Antibodies binding il4r and uses thereof

Abstract

The invention relates to the field of antibodies, and provides an antibody combined with IL4R and application thereof. In particular, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human IL4Ralpha. The invention also provides nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors, host cells and methods for expressing the antibodies or antigen-binding portions thereof. The invention further provides bispecific molecules, oncolytic viruses and pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof, as well as methods of treatment using the anti-IL4R alpha antibodies or antigen-binding portions thereof of the invention.

Description

Related Applications and Incorporation by Reference

[0001] This application claims priority to U.S. Provisional Application No. 62 / 982,521 filed on February 27, 2020. This application is a divisional application of an invention patent application with a filing date of February 25, 2021, Chinese application number 202180015046.9, and invention name “Antibodies binding to IL4R and uses thereof”.

[0002] The above-mentioned application and all documents cited in this application or during the prosecution process ("referenced documents"), all documents cited or referenced in this application (including but not limited to all documents, patents, and published patent applications cited in this application) ("referenced documents"), and all documents cited or referenced in the referenced documents in this application, together with any manufacturer's instructions, descriptions, product specifications, and product pages for any products mentioned in this application or in any document incorporated by reference in this application, are all incorporated by reference and can be used in the practice of the present invention. More specifically, all references cited in this application are incorporated by reference to the same extent as if each document was specifically and individually indicated to be incorporated by reference in this application. Any Genbank sequence mentioned in the present invention is incorporated by reference to the Genbank sequence, with the date of this reference as the earliest effective filing date of the present invention. Technical Field

[0003] The present invention relates to an isolated monoclonal antibody, in particular to a mouse, chimeric or humanized monoclonal antibody or an antigen-binding portion thereof, which binds to human IL4R, especially IL4Rα, with high affinity and functionality. The present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the present invention, expression vectors, host cells and methods for expressing the antibodies or antigen-binding portions thereof of the present 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 present invention, as well as methods for treating using the anti-IL4Rα antibodies or antigen-binding portions thereof of the present invention. Background Art

[0004] Allergic diseases associated with type 2 inflammation, such as atopic dermatitis, anaphylaxis, allergic rhinitis and allergic asthma, plague more than 3 billion people worldwide and the incidence continues to rise. According to the hygiene hypothesis, part of the reason for the high incidence is that with the improvement of living standards, the chance of contact with infectious sources decreases, making the immune system more sensitive to some originally harmless allergens (Stephen J.Galli et al., (2008) Nature 454 (7203): 445-454). Interleukin 4 (IL-4) and IL-13 are two key factors in type 2 immunity. They are required for driving most of the key hallmarks associated with type 2 inflammation, such as the production of immunoglobulin E and the recruitment of innate cells to sites of inflammation (Gruning G et al., (1998) Science 282:2261-2263; Rankin JA et al., (1996) Proc Natl Acad Sci USA 93:7821-7825; Wills-Karp M et al., (1998) Science 282:2258-2261).

[0005] IL-4 and IL-13 are located adjacent to each other on human chromosome 5 and share regulatory elements. H2), both coordinated and uncoordinated expression of these two cytokines was observed (Katherine Bao et al., (2015) Cytokine 75(1):25-37). These two cytokines bind to cell surface receptors to regulate cell function and activate transcriptional machinery. Specifically, IL-4 first binds to the IL-4Rα chain with picomolar affinity and then recruits the IL-2Rγ chainγc to form a type I receptor complex, or recruits IL-13Rα1 to form a type II receptor complex. The levels or availability of IL-2Rγγc and IL-13Rα1 determine which of them will be recruited to form a receptor complex. It has been found that non-hematopoietic cells do not express or low-express IL-2Rγγc, but highly express IL-13Rα1, while lymphocytes do the opposite. Myeloid cells are between these two types of cells. The formation of the type II IL-4 receptor complex can also be initiated by the binding of IL-13 to the IL-13Rα1 chain (binding with nanomolar affinity), and further recruiting the IL-4Rα chain. In addition to the type II IL-4 receptor, IL-13 can also bind to IL-13Rα2 with picomolar affinity, which is considered to be a decoy receptor (Irina G. Luzina et al., (2012) J Leukoc Biol 92(4): 753-764). Once the IL-4 receptor complex is formed, intracellular signaling molecules will be activated, among which STAT6 and IRS signaling will respond to the activation of the type I IL-4 receptor, while the type II IL-4 receptor cannot significantly activate the IRS (Heller NMet al., (2008) Sci Signal 1(51): ra17-ra17). STAT6 signaling is important for T H 2 cell differentiation and IL-4 production, and IRS molecules can activate PI3K and mTOR signaling pathways (Gadani SP et al., (2012) J Immunol 189: 4213-4219).

