibodies, particularly murine, chimeric, or humanized monoclonal antibodies, or antigen-binding portions thereof, that bind to human IL4R, particularly IL4Rα, with high affinity and functionality. The present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the invention, expression vectors, host cells, and methods for expressing the antibodies or antigen-binding portions thereof of the invention. The present invention further provides bispecific molecules, immunoconjugates, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof of the invention, as well as therapeutic methods using the anti-IL4Rα antibodies or antigen-binding portions thereof of the invention.

[0004] Background technology Allergic diseases associated with type 2 inflammation, such as atopic dermatitis, hypersensitivity reactions, 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 a decrease in exposure to infectious diseases as living standards improve, making the immune system better able to process certain harmless allergens (Stephen J. Galli et al., (2008) Nature 454(72) 03):445-454). Two factors central to type 2 immunity are interleukin-4 ( These are IL-4, IL-13, and IL-16. They are required to promote most of the key features associated with type 2 inflammation, such as immunoglobulin E production and innate cell recruitment to the inflammatory site (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., (199 8)Science 282:2258-2261).

[0005] IL-4 and IL-13 are adjacent to each other on human chromosome 5 and may share regulatory elements. H 2) In the present study, both coordinated and uncoordinated expression of these two cytokines was observed (Katherine Bao et al., (2015) Cytokine 75(1):25-37). IL-4 binds to receptors to regulate cell function and activate transcriptional machinery. It binds to the IL-4Rα chain with picomolar affinity and recruits the IL-2Rγ γc chain to the type I receptor complex or recruit IL-13Rα1 to form the type II receptor complex. The levels or availability of IL-2Rγγc and IL-13Rα1 regulate the receptor complex. Non-hematopoietic cells express no or low levels of IL-2Rγ γc, but highly express IL-13Rα1, whereas the opposite is true for lymphocytes. Myeloid cells lie between these two types of cells. Formation is initiated by IL-13 binding to the IL-13Rα1 chain (nanomolar affinity binding), which can also recruit the IL-4Rα chain. In addition to the type II IL-4 receptor, IL-13 can bind to the IL-13Rα2 with picomolar affinity, which is considered a decoy receptor (Irina G. Luzina et al., (2012) J Leukoc Biol 92(4):753-764). Upon assembly of the IL-4 receptor complex, intracellular signaling molecules are activated, among which STAT6 and IRS signaling respond to the activation of type I IL-4 receptor, whereas type II IL-4 receptor cannot significantly activate IRS (Heller NM et al., (2008) Sci Signal 1(51): ra17-ra17). STAT6 signaling is T H IRS molecules are important for differentiation of IL-2 cells and IL-4 production, and 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 therefore several therapeutic antibodies have been developed to modify IL-4 and IL-13-mediated signaling. These include Leprikizumab, Anrukinzumab and Tralokinumab, which combine IL-4 and Pascolizumab, which targets IL-4. Itrakinra is an IL-4Rα antagonist, and when it binds to IL-4Rα, it blocks both type I and type II IL-4 receptors (Antoniu SA (20 10) Curr Opin Investig Drugs 11:1286-1294). STAT6 inhibitors have been shown to inhibit the proliferation of prostate cancer cells, and IL-4 This suggests that targeting IL-13 may be useful in cancer treatment (N Appo 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 antibodies that have binding affinity / capacity to human and / or monkey IL4Rα and IL4Rα-IL4Rα binding affinity / capacity equivalent to or greater than that of conventional anti-IL4Rα antibodies, such as Dupilumab. Equivalent or greater effects on IL13 / IL13-IL13Rα1 interaction and corresponding intracellular signaling The present invention provides an isolated monoclonal antibody, e.g., a murine, human, chimeric, or humanized monoclonal antibody, or an antigen-binding portion thereof, that binds to IL4Rα (e.g., human IL4Rα) and has the above blocking activity.

[0008] The antibodies or antigen-binding portions thereof of the present invention can be used in a variety of 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] Thus, in one aspect, the present invention provides an isolated monoclonal antibody (e.g., a murine antibody, a chimeric antibody, or a humanized antibody) or an antigen-binding portion thereof. The monoclonal antibody or antigen-binding portion thereof binds to IL4Rα and comprises a heavy chain variable region. The heavy chain variable region comprises a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, the CDR2 region, and the CDR3 region (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 1, 5, and 10, respectively. (2) 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 to the amino acid sequences set forth in SEQ ID NOs: 1, 6 and 11, respectively; (3) 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 to the amino acid sequences set forth in SEQ ID NOs: 2, 7 and 12, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 8 and 13, respectively. (5) 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 to the amino acid sequence set forth in SEQ ID NOs: 4, 8 and 13, respectively; or (6) 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 to the amino acid sequence set forth in SEQ ID NOs: 3, 9 and 14, respectively.

[0010] In one aspect, an isolated monoclonal antibody, or antigen-binding portion thereof, of the invention comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 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=H), and 43 (X1=R, X2=A, X3=S, X4=H). 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, and amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 32, 43, 44, 47, 49, 51, or 53. The antibody or antigen-binding portion thereof binds to IL4Rα. The amino acid sequence set forth in SEQ ID NO: 32 can be encoded by the nucleotide sequence set forth in SEQ ID NO: 59 or 60. The amino acid sequence set forth in SEQ ID NO: 40 can be encoded by the nucleotide sequence set forth 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] In one aspect, an isolated monoclonal antibody or antigen-binding portion thereof of the present invention binds to IL4Rα. The monoclonal antibody or antigen-binding portion thereof comprises a light chain variable region. The light chain variable region comprises CDR1, CDR2, and CDR3 regions. wherein the CDR1 region, the CDR2 region, and the CDR3 region are (1) 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 to the amino acid sequences shown in SEQ ID NOs: 15, 22, and 26, respectively; or (2) 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 to the amino acid sequences shown in SEQ ID NOs: 16, 22, and 27, respectively. (3) 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 to the amino acid sequences set forth in SEQ ID NOs: 17, 23 and 28, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 18, 24 and 29, respectively. (5) 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 to the amino acid sequences set forth in SEQ ID NOs: 19, 24 and 30, respectively; (6) 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 to the amino acid sequences set forth in SEQ ID NOs: 20, 25 and 31, respectively. or (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 to the amino acid sequences set forth in SEQ ID NOs: 21, 25 and 31, respectively.

[0012] In one aspect, the isolated monoclonal antibody or antigen-binding portion 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 to the amino acid sequence set forth 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. The antibody or antigen-binding portion thereof binds to IL4Rα. The amino acid sequence set forth in SEQ ID NO: 35 can be encoded by the nucleotide sequence set forth in SEQ ID NO: 62 or 63. The amino acid sequence set forth in SEQ ID NO: 45 can be encoded by the nucleotide sequence set forth in SEQ ID NO: 68 or 69. The amino acid sequences shown in SEQ ID NOs: 36 (X1=F, X2=V) and 46 can be encoded by the nucleotide sequences shown in SEQ ID NOs: 64 or 70, respectively.

[0013] In one aspect, an isolated monoclonal antibody, or antigen-binding portion thereof, of the invention comprises a heavy chain variable region and a light chain variable region, each of which comprises a CDR1 region, a CDR2 region, and a CDR3 region.wherein 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 to the amino acid sequences shown in SEQ ID NOs: 1, 5, 10, 15, 22, and 26, respectively; or (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 to the amino acid sequences shown in SEQ ID NOs: 1, 6, 11, 16, 22, and 27, respectively. (3) an amino acid sequence having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 2, 7, 12, 17, 23 and 28, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 8, 13, 18, 24 and 29, respectively. (5) 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 to the amino acid sequences set forth in SEQ ID NOs: 4, 8, 13, 19, 24 and 30, respectively; (6) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 9, 14, 20, 25, respectively. and 31; or (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 to the amino acid sequences set forth in SEQ ID NOs: 3, 9, 14, 21, 25 and 31, respectively, wherein the antibody or antigen-binding portion thereof binds to IL4Rα.

[0014] In one aspect, an isolated monoclonal antibody or antigen-binding portion thereof of the invention comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region having (1) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 32 and 35, respectively; or (2) an amino acid sequence that is at least identical to the amino acid sequences set forth 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) 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 to the amino acid sequences set forth in SEQ ID NOs: 33 (X1=W, X2=S) and 37, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 33 (X1=W, X2=S) and 37, respectively; 4) 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 to the amino acid sequence set forth in SEQ ID NOs: 34 and 36, respectively (X1=L, X2=I; X1=F, X2=V; X1=F, X2=I); (5) 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 to the amino acid sequence set forth in SEQ ID NOs: 34 and 37, respectively. an amino acid sequence having 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; (6) 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 to the amino acid sequences set forth 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) 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 to the amino acid sequences set forth in SEQ ID NOs: 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% identity to the amino acid sequences set forth in SEQ ID NOs: 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 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 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 to the amino acid sequences set forth in SEQ ID NOs: 38 and 39, respectively. 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 40 and 45, respectively; (11) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 40 and 45, respectively; (12) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 40 and 45, respectively; (13) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 41 and 42 (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, respectively). , 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 to the amino acid sequence set forth 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 at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth 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 to the amino acid sequences set forth in SEQ ID NOs: 43 and 46, respectively; (15) an amino acid sequence having at least 85%, 86%, 87%, 88%, 99% or 100% identity to the amino acid sequences set forth 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 to the amino acid sequences set forth 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 to the amino acid sequences set forth 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 to the amino acid sequences set forth 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 to the amino acid sequences set forth in SEQ ID NOs: 53 and 54, respectively, wherein the antibody or antigen-binding portion thereof binds to IL4Rα.

[0015] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present invention comprises a heavy chain and a light chain. The heavy chain and light chain are linked by a disulfide bond, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, and the light chain comprising a light chain variable region and a light chain constant region. 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. The heavy chain variable region and light chain variable region comprise the aforementioned amino acid sequences, and the antibody or antigen-binding portion thereof binds to IL4Rα. The heavy chain constant region may be a human IgG4 constant region having the amino acid sequence set forth in SEQ ID NO: 55. The light chain constant region may be a human κ constant region having the amino acid sequence set forth in SEQ ID NO: 56. The amino acid sequences set forth in SEQ ID NOs: 55 and 56 may be encoded by the nucleotide sequences set forth in SEQ ID NOs: 71 and 72, respectively.

[0016] In some embodiments, the antibodies of the present invention comprise two heavy chains and two light chains, or two heavy chains and two light chains. Each heavy chain comprises the above-described heavy chain constant region, heavy chain variable region, or CDR sequence, and each light chain comprises the above-described light chain constant region, light chain variable region, or CDR sequence. The antibodies bind to IL4Rα. The antibodies of the present invention may be full-length antibodies, such as full-length IgG1, IgG2, or IgG4 isotype antibodies, preferably full-length IgG4 isotype antibodies with weak ADCC activity. The light chain constant region may be a κ constant region. In other embodiments, the antibodies of the present invention may be single-chain variable region (scFv) antibodies or antibody fragments, such as Fab or F(ab')2 fragments.

[0017] For example, compared to prior art anti-IL4Rα antibodies such as Dupilumab, the antibodies of the present invention, or antigen-binding portions thereof, have similar (if not higher) binding affinity / capacity to human IL4Rα and / or monkey IL4Rα. Equivalent (but not as significant) effects on IL13Rα1 interaction and corresponding intracellular signaling (in this case) has blocking activity.

[0018] The present invention also includes an antibody or antigen-binding portion thereof of the present invention linked to a second functional molecule (e.g., a second antibody) that has a binding specificity different from that of the antibody or antigen-binding portion thereof. The present invention also provides bispecific molecules comprising the antibodies of the present invention or antigen-binding portions thereof. The present invention also provides immunoconjugates, e.g., antibody-drug conjugates, comprising the antibodies of the present invention or antigen-binding portions thereof, wherein the antibodies or antigen-binding portions thereof are linked to a therapeutic agent (e.g., a cytotoxin). In another aspect, the antibodies of the present invention or antigen-binding portions thereof can be part of a chimeric antigen receptor (CAR). The present invention also provides immune cells, such as T cells, comprising the chimeric antigen receptor. The antibodies of the present invention or antigen-binding portions thereof can be encoded by or used in conjunction with oncolytic viruses.

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

[0020] The present invention provides nucleic acid molecules encoding the antibodies of the present invention or antigen-binding portions thereof, as well as expression vectors containing the nucleic acid molecules and host cells containing the expression vectors. The present invention also provides methods for preparing anti-IL4Rα antibodies or antigen-binding portions thereof using host cells containing the expression vectors, comprising (i) expressing the antibody in the host cells, and (ii) isolating the antibody from the host cells or cell cultures thereof.

[0021] In another aspect, a method for reducing IL4 / IL13 signaling is provided. IL4 signals through receptors including IL-4Rα and γC, and IL13 signals through receptors including 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), fibroblast proliferation, and smooth muscle proliferation, such as airway smooth muscle.

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

[0023] The disease may be an allergic disease. The allergic disease may be atopic dermatitis, allergic reaction, allergic rhinitis, or allergic asthma. In some embodiments, a method for treating an allergic disease may comprise administering to a subject an oncolytic virus encoding or carrying the composition, bispecific molecule, or 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-allergic agent. The anti-allergic agent may include an antihistamine, a corticosteroid, a beta-adrenergic receptor agonist, a cytotoxic agent, or a steroid. It may be an agent that targets LT or an agent that targets IgE.

[0024] The disease may be a tumor disease. The tumor may 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, bispecific molecule, immunoconjugate such as an antibody-drug conjugate, CAR-T cells, 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. In some embodiments, at least one additional anti-cancer antibody, such as an anti-VISTA antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, Anti-CTLA-4 antibodies, anti-TIM 3 antibodies, anti-STAT3 antibodies, and / or anti-ROR1 antibodies can be administered in conjunction with an antibody, or antigen-binding portion thereof, of the invention. In another embodiment, an antibody, or antigen-binding portion thereof, of the invention is administered in conjunction with a cytokine (e.g., IL-2, IL-21, and / or GM-CSF) or a costimulatory antibody (e.g., anti-CD137 antibody and / or anti-GIT The antibody of the present invention can be, for example, a murine antibody, a human antibody, a chimeric antibody, or a humanized antibody.

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

[0026] In another aspect, the invention provides methods, compositions, and kits for diagnosis. In some embodiments, the antibodies of the invention are 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 contents of all documents, Genbank records, patents, and published patent applications cited in this application are expressly incorporated herein by reference. Attached diagram explanation [Brief explanation of the drawings]

[0028] [Figure 1A] 1A-1C show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 and B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) to human IL4Rα. [Figure 1B] 1A-1C show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 and B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) to human IL4Rα. [Figure 1C] 1A-1C show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 and B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) to human IL4Rα. [Figure 2A] Figures 2A-2D show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 (B), B9D1D11F8D8 (C), C2C1A1A1 and C2B2F7B7 (D) to cell surface human IL4Rα. [Figure 2B] Figures 2A-2D show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 (B), B9D1D11F8D8 (C), C2C1A1A1 and C2B2F7B7 (D) to cell surface human IL4Rα. [Figure 2C] Figures 2A-2D show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 (B), B9D1D11F8D8 (C), C2C1A1A1 and C2B2F7B7 (D) to cell surface human IL4Rα. [Figure 2D]Figures 2A-2D show the binding ability of murine antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 (B), B9D1D11F8D8 (C), C2C1A1A1 and C2B2F7B7 (D) to cell surface human IL4Rα. [Figure 3] FIG. 3 shows the binding ability of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8, B9D1D11F8D8, C2C1A1A1 and C2B2F7B7 to cynomolgus monkey IL4Rα. [Figure 4A] 4A-4B show the blocking ability of murine antibodies B1D2F7D3B5, B8G11F2B7G5E8, and B9D1D11F8D8 (A), C2C1A1A1, and C2B2F7B7 (B) on the human IL4Rα-IL4 interaction. [Figure 4B] 4A-4B show the blocking ability of murine antibodies B1D2F7D3B5, B8G11F2B7G5E8, and B9D1D11F8D8 (A), C2C1A1A1, and C2B2F7B7 (B) on the human IL4Rα-IL4 interaction. [Figure 5A] 5A-5B show the blocking ability of murine antibodies B1D2F7D3B5, B8G11F2B7G5E8 and B9D1D11F8D8 (A), C2C1A1A1 and C2B2F7B7 (B) on the binding of human IL4 to a reference. [Figure 5B] 5A-5B show the blocking ability of murine antibodies B1D2F7D3B5, B8G11F2B7G5E8 and B9D1D11F8D8 (A), C2C1A1A1 and C2B2F7B7 (B) on the binding of human IL4 to a reference. [Figure 6A] 6A-6C show the blocking ability of murine antibodies B1D2F7D3B5 and B8G11F2B7G5E8 (A), B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) on the interaction of human IL4 with cell surface human IL4Rα. [Figure 6B]6A-6C show the blocking ability of murine antibodies B1D2F7D3B5 and B8G11F2B7G5E8 (A), B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) on the interaction of human IL4 with cell surface human IL4Rα. [Figure 6C] 6A-6C show the blocking ability of murine antibodies B1D2F7D3B5 and B8G11F2B7G5E8 (A), B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) on the interaction of human IL4 with cell surface human IL4Rα. [Figure 7] FIG. 7 shows the inhibitory activity of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8, B9D1D11F8D8, C2C1A1A1, and C2B2F7B7 on IL4-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. [Figure 8] Figure 8 shows the inhibitory activity of murine antibodies B1D2F7D3B5, B8G11F2B7G5E8, B9D1D11F8D8, C2C1A1A1, and C2B2F7B7 on IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. [Figure 9] FIG. 9 shows the binding ability of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 to human IL4Rα. [Figure 10] FIG. 10 shows the binding ability of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 to cell surface human IL4Rα. [Figure 11] FIG. 11 shows the ability of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 to block human IL4Rα-IL4 interaction. [Figure 12] FIG. 12 shows the inhibitory activity of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 on IL4-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. [Figure 13]Figure 13 shows the inhibitory activity of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 on IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. [Figure 14A] Figures 14A-14B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to human IL4Rα. [Figure 14B] Figures 14A-14B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to human IL4Rα. [Figure 15A] Figures 15A-15B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to cynomolgus monkey IL4Rα. [Figure 15B] Figures 15A-15B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to cynomolgus monkey IL4Rα. [Figure 16A] Figures 16A-16B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to cal-IL4Rα. [Figure 16B]Figures 16A-16B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to cal-IL4Rα. [Figure 17A] Figures 17A-17B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to cell surface human IL4Rα. [Figure 17B] Figures 17A-17B show the binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) to cell surface human IL4Rα. [Figure 18A] Figures 18A-18B show the blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on the interaction of human IL4 with 293F cells expressing human IL4Rα. [Figure 18B] Figures 18A-18B show the blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on the interaction of human IL4 with 293F cells expressing human IL4Rα. [Figure 19A] Figures 19A-19B show the blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on the human IL4Rα-IL4 interaction. [Figure 19B]Figures 19A-19B show the blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on the human IL4Rα-IL4 interaction. [Figure 20A] Figures 20A-20B show the blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on the binding of human IL4 to a reference. [Figure 20B] Figures 20A-20B show the blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on the binding of human IL4 to a reference. [Figure 21] Figure 21 shows the inhibitory activity of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on IL4-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. [Figure 22] Figure 22 shows the inhibitory activity of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14, and huC2C1A1A1-V15 (B) on IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells.