[0006] Studies have shown that excessive IL-4 / IL-13 signaling may cause allergic diseases, so some therapeutic antibodies that regulate IL-4 and IL-13-mediated signaling have been developed. For example, Leprikizumab, Anrukinzumab, and Tralokinumab, which bind to IL-13, and Pascolizumab, which targets IL-4. Dupilumab and Pitrakinra are IL-4Rα antagonists, in which Pitrakinra blocks both type I and type II IL-4 receptors after binding to IL-4Rα (Antoniu SA (2010) Curr Opin Investig Drugs 11: 1286-1294). In addition, STAT6 inhibitors have been found to inhibit the growth of prostate cancer cells, indicating that targeted therapy of IL-4 / IL-13 may be beneficial for cancer treatment (Nappo G et al., (2017) Oncogenesis 2017, 6 (5): e342). Therefore, more antibodies targeting IL-4, IL-13 and their receptors (especially IL-4Rα) with more ideal therapeutic properties are needed. SUMMARY OF THE INVENTION

[0007] The present invention provides an isolated monoclonal antibody, such as a mouse, human, chimeric or humanized monoclonal antibody or an antigen-binding portion thereof, which binds to IL4Rα (e.g., human IL4Rα) and has comparable or higher binding affinity / capacity to human IL4Rα and / or monkey IL4Rα, and comparable or higher blocking activity on IL4Rα-IL4 / IL13-IL13Rα1 interaction and corresponding intracellular signal transduction, compared to anti-IL4Rα antibodies in the prior art, such as Dupilumab.

[0008] The antibodies or antigen-binding portions thereof of the present invention have various uses, including detecting IL4Rα protein, and treating and preventing 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 mouse, chimeric or humanized antibody) or an antigen-binding portion thereof, which binds to IL4Rα and comprises a heavy chain variable region, wherein 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 comprise: (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 sequence shown in SEQ ID NOs: 1, 5 and 10, respectively; (2) 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 sequence shown in SEQ ID NOs: 1, 5 and 10, respectively; 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 1, 6 and 11; (3) amino acid sequences that are 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: 2, 7 and 12, respectively; (4) amino acid sequences that are 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: 3, 7 and 13, respectively; 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 3, 8 and 13; (5) 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: 4, 8 and 13, respectively; or (6) 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: 5, 8 and 13, respectively. The amino acid sequences shown in NOs: 3, 9 and 14 have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0010] In one aspect, the isolated monoclonal antibody or antigen binding portion thereof of the invention comprises a heavy chain variable region comprising the amino acid sequence 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=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), 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 set forth in SEQ ID NO:32, wherein the antibody or antigen binding portion thereof binds IL4Rα. The amino acid sequence set forth in SEQ ID NO:32 may be encoded by the nucleotide sequence set forth in SEQ ID NOs:59 or 60. The amino acid sequence set forth in SEQ ID NO:40 may be encoded by the nucleotide sequence set forth in SEQ ID NOs:65 or 66. The amino acid sequences shown in SEQ ID NOs: 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 NOs: 61 or 67, respectively.

[0011] In one aspect, an isolated monoclonal antibody or an antigen-binding portion thereof of the present invention binds to IL4Rα, wherein the monoclonal antibody or the antigen-binding portion thereof comprises a light chain variable region, wherein the light 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 comprise: (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 sequence shown in SEQ ID NOs: 15, 22, and 26, 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 sequence shown in SEQ ID NOs: 15, 22, and 26, respectively; 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 16, 22 and 27; (3) amino acid sequences that are 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: 17, 23 and 28, respectively; (4) amino acid sequences that are 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: 17, 23 and 28, respectively; 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 18, 24 and 29; (5) amino acid sequences that are 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: 19, 24 and 30, respectively; (6) amino acid sequences that are 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: 19, 24 and 30, respectively; or (7) 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 shown in SEQ ID NOs: 21, 25 and 31, respectively.