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

[0030] Invention Description In order to better understand the present invention, some terms are first defined. Additional definitions are provided 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 IL4Rα protein from species other than human (e.g., monkeys). In other embodiments, an antibody specific for the human IL4Rα protein may be completely specific for the human IL4Rα protein and not cross-reactive with other species or types, or it may cross-react with IL4Rα from certain other species, but not all others.

[0032] The term "human IL4Rα" refers to an IL4Rα protein having an amino acid sequence of human origin, 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 the 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 chains thereof. Full-length antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H The heavy chain constant region is composed of C H1 , C H2 and C H3 Each light chain is composed of three domains: a light chain variable region (referred to herein as V LThe light chain constant region consists of one domain, C L It is composed of: V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody binds to various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q) and can mediate the binding of immunoglobulins to host tissues or factors.

[0034] The term "antigen-binding site" of an antibody (or simply "antibody site"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an IL4Rα protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding site" of an antibody include (i) Fab fragments, V L , V H , C L and C H1 (ii) F(ab')2 fragment, a monovalent fragment consisting of a fused nucleotide sequence at the hinge region; a bivalent fragment comprising two Fab fragments linked by a sulfide bridge; (iii) V H and C H1 (iv) an Fd fragment consisting of a single arm of an antibody; V L and V H (v) V H(vi) isolated complementarity-determining regions (CDRs); and (vii) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. And, Fv fragments, which are composed of two domains, V is in L and V H Although the V are encoded by separate genes, they can be joined by a synthetic linker that allows them to be made as a single-chain protein using recombinant methods. L and V H are paired to form monovalent molecules (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242 :423-426; and Huston et al., (1988) Proc. Natl. Ac. ad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are full-length antibodies.

[0035] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., antibodies specific for the IL4Rα protein). (An isolated antibody that specifically binds to human 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 human IL4Rα protein is not An isolated antibody may have cross-reactivity to other antigens, such as IL4Rα proteins from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0036] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0037] The term "murine antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from murine germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from murine germline immunoglobulin sequences. The murine antibodies of the invention may include amino acid residues not encoded by murine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "murine 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 murine 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 with genetic material from another species.

[0039] As used herein, the term "humanized antibody" refers to antibodies from non-human species in which the protein sequence has been altered to increase similarity to antibody variants naturally produced in humans.

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

[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 protein 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," i.e., 5.0x or greater. -8 M Less than or equal to 1.0 × 10 -8 M or less, preferably 7.0 × 10 -9 K below M D It binds to the human IL4Rα protein.

[0043] As used herein, the term "does not substantially bind" to a protein or cell means that the protein or cell does not bind or does not bind with high affinity, i.e., less than 1.0 x 10 -6 M or more, preferably 1.0 × 10 -5 M or more, preferably 1.0 × 10 -4 M or more, preferably 1.0 × 10 -3 M or more, more preferably 1.0 × 10 -2 K over M D This means that the molecule binds to a protein or cell.

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

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

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

[0047] "I C 50 The term "half maximal inhibitory concentration," also known as the half maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific 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, mammals and non-mammals, including, for example, non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with mammals such as non-human primates, sheep, dogs, cats, cows, and horses being preferred.

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

[0050] As used herein, the term "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences. Sequence comparison and percent identity determination between two sequences is performed using the N ational Center For Biotechnology Institute It can be run with the default settings of the BLASTN / BLASTP algorithms available on the website.

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

[0052] Anti-IL4Rα antibody with improved binding affinity to human IL4Rα and improved IL4 / IL13 signaling blocking activity The antibodies, or antigen-binding portions thereof, of the present invention specifically bind to human IL4Rα with similar (if not higher) binding affinity / capacity compared to previously described anti-IL4Rα antibodies (such as Dupilumab).

[0053] The antibodies or antigen-binding sites thereof of the present invention bind to IL4 or IL13 of IL4Rα. It can block the binding to α1 and thereby block the corresponding intracellular signaling, which is comparable to reported anti-IL4Rα antibodies (e.g., Dupilumab). It has the same or higher blocking activity.

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

[0055] Anti-IL4Rα monoclonal antibody The antibodies of the present invention are monoclonal antibodies that are structurally and chemically characterized as described below and in the Examples. The amino acid sequence IDs of the heavy and light chain variable regions of the antibodies are summarized in Table 1 below, and some antibodies have the same V H or V L The heavy chain constant region of the antibody may be a human IgG4 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 55. The light chain constant region of the antibody may be a human kappa constant region having the amino acid sequence set forth 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 according to 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 heavy / light chain variable region sequences.

[0058] V of other anti-IL4Rα antibodies that bind to human IL4Rα H and V L The V sequence (or CDR sequence) of the anti-IL4Rα antibody of the present invention H and V L Preferably, V H and V L When the chains (or the CDRs of these chains) are mixed and paired, a particular V H / V L V from the pair H The sequence is structurally similar to V HSimilarly, a particular V H / V L V from the pair L The sequence is structurally similar to V L It is replaced by an array.

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

[0061] Furthermore, the CDR3 domain alone, independently of the CDR1 and / or CDR2 domains, can determine the binding specificity of an antibody to a homologous antigen, allowing predictable generation of multiple antibodies with the same binding specificity based on a common CDR3 sequence. See, for example, Klimka et al., British Journal of Cancer 83(2):2 52-260(2000);Beiboer et al.,, J. Mol. Biol. 296 :833-849(2000);Rader et al.,, Proc. Natl. Acad. Sci. USA 95:8910-8915(1998);Barbas et al.,, J. Am. Chem. Soc. 116:2161-2162(1994);Barbas et al.,, Proc. Natl. Acad. Sci. USA 92:2529-2533(1995);Ditzel et al.,, J. Immunol. 157:739-74 9(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. Nat See, J. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994); and Xu and Davis, Immunity 13:37-45 (2000). See also 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, See, e.g., 5,762,905 and 5,760,185, each of which is incorporated herein by reference in its entirety.

[0062] Therefore, in another embodiment, the antibody of the present invention comprises a CDR2 of the heavy chain variable region of an anti-IL4Rα antibody and a CDR3 of at least the heavy chain variable region and / or the light chain variable region of an anti-IL4Rα antibody. The antibody of the present invention may 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. 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 affinity. 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 and / or light chain variable region of an anti-IL4Rα antibody or the CDR1 of the heavy and / or light chain variable region of another anti-IL4Rα antibody, and the antibody specifically binds to human IL4Rα.

[0063] conservative qualification In another embodiment, an antibody of the present invention comprises a heavy chain variable region and / or a light chain variable region comprising CDR1, CDR2, and CDR3, respectively, and the CDR1, CDR2, and CDR3 sequences differ from the CDR1, CDR2, and CDR3 sequences of an 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 abolish antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wild t et al.,(1997)Prot. Eng. 10:835-41; v et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12;Kel ley and O'Connell (1993)Biochem.32:6862-35;A dib-Conquy et al.,(1998)Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43 Please refer to.

[0064] Thus, in some embodiments, an antibody of the invention comprises a heavy chain variable region and / or a light chain variable region comprising CDR1, CDR2, and CDR3, respectively, wherein: (a) the CDR1 of the heavy chain variable region comprises a sequence listed in Table 1, and / or a conservative modification thereof; and / or (b) CDR2 of the heavy chain variable region comprises a sequence listed in Table 1, and / or a conservative modification thereof; and / or (c) the CDR3 of the heavy chain variable region comprises a sequence listed in Table 1, and / or a conservative modification thereof; and / or (d) CDR1, and / or CDR2, and / or CDR3 of the light chain variable region comprise a sequence listed in Table 1, and / or a conservative modification thereof; and (e) This antibody specifically binds to human IL4Rα.

[0065] The antibodies of the present invention exhibit high affinity binding to human IL4Rα and IL4Rα-IL4 binding or It has one or more functional properties, such as blocking activity against 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 modifications" is intended to refer to amino acid alterations that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as point mutation and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to amino acid residues "Replacement" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are known in the art. These amino acid residue families include those with 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.), and those with 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, phenyl These include amino acids with beta-branched side chains (e.g., alanine, methionine), beta-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 an antibody of the invention can be substituted with another amino acid residue from the same side chain family, and the altered antibody can be tested for retained function (i.e., the above-mentioned functions) using the functional assays described herein.

[0068] Engineered and modified antibodies The antibody of the present invention is a V H / V L An antibody having one or more of the sequences can be used as a starting material to prepare engineered antibodies. The antibody can have one or both variable regions (i.e., V H and / or V L ) (e.g., in one or more CDR regions and / or in one or more framework regions).

[0069] In certain embodiments, CDR grafting can be used to alter the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six complementarity-determining regions (CDRs) of their heavy and light chains. Therefore, the amino acid sequences within the CDRs are more diverse among different antibodies than those outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific natural antibody by constructing an expression vector in which the CDR sequences of a specific natural antibody can be grafted onto the framework sequences of a different antibody (see, e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; 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 present invention relates to an isolated monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2, and CDR3 of the present invention as described above, and the light chain variable region comprises the CDR1, CDR2, and CDR3 of the present invention as described above. These antibodies are similar to the V of the monoclonal antibodies of the present invention. H and V L It contains CDR sequences but may contain different framework sequences.