[0012] In one aspect, an isolated monoclonal antibody of the invention, or an antigen binding portion thereof, comprises a light chain variable region comprising 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 sequence set forth in SEQ ID NOs: 35, 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I), 37, 39, 45, 46, 48, 50, 52 or 54, wherein the antibody or antigen binding portion thereof binds IL4Rα. The amino acid sequence set forth in SEQ ID NO: 35 can be encoded by the nucleotide sequence set forth in SEQ ID NOs: 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 NOs: 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, the isolated monoclonal antibody or antigen-binding portion thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a CDR1 region, a CDR2 region, and a CDR3 region, respectively, wherein the heavy chain variable region CDR1, CDR2, and CDR3 and the light chain variable region CDR1, CDR2, and CDR3 comprise: (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 sequence shown in SEQ ID NOs: 1, 5, 10, 15, 22, and 26, 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 sequence shown in SEQ ID NOs: 1, 5, 10, 15, 22, and 26, respectively; 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 1, 6, 11, 16, 22 and 27; (3) amino acid sequences that are 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: 2, 7, 12, 17, 23 and 28, respectively; (4) amino acid sequences that are 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: 3, 4, 5 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 3, 8, 13, 18, 24 and 29; (5) amino acid sequences that are 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: 4, 8, 13, 19, 24 and 30, respectively; (6) amino acid sequences that are 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: 5, 8, 13, 19, 24 and 30, respectively; NOs:3, 9, 14, 20, 25 and 31 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 shown in SEQ ID NOs:3, 9, 14, 21, 25 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 shown in SEQ ID NOs:3, 9, 14, 21, 25 and 31, respectively, wherein the antibody or its antigen-binding portion binds to IL4Rα.

[0014] In one aspect, the isolated monoclonal antibody or antigen-binding portion thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise: (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 sequence set forth in SEQ ID NOs: 32 and 35, 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 sequence set forth in SEQ ID NOs: 32 and 35, respectively; NOs:33 (X1=W, X2=S) and 36 (X1=L, X2=I; X1=F, X2=V; X1=F, X2=I) 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 36 (X1=L, X2=I; X1=F, X2=V; X1=F, X2=I); (3) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:33 (X1=W, X2=S) and 37, respectively; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: NOs:34 and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) 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) 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:34 and 37, respectively; (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:34 and 37, respectively; NOs:33 (X1 = L, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) 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); (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:33 (X1 = L, X2 = A) and 37, respectively; (8) 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: NOs: 33 (X1 = W, X2 = A) and 36 (X1 = L, X2 = I;91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 33 (X1=W, X2=A) and 37, respectively; (10) amino acid sequences set forth in SEQ ID NOs: 34 (X1=W, X2=A) and 38 (X1=F, X2=V); (11) amino acid sequences set forth in SEQ ID NOs: 35 (X1=W, X2=A) and 39 (X1=F, X2=V); (12) amino acid sequences set forth in SEQ ID NOs: 36 (X1=W, X2=A) and 37, respectively; (13) amino acid sequences set forth in SEQ ID NOs: 37 (X1=W, X2=A) and 38 (X1=F, X2=V); NOs:38 and 39 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:38 and 39; (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; 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 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; (13) respectively with 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 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; (14) respectively with SEQ ID The amino acid sequences set forth in NOs: 43 and 46 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;(15) amino acid sequences that are 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: 44 and 46, respectively; (16) amino acid sequences that are 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: 47 and 48, respectively; (17) amino acid sequences that are 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: 48 and 49, 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: 49 and 50; (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: 51 and 52, respectively. 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 IDNOs: 53 and 54, wherein the antibody or antigen binding portion thereof binds to IL4Rα. ;

[0015] In one embodiment, the isolated monoclonal antibody of the present invention or its antigen binding portion comprises a heavy chain and a light chain, wherein the heavy chain and the light chain are connected by a disulfide bond, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the above-mentioned amino acid sequence, and the antibody or its antigen binding portion binds to IL4Rα. The heavy chain constant region can be a human IgG4 constant region having an amino acid sequence as shown in SEQ ID NO: 55, and the light chain constant region can be a human kappa constant region having an amino acid sequence as shown in SEQ ID NO: 56. The amino acid sequences shown in SEQ ID NOs: 55 and 56 can be encoded by the nucleotide sequences shown in SEQ ID NOs: 71 and 72, respectively.

[0016] In some embodiments, the antibodies of the present invention comprise two heavy chains and two light chains, or consist of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region, the above-mentioned heavy chain variable region or CDR sequence, and each light chain comprises the above-mentioned light chain constant region, the above-mentioned light chain variable region or CDR sequence, wherein the antibody binds to IL4Rα. The antibodies of the present invention may be full-length antibodies, such as full-length antibodies of the IgG1, IgG2 or IgG4 isotypes, preferably full-length antibodies of the IgG4 isotype with weak ADCC activity. The light chain constant region may be a kappa 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 Fragment.