[0071] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase); Kabat et al., (1991) supra; Tomlinson et al., (1992) J. Mo l. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836, which are expressly incorporated herein by reference. In another embodiment, germline DNA sequences of human heavy chain variable region genes and human light chain variable region genes are available in the Genbank database. For example, the following heavy chain germline sequences from the HCo7 HuMAb mouse are available in 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 HCo12 HuMAb mice are available in Genbank with 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 can be identified using Gapped BLAST (Altschul BLAST) algorithms well known to those skilled in the art. et al., (1997), supra), a sequence similarity search method was used to The sequences are compared to a piled protein sequence database.

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

[0074] Another type of variable region modification is the V H and / or V L The goal of this method is to mutate amino acid residues within the CDR1, CDR2, and / or CDR3 regions of an antibody of interest, thereby improving one or more binding characteristics (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 property of interest can be assessed in in vitro or in vivo assays, as known in the art. Preferably, conservative modifications (known in the art) are introduced. Mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically, no more than one, two, three, four, or five residues within the CDR regions are altered.

[0075] Accordingly, in another embodiment, the present invention provides an isolated anti-IL4Rα monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) a V H CDR1 sequence, or 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions V containing an amino acid sequence having H (b) the CDR1 region of the present invention; HV including a CDR2 sequence or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions H (c) the CDR2 region of the present invention; H V including a CDR3 sequence or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions H (d) the V of the present invention; L V including 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) V of the present invention L CDR2 sequence, or 1, 2, 3, 4 or 5 amino acid substitutions; V containing an amino acid sequence with deletions or additions L CDR2 region; and (f) the present invention Ming V L CDR3 sequence or 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions V containing an amino acid sequence having L CDR3 region.

[0076] The engineered antibodies of the present invention may include, for example, V H and / or V L These include those in which framework region residues of the antibody have been modified. Generally, such framework region modifications can be used to reduce the immunogenicity of the antibody. For example, one or more framework region residues are "backmutated" to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues may be present in the antibody framework. The working sequence can be identified by comparison to the germline sequence from which the antibody is derived.

[0077] Another type of framework modification is a modification within the framework region or between one or more CD One or more residues within the R region are mutated to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method, also known as "deimmunization," is described in further detail in U.S. Patent Publication No. 20030153043.

[0078] Additionally, or as an alternative to modifications made within the framework or CDR regions, antibodies of the invention typically contain modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, again to alter one or more functional properties of the antibody.

[0079] In some embodiments, C H1 - Hinge region: Cysteine ​​residues in the hinge region The method is further described in U.S. Patent No. 5,677,425. H1 The number of cysteine ​​residues in the hinge region is altered to, for example, facilitate assembly of the light and heavy chains 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 made in the C of the Fc hinge fragment to impair binding to Staphylococcus aureus protein A (SpA) compared to the native Fc hinge domain. H2 -C H3 This method is described in U.S. Pat. Further details are described in Os. No. 6,165,745.

[0081] In another embodiment, the glycosylation of the antibody is modified. For example, a glycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an 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 remove a glycosylation site in one or more variable region frameworks, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for an antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861. It's lame.

[0082] Additionally, or alternatively, antibodies can be produced with altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. 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 with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), and therefore antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their sugar chains. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were transfected using two replacement vectors. This was generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using the IFN-γ gene (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87: 614-22). As another example, EP 1,176,195 describes fucosyltransferases. A cell line with a functionally disrupted FUT8 gene encoding the enzyme has been described, Antibodies expressed in such cell lines exhibit hypofucosylation due to the reduction or elimination of α-1,6 bond-related enzymes. EP 1,176,195 also describes cell lines, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662), in which the enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of an antibody is reduced or eliminated. PCT Publication WO 03 / 035835 discloses a method for attaching fucose to an Asn(297)-linked glycan. Mutants that result in reduced ability to bind to fucosylated antibodies and also result in hypofucosylation of antibodies expressed in those host cells described the CHO cell line Lec13 cells (see also Shields et al. See I., (2002) J. Biol. Chem. 277:26733-26740. Antibodies with altered glycosylation profiles are described in PCT Publication WO 06 / 066994. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as duckweed. Methods for producing antibodies in plant systems are described in Alston & Bird LLP's Attorney Docket No. 040989 / 314911, filed 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. 14:5516-5517). twenty three).

[0083] Another modification of the antibodies of the present invention is pegylation. Antibodies 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 activated ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with an activated PEG molecule (or an analogous activated water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono(C1-C10)alkoxy- or aryloxy-polyethyleneglycol. for derivatizing other proteins with methylpropional or polyethylene glycol-maleimide The term "PEG" is intended to encompass any form of PEG that has been used in the art. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. See, e.g., EP0154316 and EP0401384.

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

[0085] For example, an antibody can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or alteration of the antibody's pK, resulting in altered antigen binding. (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and d 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 al.,(2000)Mol I Glycosylation is mediated by NXS / T sequences. In some cases, anti-IL4Rα antibodies that do not contain variable region glycosylation are preferred. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0086] In a preferred embodiment, the antibody does not contain an asparagine isomerization site. Asparagine deamidation occurs at an NG or DG sequence and creates a link in the polypeptide chain. This leads to the formation of isoaspartic acid residues that reduce its stability (isoaspartic acid residues). lagic acid effect).

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

[0088] Nucleic acid molecules encoding antibodies of the invention In another aspect, the present invention provides nucleic acid molecules encoding the heavy and / or light chain variable regions, or CDRs, of the antibodies of the present invention. The nucleic acids can be present in whole cells, in a cell lysate; or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques. The nucleic acids 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] Nucleic acids of the 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, as described further below), 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 an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0090] Preferably, the nucleic acid molecule of the present invention contains the V H and V L These include those encoding the sequences or CDRs. H and V L Once the DNA fragments encoding the 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. H or V LA DNA fragment encoding an antibody constant region is operably linked to another DNA fragment encoding another protein via an antibody constant region or a flexible linker. As used in this case, the term "operably linked" is intended to mean that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

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

[0092] V L The DNA encoding the light chain constant region C L by operably linking to another DNA molecule encoding V L The isolated DNA encoding the full-length light chain gene (and The sequences of human light chain constant region genes can be found in the art. It is 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 may be a kappa or lambda constant region.

[0093] To prepare the scFv gene, Hand V L The DNA fragment encoding the V is operably linked to another fragment encoding a flexible linker, e.g., another fragment encoding the amino acid sequence (Gly-Ser), H and V L The array is split into consecutive units. V L Area and V H The domains are connected by this flexible linker (see, e.g., Bird et al., (1988) Science 24 2:423-426;Huston et al.,(1988)Proc. Natl. Aca d. Sci. USA 85:5879-5883; see McCafferty et al., (1990) Nature 348:552-554).

[0094] Production of monoclonal antibodies of the invention The monoclonal antibodies (mAbs) of the present invention are prepared according to the method described by Kohler and Milstein ( 1975) Nature 256: 495 well-known somatic cell hybridization (hybridization) Monoclonal antibodies can be produced using clonal antibody (antibody) gene (antibody) or humanized antibody (antibody) technologies. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display technology. Chimeric or humanized antibodies are also well known in the art, as described, for example, 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 specifically incorporated herein by reference in their entirety.

[0095] Generation of transfectomas producing monoclonal antibodies of the invention Antibodies of the present invention can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology 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 genes are operably linked to transcriptional and translational control sequences. In this case, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their prescribed function of regulating the transcription and translation of the antibody gene.

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

[0097] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. H Segment in vector C H operatively connected to the V L Segment in vector C L Full-length antibody genes of any antibody isotype can be produced by inserting the variable regions, operably linked to the segments, into an expression vector encoding heavy and light chain constant regions of the desired isotype. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a 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 from a non-immunoglobulin protein).

[0098] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" can refer to a variety of 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 are intended to encompass such processes. While it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic, and most preferably mammalian, host cells is most preferred, as eukaryotic cells, and particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunocompetent antibodies.

[0099] Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO) cells (dhfr-CH for use with the DHFR selectable marker). O cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, and the DHFR selection marker, described, for example, by RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, CO Other preferred expression systems, particularly for use with NSO myeloma cells, include those described in WO 87 / 04462, WO 89 / 01036 and EP 338,841. The disclosed GS gene expression system is a recombinant expression vector encoding an antibody gene that is introduced into mammalian host cells, and the antibody is prepared by culturing the host cells for a time sufficient to express the antibody within the host cells, or preferably for a time sufficient to secrete the antibody into the medium in which the host cells grow. The antibody can be recovered from the medium using standard protein purification methods.

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

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

[0102] Bispecific molecules can come in many different shapes and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody format, except that the two binding arms have different specificities rather than the same specificity. At the other end, they consist of two single-chain antibody fragments (scFvs) linked by a peptide chain. Intermediate-sized bispecific molecules include two different F(ab) fragments linked by a peptidyl linker, so-called Bs(scFv)2 constructs. These and other formats of bispecific molecules 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 therapeutic agent are preferably attached via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferred linkers are peptide linkers such as, for example, 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. , U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PC Publications WO02 / 096910; WO07 / 038,658; WO07 / 051,081; WO07 / 059,404; WO08 / 083,312; and WO08 / 103,693; and 20060247295, the disclosures of which are incorporated herein by reference.