[0017] Compared to the anti-IL4Rα antibodies in the prior art, such as Dupilumab, the antibodies or antigen-binding portions thereof of the present invention have comparable (if not higher) binding affinity / ability to human IL4Rα and / or monkey IL4Rα, and comparable (if not higher) blocking activity on IL4Rα-IL4 / IL13-IL13Rα1 interaction and corresponding intracellular signal transduction.

[0018] The present invention also provides bispecific molecules comprising antibodies or antigen-binding portions thereof of the present invention, wherein the antibodies or antigen-binding portions thereof are connected to a second functional molecule (e.g., a second antibody) having different binding specificities therewith. The present invention also provides immunoconjugates comprising antibodies or antigen-binding portions thereof of the present invention, such as antibody-drug conjugates, wherein the antibodies or antigen-binding portions thereof are connected to therapeutic agents (e.g., cytotoxins). On the other hand, the antibodies or antigen-binding portions thereof of the present invention may be part of a chimeric antigen receptor (CAR). The present invention also provides immune cells, such as T cells, comprising chimeric antigen receptors. The antibodies or antigen-binding portions thereof of the present invention may also be encoded by oncolytic viruses or used together with oncolytic viruses.

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

[0020] The present invention also provides a nucleic acid molecule encoding the antibody of the present invention or its antigen-binding portion, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector. The present invention also provides a method for preparing an anti-IL4Rα antibody or its antigen-binding portion using a host cell comprising an expression vector, comprising the following steps: (i) expressing the antibody in a host cell, and (ii) isolating the antibody from the host cell or its cell culture.

[0021] In another aspect, the present invention provides a method of reducing IL4 / IL13 signaling. IL4 signals through receptors comprising IL-4Rα and γC, while IL13 signals through receptors comprising IL-4Rα and IL13 Rα1. Non-limiting examples of IL4 / IL13 signaling include activation and / or proliferation of B cells, eosinophils, macrophages (e.g., activated macrophages), proliferation of fibroblasts, and proliferation of smooth muscle, such as proliferation of airway smooth muscle cells.

[0022] In another aspect, the invention provides a method of 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 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, the method for treating an allergic disease comprises administering to the subject a composition of the present invention, a bispecific molecule, or an oncolytic virus encoding an antibody or carrying an antibody, or a nucleic acid molecule or vector capable of expressing the same components in the subject. The method further comprises administering an antiallergic agent. The antiallergic agent may be an antihistamine, a corticosteroid, a β-adrenergic receptor agonist, a drug targeting cyc-LTs, or a drug targeting 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 includes administering to the subject a composition of the present invention, a bispecific molecule, an immunoconjugate such as an antibody drug conjugate, a CAR-T cell, or an oncolytic virus encoding an antibody or carrying an antibody, or a nucleic acid molecule or vector capable of expressing the same components in the subject. In some embodiments, at least one additional anti-cancer antibody may be administered together with an antibody of the present invention or its antigen-binding portion thereof, such as an anti-VISTA antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM 3 antibody, an anti-STAT3 antibody, and / or an anti-ROR1 antibody. In another embodiment, the antibody of the present invention or its antigen-binding portion thereof may be administered together with a cytokine (e.g., IL-2, IL-21, and / or GM-CSF) or a co-stimulatory antibody (e.g., anti-CD137 and / or anti-GITR antibody). The antibody of the present invention may be, for example, a mouse, a human, a chimeric or a humanized antibody.

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

[0026] In another aspect, the invention provides diagnostic methods, compositions and kits. In one embodiment, 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] Based on the following specific description and examples, other features and advantages of the present invention will become clear, and specific description and examples should not be interpreted as limiting. The contents of all documents, Genbank records, patents, and published patent applications cited in this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1C The binding abilities of mouse antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 and B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) to human IL4Rα are shown.

[0029] Figures 2A-2DThe binding ability of mouse antibodies B1D2F7D3B5 (A), B8G11F2B7G5E8 (B), B9D1D11F8D8 (C), C2C1A1A1, and C2B2F7B7 (D) to human IL4Rα on the cell surface is shown.

[0030] Figure 3 The binding ability of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8, B9D1D11F8D8, C2C1A1A1 and C2B2F7B7 to cynomolgus monkey IL4Rα is shown.

[0031] Figure 4A-4B Shown are the blocking abilities of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8 and B9D1D11F8D8 (A), C2C1A1A1 and C2B2F7B7 (B) on human IL4Rα-IL4 interaction.

[0032] Figure 5A-5B The blocking ability of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8 and B9D1D11F8D8 (A), C2C1A1A1 and C2B2F7B7 (B) on the binding of the reference article to human IL4Rα is shown.

[0033] Figures 6A-6C Shown are the blocking abilities of mouse antibodies B1D2F7D3B5 and B8G11F2B7G5E8 (A), B9D1D11F8D8 (B), C2C1A1A1 and C2B2F7B7 (C) on the interaction of human IL4 with human IL4Rα on the cell surface.

[0034] Figure 7 Shown are the inhibitory activities of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8, B9D1D11F8D8, C2C1A1A1, and C2B2F7B7 on IL4-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells.

[0035] Figure 8 Shown are the inhibitory activities of mouse antibodies B1D2F7D3B5, B8G11F2B7G5E8, B9D1D11F8D8, C2C1A1A1, and C2B2F7B7 on IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells.

[0036] Fig. 9 The chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 show their ability to bind to human IL4Rα.

[0037] Fig.10 The chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 show their ability to bind to human IL4Rα on the cell surface.

[0038] Fig.11 The blocking ability of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 on human IL4Rα-IL4 interaction is shown.

[0039] Fig.12 The inhibitory activity of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 on IL4-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells is shown.

[0040] Fig.13 The inhibitory activities of chimeric antibodies B8G11F2B7G5E8 and C2C1A1A1 on IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells are shown.

[0041] Figures 14A-14B The binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) to human IL4Rα is shown.

[0042] Figures 15A-15B The binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) to cynomolgus monkey IL4Rα is shown.

[0043] Figures 16A-16B The binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) to cal-IL4Rα is shown.

[0044] Figures 17A-17BThe binding ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) to human IL4Rα on the cell surface is shown.

[0045] Figures 18A-18B Shown are the ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) to block the interaction of human IL4 with 293F cells expressing human IL4Rα.

[0046] Figures 19A-19B Shown are the blocking abilities of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) on human IL4Rα-IL4 interaction.

[0047] Figures 20A-20B The blocking ability of humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4 and huB8G11F2B7G5E8-V14 (A), huC2C1A1A1-V14 and huC2C1A1A1-V15 (B) on the binding of the reference article to human IL4Rα is shown.

[0048] Fig.21 Shown are the inhibitory activities 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.

[0049] Fig. 22 Shown are the inhibitory activities 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.

[0050] The invention will best be understood from the following detailed description taken in conjunction with the accompanying drawings which are presented by way of example only and should not be construed as being limited to the particular embodiments described. DETAILED DESCRIPTION OF THE INVENTION

[0051] In order to better understand the present invention, some terms are first defined. Other definitions are listed throughout the detailed description.

[0052] The term "IL4Rα" refers to the interleukin 4 receptor alpha subunit. The term "IL4Rα" includes variants, subtypes, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific for human IL4Rα protein may cross-react with IL4Rα proteins of species other than humans (e.g., monkeys). In other embodiments, antibodies specific for human IL4Rα protein may be completely specific for human IL4Rα protein and may not cross-react with proteins of other species or other types, or may cross-react with IL4Rα derived from certain other species but not all other species.

[0053] The term "human IL4Rα" refers to an IL4Rα protein having a human amino acid sequence, such as the amino acid sequence of human IL4Rα with Genbank accession number NP_001244335.1. The terms "cynomolgus monkey IL4Rα" and "marmoset IL4Rα" refer to IL4Rα sequences, such as the amino acid sequences with Genbank accession numbers NOs. EHH60265.1 and NP_001244161.1, respectively.

[0054] The term "antibody" as used herein includes full-length antibodies and any antigen-binding fragments thereof (i.e., antigen-binding portions) or single chains thereof. Full-length antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely, C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V domains can also be divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by more conserved framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including a variety of immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0055] As used herein, the "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL4Rα protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed in the "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is composed of V L 、V H , C L and C H1 domain-consisting monovalent fragment; (ii) F(ab') 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) a fragment consisting of V H and C H1 (iv) an Fd fragment consisting of an antibody single-arm V L and V H Fv fragment composed of V H (vi) a separate complementarity determining region (CDR); and (vii) a nanobody, i.e., a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains V of the Fv fragment are L and V H Encoded by different genes, they can be recombined to form a single protein chain through a synthetic linker. L and V H The domains are paired to form monovalent molecules (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained by common techniques known to those skilled in the art, and can be functionally screened in the same manner as full-length antibodies.