[0104] Antibody-encoding or antibody-carrying oncolytic viruses Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the present invention can be used in conjunction with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies of the present invention can be introduced into humans.

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

[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] CAR contains a signal at the N-terminus of the extracellular antigen-binding domain that targets the nascent receptor to the endoplasmic reticulum. The CAR may contain a peptide and a hinge peptide at the N-terminus of the extracellular antigen-binding domain, which makes it easier to bind to a receptor. Preferably, the intracellular signaling domain of the CAR contains a primary intracellular signaling domain and one or more costimulatory signaling domains. The most commonly used and most potent primary intracellular signaling domain is the CD3- The zeta cytoplasmic domain, whose ITAM phosphorylation leads to T cell activation, and the costimulatory signaling domain are derived from costimulatory proteins such as CD28, CD137, and OX40.

[0108] CARs may further include factors that enhance T cell proliferation, persistence, and anti-tumor activity, such as cytokines and costimulatory ligands.

[0109] Also provided are engineered immune effector cells comprising the CARs 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 pharmaceutical compositions comprising one or more antibodies (or antigen-binding portions thereof, or bispecific molecules, CAR-T cells, oncolytic viruses, or immunoconjugates) of the invention formulated together with a pharmaceutically acceptable carrier. antibody (or antigen-binding portion thereof, or bispecific molecule, CAR-T cell, , oncolytic virus, immunoconjugate) can be administered separately. The composition can optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug, such as an anti-tumor or anti-allergic agent.

[0111] Pharmaceutical compositions can contain any number of excipients. Excipients that can be used include vectors, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending agents, solubilizing agents, colorants, flavoring agents, coating agents, disintegrating agents, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is discussed in detail in Gennaro, ed., Rem ington: The Science and Practice of Pharmacy, 20th Ed.(Lippincott Williams & Wilkins 20 03), 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 may 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" refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the antibodies of the present invention can be administered via parenteral routes, such as topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

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

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

[0115] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus dose may be administered, or multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenterally administered compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages 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 vector. Alternatively, antibodies may be administered as sustained-release formulations, in which case less frequent administration is required.

[0116] When administering the composition, the dosage can range from about 0.0001 to 100 mg / kg of host body weight, more usually 0.01 to 5 mg / kg. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. Preferred dosing regimens for the anti-IL4Rα antibodies of the invention include intravenous administration at 1 mg / kg or 3 mg / kg body weight, with the antibody administered in one of the following dosing regimens: (i) six doses every four weeks, followed by every three months; (ii) every three weeks; or (iii) one dose at 3 mg / kg body weight, followed by 1 mg / kg body weight every three weeks. In some methods, 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 an anti-IL4Rα antibody or antigen-binding portion thereof, bispecific molecule, CAR-T cell, oncolytic virus, or immunoconjugate of the invention can preferably result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-asymptomatic periods, or prevention of damage or disability caused by the disease affliction. For example, in treating a subject with a tumor, a "therapeutically effective amount" is an amount that preferably inhibits tumor growth by at least about 20%, preferably by at least about 40%, more preferably by at least about 60%, and more preferably by at least about 88%, compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or otherwise ameliorate symptoms in a subject, which can typically be a human or another mammal.

[0118] The pharmaceutical composition may 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 may 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, for example, (1) a needleless hypodermic injection device (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 Pon (3) Transdermal Drug Delivery Devices (U.S. Patent No. 4 ,486,194); (4) injection devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patent Nos. 4,439,196 and 4,47 5,196), the disclosures of which are incorporated herein by reference. INCORPORATED INTO THE SPECIFICATION.

[0120] In certain embodiments, the monoclonal antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present invention cross the blood-brain barrier, they can be formulated into liposomes and can further include targeting moieties to enhance selective delivery to specific cells or organs. See, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,000; 16; and 5,399,331; VV Ranade (1989) J. Clin. Phar macol.29:685;Umezawa et al.,(1988)Biochem.B iophys. Res. Commun. 153:1038;Bloeman et al.,(1995)FEBS Lett.357:140;M. Owais et al.,(1995)Antimicrob. Agents Chemother. 39:180;Brisco oe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. Killion and Fidler (1994) Immunomethods 4:2 Please refer to 73.

[0121] Uses and methods of the present invention Antibodies or antigen-binding portions thereof of the present invention, or bispecific, CAR-T cell, oncolytic Compositions containing the active virus, immunoconjugates, have numerous in vitro and in vivo utilities, including, for example, involvement in the treatment of allergic diseases associated with excessive IL4 and / or IL13 signaling.

[0122] The anti-IL4Rα antibody of the present invention binds IL4Rα to IL4 or IL13-IL13Rα1. Because of its ability to block binding and reduce type 2 immunity, the present invention provides a method for treating an allergic disease associated with type 2 immunity, comprising administering the composition of the present invention to a subject. The allergic disease may be atopic dermatitis, allergic reaction, allergic rhinitis, or allergic asthma.

[0123] In another aspect, since IL4 or IL13 signaling can activate STAT6 and STAT6 inhibitors have been found to inhibit cancer cell growth, the present invention provides a method for inhibiting tumor cell growth in a subject, comprising administering to the subject a composition of the present invention, thereby inhibiting tumor growth in the subject. Non-limiting examples of tumors that can be treated with the antibodies of the present invention include melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer (varian cancer), and urothelial cancer. Including, but not limited to, cancer.

[0124] In another aspect, the present invention provides a method for reducing or inhibiting activation of cells responsive to IL-4 or IL-13. In some embodiments, inhibiting activation comprises inhibiting cytokine production or secretion. In some embodiments, inhibiting activation comprises inhibiting proliferation. Stimulation of hybrid IL-4Rα / γC receptors Cells that respond to IL-4 via stimulation include B cells, eosinophils, and macrophages. Cells that respond 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 smooth muscle cell proliferation. In another embodiment, the present invention provides a method for inhibiting fibroblast proliferation. Provide a way to harm.

[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, the diagnosis indicates a 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 correlated with the occurrence and multiple recurrence of oral cancer. In some embodiments, the antibodies of the present invention are used in oral cancer diagnostic kits or methods for determining prognosis and appropriate treatment and follow-up. IL-4Ra expression in tumors has been shown to be significantly elevated in patients with epithelial malignant pleural mesothelioma (MPM) following surgical resection. In some embodiments, the antibodies of the invention are used in diagnostic kits or methods 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 portion, bispecific molecule, or oncolytic virus of the present invention with one or more other drugs effective in ameliorating type 2 immune-related allergic diseases. The drugs can be antihistamines (targeting H1 histamine receptors) clinically used to treat allergic rhinitis, or corticosteroids, beta-adrenergic receptor agonists, and drugs targeting cyc-LTs clinically used to treat asthma. Omalizumab, an anti-IgE antibody, is also used. can also be used to treat allergic diseases with the antibodies or antigen-binding portions thereof, bispecific molecules, or oncolytic viruses of the invention. In certain embodiments, the subject is a human.

[0127] In another aspect, the present invention provides a method for administering an anti-IL4Rα antibody or antigen-binding portion thereof, bispecific molecule, CAR-T cell, oncolytic virus, or immunoconjugate of the invention to a subject in need of treatment with tumor growth inhibitors (TGF-β) or other TGF-β inhibitors (TGF-β) for tumors in a subject. In some embodiments, the present invention provides a method for inhibiting tumor growth in a subject, comprising administering to the subject an anti-IL4Rα antibody (or an antigen-binding portion thereof, a bispecific molecule, an oncolytic virus, a CAR-T cell, or an immunoconjugate) and one or more other antibodies, such as an anti-OX40 antibody, an anti-TIM-3 antibody, an anti-CD137 antibody, an anti-GITR antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, and an anti-PD-1 antibody. In certain embodiments, the subject is a human. The IL4Rα pathway inhibitor can be further combined with a standard of care for cancer. For example, the IL4Rα pathway inhibitor can be combined with an LAG-3 and / or PD-1 inhibitor and a chemotherapy regimen. The anti-IL4Rα antibody can be administered with a chemotherapeutic agent, which may be a cytotoxic agent. For example, epirubicin, oxaliplatin, and 5-fluorouracil can be administered to a patient undergoing anti-IL4Rα therapy. If desired, 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, such as cancer cells, purified tumor antigens (recombinant proteins, peptides, and transfected with genes encoding immunostimulatory cytokines, including glycomolecules, and These cells were isolated (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 antigens, Trp-2, MART1, and / or tyrosinase, or cytokines. These include tumor cells transfected to express the anti-inflammatory cytokine GM-CSF. Other treatments that can be used in combination with L4Rα antibodies include, but are not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.

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

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

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

[0131] Example Example 1: Generation of mouse anti-IL4Rα monoclonal antibody using hybridoma technology immunization Mice were immunized according to the method described in E. Harlow and D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. Recombinant human IL4Rα-his protein (Sino Biotechnology) was used. A homemade human IL4Rα-his protein (amino acid sequence shown in SEQ ID NO: 57) was used as an immunogen to determine the titer of antisera and to screen for hybridomas secreting antigen-specific antibodies. The immunization dose for primary and booster immunizations was 20 μg of human IL4Rα-his protein per injection per mouse. To enhance the immune response, a complete immunization was performed. Freund's adjuvant and incomplete Freund's adjuvant (Sigma, St. Louis, Mo., USA) were used for the primary and booster immunizations, respectively. To prepare the adjuvant and immunogen mixture, the adjuvant was gently mixed in a vial by vortexing. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL microcentrifuge tube. The antigen was prepared in PBS or saline to a concentration ranging from 0.2 to 0.3 mg / mL. Next, the calculated amount of antigen was added to the microcentrifuge tube along with the adjuvant, and the resulting mixture was gently vortexed for 2 minutes to create a water-in-oil emulsion. The adjuvant-antigen mixture was then mixed. The raw emulsion was drawn 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 two to three times depending on the antiserum titer. Animals with better titers were given a final booster by intraperitoneal injection before cell fusion.