[0056] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to an 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 a human IL4Rα protein may have cross-reactivity to other antigens, such as IL4Rα proteins of other species. In addition, the isolated antibody is substantially free of other cellular materials and / or chemicals.

[0057] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to an antibody molecule preparation of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0058] As used herein, the term "mouse antibody" refers to an antibody in which the framework region and CDR region in the variable region are both from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also from a mouse germline immunoglobulin sequence. The mouse antibody of the present invention may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo). However, the term "mouse antibody" used herein does not include antibodies in which CDR sequences from other mammalian species are inserted into mouse framework region sequences.

[0059] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally, a chimeric antibody refers to an antibody that combines genetic material from one species with genetic material from another species.

[0060] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to a natural human antibody.

[0061] The term "isotype" refers to the antibody class (eg, IgM or IgGl) encoded by the heavy chain constant region genes.

[0062] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0063] 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 of one or more non-human species) but does not substantially bind to non-IL4Rα proteins. Preferably, the antibody binds to human IL4Rα protein with "high affinity", i.e., with an affinity of 5.0× -8 M or less, preferably 1.0×10 -8 M or less, more preferably 7.0×10-9 M or smaller K D Binds to human IL4Rα protein.

[0064] As used herein, the term "substantially not binding" a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., with an affinity of 1.0×10 -6 M or more, preferably 1.0×10 -5 M or more, more preferably 1.0×10 -4 M or more, more preferably 1.0×10 -3 M or more, more preferably 1.0×10 -2 M or larger K D Binding proteins or cells.

[0065] The term "high affinity" for IgG antibodies means that the antibody has a binding affinity of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or less, more preferably 7.0×10 -9 M or less, more preferably 1.0×10 -9 M or smaller K D . But for other antibody isotypes, “high affinity” binding may be different. For example, for the IgM isotype, “high affinity” means that the antibody has a 1.0×10 -6 M or less, preferably 1.0×10 -7 M or less, more preferably 1.0×10 -8 M or smaller K D .

[0066] The term "K assoc ” or “K a " refers to the association rate of a specific antibody-antigen interaction, and the term "K" as used herein dis ” or “K d " refers to the dissociation rate of a specific antibody-antigen interaction. The term "K" as used herein D ” refers to the dissociation constant, which is K d With K a The ratio (K d / K a ) and expressed as molar concentration (M). D The value can be determined using methods well known in the art. D A preferred method of quantifying the value is by using surface plasmon resonance, preferably using a biosensor system such as Biacore TM The system is measured.

[0067] The term “EC 50 ”, also known as the half-maximal effect concentration, is the antibody concentration that induces a response halfway between baseline and maximum after a specific exposure time.

[0068] The term "IC 50 ", also known as the half-maximal inhibitory concentration, refers to the antibody concentration that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0069] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles; preferably mammals, such as non-human primates, sheep, dogs, cats, cows and horses.

[0070] The term "therapeutically effective amount" refers to an amount of an antibody of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer), and / or to reduce the severity of a disease or condition. It should be understood that the therapeutically effective amount is related to the disease being treated, wherein those skilled in the art can easily discern the actual effective amount.

[0071] The term "identity" used in the present invention refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. The sequence comparison and identity percentage between two sequences can be measured by the default settings of the BLASTN / BLASTP algorithm on the website of the National Center for Biotechnology Information of the United States.

[0072] Various aspects of the invention are described in more detail below.

[0073] Anti-IL4Rα antibodies have stronger binding affinity to human IL4Rα and stronger blocking ability on IL4 / IL13 signaling force

[0074] The antibodies or antigen-binding portions thereof of the present invention specifically bind to human IL4Rα with comparable (if not better) binding affinity / ability than reported anti-IL4Rα antibodies (such as Dupilumab).

[0075] The antibodies or antigen-binding portions thereof of the present invention can block the binding of IL4Rα to IL4 or IL13-IL13Rα1, thereby blocking the corresponding intracellular signal transduction, and have comparable or higher blocking activity than reported anti-IL4Rα antibodies (such as Dupilumab).

[0076] Preferably, the antibody of the present invention is a humanized monoclonal antibody. In addition, or alternatively, the antibody of the present invention may be, for example, a chimeric monoclonal antibody.

[0077] Anti-IL4Rα monoclonal antibody

[0078] The antibodies of the present invention are monoclonal antibodies whose structures and chemical properties are described below and in the Examples. The amino acid sequence IDs of the heavy / light chain variable regions of the antibodies are summarized in Table 1 below. 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 an amino acid sequence as shown in SEQ ID NO:55, and the light chain constant region of the antibody may be a human kappa constant region having an amino acid sequence as shown in SEQ ID NO:56. Table 1. Amino acid sequence IDs of heavy / light chain variable regions

[0079] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, as is well known in the art, the CDR region can also be determined based on the heavy / light chain variable region sequence by other numbering systems such as Chothia, IMGT, AbM or Contact numbering systems / methods.

[0080] V of other anti-IL4Rα antibodies binding to human IL4Rα H and V L The sequence (or CDR sequence) may be the same as the V sequence of the anti-IL4Rα antibody of the present invention. H and V L Sequences (or CDR sequences) are "mixed and paired". Preferably, when V H and V L When chains (or CDRs within those chains) are mixed and paired, H / V L Centered V H The sequence is structurally similar to V H Likewise, it is preferred to replace the specific V H / V L Centered V L Sequence replaced with a structurally similar V L sequence.

[0081] Thus, in one embodiment, an antibody or antigen binding portion thereof of the invention comprises: (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1; and (b) a light chain variable region comprising the amino acid sequence listed in Table 1, or a V region of another anti-IL4Rα antibody L , wherein the antibody specifically binds to human IL4Rα.

[0082] 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 the light chain variable region listed in Table 1, or CDRs of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα.

[0083] In another embodiment, an antibody of the invention or an antigen-binding portion thereof comprises CDR2 of the heavy chain variable region of an anti-IL4Rα antibody and CDRs of other antibodies that bind to human IL4Rα, such as 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.

[0084] Furthermore, it is well known in the art that, independent of the CDR1 and / or CDR2 domains, the CDR3 domain alone can determine the binding specificity of an antibody to a cognate antigen, and that multiple antibodies with the same binding specificity can be predictively generated based on a common CDR3 sequence. See, eg, Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Rader et al. al.,, Proc.Natl.Acad.Sci.USA95:8910-8915 (1998); Barbas et al.,, J.Am.Chem.Soc.116:2161-2162 (1994); Barbas et al.,, Proc.Natl.Acad.Sci.USA92:2529-2533 (1995); Ditzel et al., al.,,J.Immunol.157:739-749(1996);Berezov et al.,,BIAjournal 8:Scientific Review 8(2001); Igarashi et al.,,J.Biochem(Tokyo)117:452-7(1995);Bourgeois et al.,,J.Virol 72:807-10(1998);Levi et al.,,Proc.Natl.Acad.Sci.USA90:4374-8(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; 5,762,905 and 5,760,185. Each of these references is incorporated herein by reference in its entirety.

[0085] Therefore, in another embodiment, the antibody of the present invention comprises the CDR2 of the heavy chain variable region of the anti-IL4Rα antibody and at least the CDR3 of the heavy chain variable region and / or the light chain variable region of the anti-IL4Rα antibody, or the CDR3 of the heavy chain variable region and / or the light chain variable region of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα. These antibodies preferably 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 the anti-IL4Rα antibody, or the CDR2 of the light chain variable region of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα. In another embodiment, the antibody of the present invention may further include CDR1 of the heavy chain variable region and / or light chain variable region of an anti-IL4Rα antibody, or CDR1 of the heavy chain variable region and / or light chain variable region of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα.

[0086] Conservative modification

[0087] In another embodiment, the antibody of the present invention comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 sequences are different from the CDR1, CDR2 and CDR3 sequences of the anti-IL4Rα antibody of the present invention, and the difference is due to one or more conservative modifications. It should be understood in the art that certain conservative sequence modifications will not eliminate antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al. al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10: 341-6 and Beers et al. al., (2000) Clin. Can. Res. 6:2835-43.

[0088] Therefore, in one embodiment, an antibody of the invention comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise CDR1, CDR2 and CDR3, respectively, wherein: (a) CDR1 of the heavy chain variable region comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or (b) CDR2 of the heavy chain variable region comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or (c) the CDR3 of the heavy chain variable region comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or (d) CDR1, and / or CDR2, and / or CDR3 of the light chain variable region comprise the sequences listed in Table 1, and / or conservative modifications thereof; and (e) The antibody specifically binds to human IL4Rα.