[0132] Hybridoma fusion and screening Immediately prior to fusion, mouse myeloma cell line (SP2 / 0-Ag14, ATCC#CRL-158 1) Cells were cultured to reach the logarithmic phase. Spleen cells from immunized mice were prepared aseptically and cultured according to the method of Kohler G, and Milstein C, "Continuous culture of spleen cells." tures of fused cells secreting antibody of The hybridoma cells were fused with myeloma cells according to the method described in "Predefined specificity," Nature, 256: 495-497 (1975). The fused "hybrid cells" were then dispensed into 96-well plates containing DMEM / 20% FCS / HAT medium. Surviving hybridoma colonies were observed under a microscope 7 to 10 days after fusion. Two weeks later, the supernatant from each well was incubated with recombinant human IL4Rα-his protein. The hybridomas were subjected to ELISA-based screening. Briefly, human IL4Rα-his protein (2.0 μg / mL) in PBS was coated onto an ELISA plate at 60 μL / well and incubated overnight at 4°C. The plate was washed four times with PBST and then blocked with 200 μL of blocking buffer (5% w / v skim milk in PBST). Diluted hybridoma supernatant (60 μL) was added to each well and incubated at 37°C for 40 minutes. The plate was then washed four times, and HRP goat anti-mouse IgG (Jackson Immunoresearch, Cat. No. 115-036-071) was used for detection, and the binding OD value was observed at 450 nm. Positive hybridomas secreting antibodies capable of binding to human IL4Rα-his protein were then selected and transferred to a 24-well plate. Highly specific human IL4Rα binding and IL4RαIL4 or IL4RαIL4 were detected. Hybridoma clones producing antibodies exhibiting L4Rα-13Rα1-IL13 blocking activity The monoclonal antibody was purified by limiting dilution to ensure the clonality of the cell line. Briefly, the monoclonal antibody was purified by a Protein A Sepharose column (Bestchrom (Shanghai) Biosciences, Cat. No. AA0273) for 5 min. The column was washed with ~10 column volumes of PBS buffer. The cell supernatant was passed through the column, and then the column was washed with PBS buffer until the protein absorbance reached baseline. The column was then eluted with elution buffer (0.1 M glycine-HCl, pH 2.7). The eluate was immediately collected in a 1.5 mL tube and neutralized with neutralization buffer (1 M Tris-HCl The immunoglobulin-containing fractions were pooled and neutralized with PBS at 4°C overnight. The purified monoclonal antibodies were then characterized for in vitro functional activity as follows.

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

[0134] Briefly, Biacore (GE Healthcare, Pittsburgh, USA) Goat anti-mouse IgG (GE healthcare, Cat. No. BR100838, Mou) was prepared using a standard amine coupling kit provided by GE Healthcare, PA, USA. The biosensor (Se Antibody Capture Kit) was covalently coupled to a CM5 chip (carboxymethylated dextran-coated chip) via primary amine groups. Unreacted areas on the biosensor surface were blocked with ethanolamine. Then, a purified anti-IL4Rα antibody of the present invention at a concentration of 66.67 nM and 10 μg / ml of anti-IL4Rα benchmark (also known as Dupilumab®, BM) were flowed over the chip at a flow rate of 10 μL / min. Next, recombinant human IL4Rα-his (created in-house, amino acid sequence shown in SEQ ID NO: 57), cynomolgus monkey IL4Rα-h in HBS EP buffer (provided by Biacore) were analyzed. Serial dilutions of cal-IL4Rα-his protein (Sino Biological Inc., Cat. No. 90897-C08H) or marmoset IL4Rα-his protein (a product ordered from Sino Biological Inc., also known as cal-IL4Rα-his, the amino acid sequence of which is shown in SEQ ID NO: 58) were flowed over the chip at a flow rate of 30 μL / min. Antibody binding kinetics was followed for 2 min, and dissociation kinetics was followed for 10 min. BIA evaluation software was used to fit the binding and dissociation curves to a 1:1 Langmuir binding model. 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 antibodies 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 in PBS (Jackson Immuno Research, Catalog No. 115-005-00 8) was coated onto a 96-well plate at 100 μl / well and incubated overnight at 4°C. The plate was washed once with washing buffer (PBS + 0.05% w / v Tween-20, PBST), and then 200 μl / well of blocking buffer (PBST containing 5% w / v skim milk) was added and blocked for 2 hours at 37°C. The plate was washed again. 100 μl / well of serial dilutions (5-fold dilutions, 6-fold dilutions with 2.5% w / v skim milk in PBST) were added. The anti-IL4Rα antibody of the present invention, which was tested starting from 6.7 nM, benchmark or negative control hIgG (human immunoglobulin (pH 4) for intravenous injection, Hualan Biological Engineering) Incubate the plate with PBS (Neering Inc.) at 37°C for 40 minutes, then re-incubate the plate. The plate was washed twice. 100 μL / well of biotin-labeled human IL4Rα-his protein (synthetic In column 57, 0.14 nM of a homemade antibody (in 2.5% w / v nonfat milk in PBST) was added to the 96-well plate containing the captured anti-IL4Rα antibody. After incubation at 37°C for 40 minutes, the plate was washed four times and 100 μL / well of HRP-conjugated streptavidin (diluted 1:10,000 in PBST, Jackson Immuno Research, catalog number 016-030-084) was added and incubated at 37°C for 40 minutes. After the final wash, 100 μL / well of ELISA substrate TMB (Innoreagen) was added. ts, Catalog No. MB-S-002) was added and incubated. After 10 minutes, The reaction was stopped by adding 50 μL / well of 1 M H2SO4 at 5°C, and the absorbance was measured at 450 nm. Data were analyzed using Graphpad Prism software. EC 50 The value was obtained.

[0139] 3.2 Cell-based coupled FACS Mouse anti-IL4Rα antibody against IL4Rα expressed on the surface of 293F-IL4Rα cells The binding activity of the antibody was tested by flow cytometry (FACS). 293F cells (Thermofisher Inc., catalog number 11625019) were infected with a nucleotide sequence encoding amino acid residues 1-82 of human IL4Rα (uniprot #P24394-1) between EcoRI and XbaI. 5) was transfected with a pCMV-TP plasmid construct containing nucleotides encoding The transfected and stable cell pool (designated 293F-IL4Rα) was then Cell-based binding FACS and cell-based ligand blocking FACS analysis of 293F-IL4Rα cells were selected. 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and subjected to FACS analysis. The cells were resuspended in buffer (phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum). Next, serial dilutions of anti-IL4Rα antibody or control substance in FACS buffer (starting from 80 nM, 4-fold serial dilutions) were added to 2 × 10 5 The cells were added to a 96-well plate containing 100 μL of R-phycoerythrin-labeled affinity-purified F(ab') per well and incubated on ice for 40 minutes. The cells were washed twice with FACS buffer and then added to the plate with 100 μL of R-phycoerythrin-labeled affinity-purified F(ab') per well. 2 Fragmented goat anti-mouse IgG (H+L) (diluted 1:1000 in FACS buffer, J After incubation for 40 minutes at 4°C in the dark, cells were washed three times and resuspended in FACS buffer. Becton Dickinson FACS Canto II- Fluorescence values ​​were measured using HTS. Graphpad Prism software was used. analyze the data and 50 The value was obtained.

[0140] 3.3 Indirect ELISA The cross-reactivity of anti-IL4Rα antibodies with cynomolgus monkey IL4Rα protein or cal-IL4Rα-his protein was measured. Briefly, the antibodies were incubated in carbonate / bicarbonate buffer (pH 9. 6) cynomolgus monkey IL4Rα-his protein (Sino biological inc., Cat. No. 90897-C08H) 2 μg / mL in carbonate / bicarbonate buffer (pH 9.6) cal-IL4Rα-his protein (outsourced from Sinobiological Inc., product catalog BAX2) 0.2 μg / mL was coated onto a 96-well plate at 100 μL / well and incubated at 37°C for 2 hours. After washing once with washing 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 for 2 hours at 37°C. The plate was washed again, and 100 μL of serially diluted anti-IL4Rα antibodies of the present invention or control substances (0.004 to 66.7 nM, starting from 66.7 nM, diluted 5-fold in PBST containing 2.5% w / v skim milk) was added to each well and incubated for 40 minutes at 37°C. After washing the plate four times, 100 μL / well of peroxidase-labeled affinity-purified goat anti-mouse IgG (Fcγ fragment-specific) (diluted 1:5000 in PBST buffer, Jackson Immunosphere) was added. Research, Catalog Number: 115-036-071) at 37°C for 40 minutes. After the final wash, 100 μL / well of TMB (Innoreagents) was added and incubated. After 3 to 10 minutes, 50 μL / well of 1M H2SO4 was added at 25°C. The reaction was stopped by adding 0.5% CO₂Cl to the wells and the absorbance was read at 450 nm. Data were analyzed using Graphpad Prism software and EC 50 The value was obtained.

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

[0142] The results showed that the mouse anti-IL4Rα antibodies of the present invention specifically bound to human IL4Rα with high binding affinity, and some of them bound to monkey IL4Rα protein with higher binding activity than the benchmark.

[0143] [Table 3]

[0144] Example 4: Blocking activity of murine anti-IL4Rα antibodies against IL4Rα-benchmark or IL4Rα-IL4 interactions 4.1 Ligand-blocking ELISA The ability of the anti-IL4Rα antibodies of the present invention to block IL4-IL4Rα interaction was determined by competitive ELISA. Briefly, human IL4Rα-his protein (SEQ ID NO: 57, prepared in-house) at a concentration of 2 μg / mL in PBS was coated onto a 96-well plate at 100 μL / well 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 nonfat milk in PBST at 37°C for 2 hours. The plate was then washed again using wash buffer.

[0145] Anti-IL4Rα antibody or control substance was incubated in PBST buffer containing 2.5% w / v skim milk. Serial dilutions (starting from 80 nM, 4-fold serial dilutions) were performed. Controls were added to the IL4Rα-coated plates at 100 μL / well and incubated with human IL4Rα-his protein at 37°C for 40 minutes. The plate was washed four times with buffer, and then 100 μL of biotin-labeled human IL4 protein (Sinobiological Inc., Cat. No. 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 (PBS Diluted 1:10000 in T buffer, Jackson Immunoresearch (Catalog No. 016-030-084) was added and incubated at 37°C for 40 minutes. The plate was washed again with washing buffer. Finally, TMB was added and the reaction was washed with 1M H2SO4. The reaction was stopped and the absorbance was read at 450 nm. Data were analyzed using Graphpad Prism software and IC 50 The value was obtained.

[0146] 4.2 Benchmark blocking ELISA The ability of the anti-IL4Rα antibodies of the present invention to block benchmark-human IL4Rα binding was measured in a competitive ELISA assay. Briefly, 96-well microplates were 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 plates were washed with wash buffer (PBS + 0.05% w / v Tween-20, PBST) and 37 min with 5% w / v non-fat milk in PBST. During plate blocking, the anti-IL4Rα antibody of the present invention or a control was added to the plate using biotin-labeled human IL4Rα-his protein (SEQ ID NO: 57, produced in-house, in PBS). (prepared at 0.55 nM in 2.5% w / v nonfat milk in T) starting at 100 nM in a 4-fold series The plate was washed and incubated for 40 minutes at 25°C. The IL4Rα-his mixture was added to the Benchmark-coated plate at 100 μl per well. After incubation at 37°C for 40 minutes, the plate was washed using washing buffer. Then, 100 μl / well of HRP-labeled streptavidin was added to the plate and incubated at 37°C for 40 minutes to detect the biotin-labeled human IL4Rα-his bound to the plate. The plate was washed again with washing buffer. Finally, TMB was added. The reaction was stopped with 1M H2SO4 and the absorbance was read at 450 nm. Analyze the data using pad Prism software and IC 50 The value was obtained.

[0147] 4.3 Cell-based ligand-blocking FACS Using the 293F-IL4Rα cells prepared above, IL4 tagging to cell surface IL4Rα was performed. The activity of anti-IL4Rα antibodies to block protein binding was measured by flow cytometry (FAC). S).

[0148] Briefly, 293F-IL4Rα cells were harvested from cell culture flasks and washed twice. The cells were resuspended in FACS buffer (PBS containing 2% v / v fetal bovine serum). Next, serially diluted anti-IL4Rα antibody or control substance in FACS buffer (starting from 80 nM, 4-fold serial dilutions) was added to the wells at 100 μL / well. 5 The plate was washed twice with FACS buffer, and 100 μl / well of 1.67 nM biotin-labeled human IL4 protein (Sino biological inc., Cat. No. 11846-HNAE) was added and incubated in the dark at 4°C for 40 minutes. The plate was washed twice with FACS buffer, and then 100 μl / well of R- Phycoerythrin-streptavidin (1:500 dilution in FACS buffer, Jac The cells were washed twice and resuspended in FACS buffer. A Becton Dickinson FACS Canto II-HTS instrument was used. Fluorescence was measured using Graphpad Prism software. Analyze and IC 50 The value was obtained.

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

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

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

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

[0153] [Table 4]

[0154] Example 5: Cell-based functional assay of mouse anti-IL4Rα antibodies IL4 and IL13 bind to IL4Rα and HEK293T-IL4Rα-STAT6-S STAT6 phosphorylation was induced in TAT6LUC-LB2 cells. The steps are important in the IL4 / IL13 signaling pathway.

[0155] Briefly, pcDNA3.1-Puro (YouBio biological Inc., Cat. No. VT9222) Plasmid construct (BamHI and Xh oI) containing the nucleotides encoding human IL4Rα, STAT6 plasmid (Sino biological inc., Catalog No. HG13190-NH)(Kp The plasmid contains the nucleotides encoding human STAT6 between nI and XbaI, and the STAT6 luciferase reporter gene plasmid STAT6-Luc (Yeasen b Biol. Inc., Cat. No. 11588ES03) was used to identify IL13R HEK293T cells (ATCC CRL-11268) natively expressing IL4Rα were stably transfected to generate HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells in-house. A single cell clone, LB2, was then selected for all subsequent functional assays.

[0156] The anti-IL4Rα antibodies of the present invention were tested for their inhibitory effects on IL4- and IL13-induced STAT6 phosphorylation.

[0157] Briefly, log-phase HEK293T-IL4Rα-STAT6-STAT6LU Resuspend C-LB2 cells in medium (RPMI1640 + 10% FBS) and add 100 μL / well. 96-well plates were seeded with 5 x 10 cells per well. 5 Next, 50 μL of serially diluted anti-IL4Rα antibody or control (anti-CD22 antibody prepared in-house) was added to the cells. (including 100 nM, 5-fold serial dilutions) 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 No. 11846-HNAE) or IL13 protein (50 ng / mL, Sino biological inc., Catalog No. 10369-HNAC) was added to each well and incubated at 37°C for 20 minutes. The plate was centrifuged and staining buffer (prepared in-house, DPBS + 0.5% w / v BSA + 2 mM E After washing twice with BDTA, 50 μL of fixation buffer (BD Biosciences Inc., Cat. 5545655) was added to each well and incubated at 4°C for 30 minutes. The cells were washed twice, and 200 μL of permeabilization buffer (BD biosciences inc., Catalog No. 558050) was added to each well, followed by incubation in an ice bath for 30 minutes. After washing three times with staining buffer, anti-pSTAT6 antibody (20-fold dilution of pSTAT6 stock solution, BD Biosciences Inc., Cat. No. 562079) was applied. The plate was then washed twice and resuspended in staining buffer. Fluorescence values ​​were measured using Graphpad Prism software. Analysis and IC 50 The value was obtained.

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

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

[0160] [Table 5]

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

[0162] The heavy and light chain variable domains of 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 kappa light chain constant region (SEQ ID NO: 56), respectively, in which the C-terminus of the variable region is linked 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 293F suspension cells in 1 mg / mL PEI at a ratio of 60%:40% light chain construct:heavy chain construct.

[0164] After 6 days of culture in shake flasks, the cell supernatant was collected, spun down to pellet the cells, and filtered through a 0.22 μm filter for immunoglobulin isolation. The chimeric antibody was purified by Protein A affinity chromatography. Briefly, a Protein A Sepharose column (Bestchrom (Shanghai) Biosciences, catalog number AA0273) was washed with 5 to 10 column volumes using PBS buffer. The cell supernatant was passed through the Protein A Sepharose column, and the column was washed with PBS buffer until the protein absorbance reached 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. Fractions containing immunoglobulin were pooled and dialyzed overnight at 4°C against PBS.

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

[0166] For the Capture ELISA, 2 μg / ml of goat anti-human IgG (affinity-purified goat anti-human IgG, Fcγ fragment specific, Jackson Immunoresearch, Cat. No. 109-005-098) was used at 100 μL / well.

[0167] For indirect ELISA, peroxidase-conjugated affinity-purified goat anti-mouse IgG ( 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 instead of the Fcγ fragment specific antibody.

[0168] In BIAcore, goat anti-human IgG (GE hea) was used instead of goat anti-mouse IgG. The antibody was covalently coupled to a CM5 chip using a Human Antibody Capture Kit (Latest Care, Cat. No. BR100839).

[0169] For cell-based coupled FACS, R-phycoerythrin-labeled affinity-purified goat anti-human IgG (Fcγ fragment specific, Jackson Immunore) was used instead of R-phycoerythrin-labeled affinity-purified F(ab')2 fragmented goat anti-mouse IgG (H+L). search, 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 to 13. The data demonstrate that the chimeric antibodies have similar binding affinity / capacity and blocking activity as their parental murine antibodies.

[0171] [Table 6]

[0172] Example 7: Humanization of anti-IL4Rα monoclonal antibodies B8G11F2B7G5E8 and C2C1A1A1 Humanized mouse anti-IL4Rα antibodies B8G11F2B7G5E8 and C2C1A1A1 Humanization of the murine antibody was carried out using established CDR grafting methods as described below.

[0173] To select acceptor frameworks for humanization of the 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 heavy and light chain variable region CDRs of the mouse antibody were inserted into the selected framework, and the framework residues were further backmutated to obtain more candidate heavy and light chain variable regions. A total of 13 exemplary humanized B8G11F2B7G5E8 antibodies, namely, huB8G11F2B7G5E8-V1 through huB8G11F2B7G5E8-V11, huB8G11F2B7G5E8-V13 and huB8G11F2B7G5E8-V14, and 16 exemplary humanized C2C1A1A1 antibodies, namely, huC2C1A1A1-V1 through huC2C1A1A1-V1. The sequences from huC2C1A1A1 to huC2C1A1A1-V16 were obtained, and the sequence IDs of the heavy chain / light chain variable regions are shown below. Shown in 1.

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

[0175] After 6 days of culture in shake flasks, the cell supernatant was harvested, the cells in the supernatant were pelleted by centrifugation, and the immunoglobulins were isolated by filtration through a 0.22 μm filter. The antibodies were purified by Protein A affinity chromatography. Briefly, a Protein A Sepharose column (Bestchrom (Shanghai) Biosciences, catalog number AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was passed through the Protein A Sepharose column, and the column was washed with PBS buffer until the protein absorbance reached 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 immunoglobulin-containing fractions were pooled and dialyzed overnight against PBS at 4°C.

[0176] Example 8: Characterization of humanized antibodies

[0177] [Table 7]

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

[0179] [Table 8]

[0180] The results show that the humanized antibodies have similar binding affinity to human IL4Rα as the chimeric antibodies, and all humanized huC2C1A1A1 antibodies exhibit higher binding affinity to human IL4Rα compared to the reference.

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

[0182] For Capture ELISA, goat anti-mouse IgG (Fcγ fragment specific) Instead of 2 μg / mL goat anti-human IgG (affinity purified goat anti-human IgG, Fcγ fragment specific, Jackson Immunoresearch, catalog No. 109-005-098) was used at 100 μL / well.

[0183] For indirect ELISA, peroxidase-conjugated affinity-purified goat anti-mouse IgG ( Instead of peroxidase-labeled affinity-purified F(ab')2 fragmented goat anti-human IgG (Fcγ fragment specific, Jackson Immunol. munoresearch, catalog 109-036-098) Use at 100 μl / well Used.

[0184] In BIAcore, goat anti-human IgG (GE he) was used instead of goat anti-mouse IgG. The antibody was covalently coupled to a CM5 chip using a Human Antibody Capture Kit (Healthcare, Cat. No. BR100838).

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

[0186] The thermal stability of the humanized antibodies huB8G11F2B7G5E8-V14 and huC2C1A1A1-V15 was also tested. TM Thermal Shift Protein Stability Kit (Biotium, Cat. No. 33 Protein thermal shift assays were performed using a 022-T (Cat. No. 181214) The melting temperature (Tm) was measured using the GloMelt TM The dye was thawed at room temperature. The vial containing the dye was vortexed and centrifuged. 5 μL 200x dye was added to 95 μL PBS to prepare 10x dye. 2 μL 10x dye and 10 μg humanized antibody were added to the reaction, and PBS was added to bring the total reaction volume to 20 μL. The microcentrifuge tubes containing the dye and antibody were briefly centrifuged and run in a real-time PCR thermal cycler (Roche, ) using the melting curve program set using the parameters in Table 9. The plate was placed in a LightCycler 480 II.

[0187] [Table 9]

[0188] The results are shown in Tables 10-1 to 10-3 and FIGS. 14A to 14B through FIG.

[0189] [Table 10-1]

[0190] [Table 10-2]

[0191] [Table 10-3]

[0192] The data show that the humanized C2C1A1A1 antibody has comparable (if not better) binding affinity / activity to human IL4Rα and IL4Rα-IL4 / IL13 compared to the reference. Although the humanized B8G11F2B7G5E8 antibody exhibits blocking ability, the IL4 / IL13-IL It showed significantly superior blocking ability against the 13Rα1-IL4Rα interaction.

[0193] While 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

1. 1. An isolated monoclonal antibody or antigen-binding portion thereof that binds to the interleukin-4 receptor alpha subunit (IL4Rα), comprising a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, (2) 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 to the amino acid sequences set forth in SEQ ID NOs: 1, 6 and 11, respectively; (3) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% identity to the amino acid sequences set forth in SEQ ID NOs: 2, 7 and 12, respectively. , 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 3, 8 and 13, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 8 and 13, respectively; (5) an amino acid sequence having at least 85%, 86%, 87%, 88%, (6) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 9 and 14, respectively; or an isolated monoclonal antibody or an antigen-binding portion thereof, comprising:

2. The heavy chain variable region has SEQ ID NOs: 32, 33 (X1=W, X2=S; X1=L, X2=A; X1=W, X2=A), 34, 38, 40, 41 (X1=A, =Q, X3=V, X4=H; X1=A, X2=K, X3=M, X4=H; 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, comprising 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 to the amino acid sequence set forth in any one of the following: X1 = K, X2 = A, X3 = T, X4 = N; X1 = K, X2 = A, X3 = S, X4 = D; X1 = R, X2 = V, 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.

3. The light chain variable region comprises a CDR1, a CDR2, and a CDR3, and the CDR1, the CDR2, and the CDR3 are (1) 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 to the amino acid sequences shown in SEQ ID NOs: 15, 22, and 26, respectively; or (2) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, or 100% identity to the amino acid sequences shown in SEQ ID NOs: 16, 22, and 27, respectively. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 17, 23 and 28, respectively; (3) 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 to the amino acid sequences set forth in SEQ ID NOs: 17, 23 and 28, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 18, 24 and 29, respectively. (5) 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 to the amino acid sequences set forth in SEQ ID NOs: 19, 24 and 30, respectively; (6) 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 to the amino acid sequences set forth in SEQ ID NOs: 20, 25 and 31, respectively.

9. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, comprising: (1) 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 to the amino acid sequences set forth in SEQ ID NOs: 21, 25 and 31, respectively.

4. 4. The isolated monoclonal antibody or antigen-binding portion thereof of claim 3, wherein 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 to the amino acid sequence set forth 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.

5. The heavy chain variable region and the light chain variable region are selected from the group consisting of: (1) 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: 1, 5, 10, 15, 22 and 26, respectively; and (2) 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: 1, 6, 11, 16, 22 and 27, respectively. (3) 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 to the amino acid sequences set forth in SEQ ID NOs: 2, 7, 12, 17, 23 and 28, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 8, 13, 18, 24 and 29, respectively. (5) 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 to the amino acid sequences set forth in SEQ ID NOs: 4, 8, 13, 19, 24 and 30, respectively; (6) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 9, 14, 20, 25 and 31, respectively; 8. The isolated monoclonal antibody or antigen-binding portion thereof of claim 3, comprising: (1) an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 3, 9, 14, 21, 25 and 31, respectively; or (2) 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 to the amino acid sequences set forth in SEQ ID NOs: 3, 9, 14, 21, 25 and 31, respectively.

6. The heavy chain variable region and the light chain variable region are selected from the group consisting of: (1) 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 to the amino acid sequences shown in SEQ ID NOs: 32 and 35, respectively; and (2) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% or 100% identity to 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) 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 to the amino acid sequences set forth in SEQ ID NOs: 33 (X1 = W, X2 = S) and 37, respectively; (4) 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 to the amino acid sequences set forth in SEQ ID NOs: 34 and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), respectively. (5) 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 to the amino acid sequences set forth in SEQ ID NOs: 34 and 37, respectively; (6) 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 to the amino acid sequences set forth 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) 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 NOs: 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 sequence shown in SEQ ID NOs: 33 (X1 = W, X2 = A) and 36 (X1 = X1 = F, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I); (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 to the amino acid sequence shown in SEQ ID NOs: 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 set forth in SEQ ID NOs: 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 set forth in SEQ ID NOs: 40 and 45, respectively. (12) At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 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 NOs: 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 46, respectively; (1 4) 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 to the amino acid sequences set forth 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 to the amino acid sequences set forth in SEQ ID NOs: 44 and 46, respectively;(16) At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequences set forth 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 set forth in SEQ ID NOs: 49 and 50, respectively; (18) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identity with the amino acid sequences set forth in SEQ ID NOs: 51 and 52, respectively. 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 53 and 54, 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 to the amino acid sequences set forth in SEQ ID NOs: 53 and 54, respectively.

7. 7. The isolated monoclonal antibody or antigen-binding portion thereof of claim 6, 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 set forth in SEQ ID NO: 55 and the light chain constant region has the amino acid sequence set forth in SEQ ID NO:

56.

8. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the monoclonal antibody or antigen-binding portion thereof (a) binds to human IL4Rα; (b) binds to simian IL4Rα; (c) blocks IL4Rα-IL4 interaction; and (d) blocks IL4Rα-IL13-IL13Rα1 interaction.

9. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the monoclonal antibody or antigen-binding portion thereof is a murine antibody, a human antibody, a chimeric antibody, or a humanized antibody.

10. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the monoclonal antibody or antigen-binding portion thereof is an IgG1, IgG2, or IgG4 isotype.

11. A nucleotide encoding the isolated monoclonal antibody or its antigen-binding portion according to claim 1.

12. An expression vector comprising the nucleotide of claim 11.

13. A host cell comprising the nucleotide of claim 11 or the expression vector of claim 12.

14. A pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding portion thereof described in claim 1, a nucleotide described in claim 11, an expression vector described in claim 12, or a host cell described in claim 13, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, further comprising an antiallergic agent or an antitumor agent.

16. The pharmaceutical composition according to claim 15, wherein the antiallergic agent is an antihistamine, a corticosteroid, a β-adrenergic receptor agonist, a drug that targets cyc-LTs, or a drug that targets IgE.

17. A method for treating an allergic disease associated with excessive IL4 and / or IL13 signaling, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 14 or 16. The method comprising:

18. 18. The method of claim 17, wherein the allergic disease is atopic dermatitis, an allergic reaction, allergic rhinitis, or allergic asthma.

19. A method for treating a tumor associated with increased STAT6 activation in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 14.

20. 20. The method of claim 19, wherein the tumor is a solid tumor.

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