[0089] The antibodies of the present invention have one or more of the above-mentioned functional properties, such as high affinity for human IL4Rα and blocking activity against IL4Rα-IL4 binding or IL4Rα-IL13-IL13Rα1 binding.

[0090] In various embodiments, the antibody can be, for example, a mouse, human, humanized, or chimeric antibody.

[0091] As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the antibody binding properties. Such conservative modifications include amino acid replacements, additions and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacement refers to replacing amino acid residues with amino acid residues with similar side chains. Amino acid residue groups with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of an antibody of the invention can be replaced with other amino acid residues from the same side chain group, and the resulting antibody can be tested for retention of function (i.e., the function described above) using the functional assays described herein.

[0092] Engineered and modified antibodies

[0093] The antibodies of the present invention can be used to comprise one or more V of the anti-IL4Rα antibodies of the present invention. H / V LThe antibody sequence is used as the starting material to engineer the modified antibody. The antibody can be modified by modifying one or two variable regions (i.e., V H and / or V L ) (e.g., in one or more CDR regions and / or one or more framework regions) to carry out engineering modifications. In addition, or, the residues in the constant region can be modified, for example, to change the effector function of the antibody.

[0094] In certain embodiments, CDR transplantation can be used to modify the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six complementary determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are more diverse between various antibodies than the amino acid sequences outside the CDRs. Because CDR sequences are responsible for major antibody-antigen interactions, recombinant antibodies that mimic the properties of specific natural antibodies can be expressed by constructing expression vectors in which the CDR sequences of a specific natural antibody are transplanted into the framework region sequences of another antibody with different properties (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. See also USA 86:10029-10033; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0095] Therefore, another embodiment of the present invention relates to an isolated monoclonal antibody or an antigen-binding portion thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the above-mentioned CDR1, CDR2 and CDR3 of the present invention, and the light chain variable region comprises the above-mentioned CDR1, CDR2 and CDR3 of the present invention. Although these antibodies comprise the monoclonal antibody V H and V L CDR sequences, which may contain different framework sequences.

[0096] Such a framework sequence can be obtained from a public DNA database or published reference that includes germline antibody gene sequences. For example, the germline DNA sequences of human heavy chain variable region genes and human light chain variable region genes can be obtained in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat et al., (1991), supra; Tomlinson et al., (1992) J. Mol. Biol. 227: 776-798; and Cox et al., (1994) Eur. J. Immunol. 24: 827-836, the contents of which are expressly incorporated herein by reference. In another embodiment, the germline DNA sequences of human heavy chain variable region genes and human light chain variable region genes can be obtained in the Genbank database. For example, the following heavy chain germline sequences from the HCo7 HuMAb mouse have Genbank accession numbers: 1-69 (NG--0010109, NT--024637 & BC070333), 3-33 (NG--0010109 & NT--024637), and 3-7 (NG--0010109 & NT--024637). In another embodiment, the following Genbank accession numbers of heavy chain germline sequences from HCo12 HuMAb mice are: 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).

[0097] The antibody protein sequence was compared with the compiled protein sequence database using Gapped BLAST (Altschul et al., (1997) supra), one of the sequence similarity search methods well known to those skilled in the art.

[0098] The antibody framework sequences used in the present invention are preferably those that are structurally similar to the antibody framework sequences of the present invention. H The CDR1, CDR2 and CDR3 sequences of the present invention can be transplanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequence can be transplanted into a framework region comprising one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues in the framework region to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0099] Another type of variable region modification is to modify V H and / or V L The amino acid residues in the CDR1, CDR2 and / or CDR3 regions of the target antibody are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Mutations can be introduced by point mutations or PCR-mediated mutations, and the effects of mutations on antibody binding or other functional properties can be evaluated by in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, no more than one, two, three, four or five residues in a CDR region are usually changed.

[0100] Thus, in another embodiment, the present invention provides an isolated anti-IL4Rα monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, comprising: (a) V H The CDR1 region comprises the V H CDR1 sequence, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) V H The CDR2 region comprises the V H CDR2 sequence, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (c) V H The CDR3 region comprises the V H CDR3 sequence, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (d) V L The CDR1 region comprises the V L CDR1 sequence, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) V L The CDR2 region comprises the V L CDR2 sequence, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; and (f) V L The CDR3 region comprises the V L CDR3 sequence, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions.

[0101] The engineered antibodies of the present invention include V H and / or V LIn some embodiments, the present invention relates to antibodies that have been modified in the framework region residues of the antibody to improve the antibody properties. Generally speaking, such framework region modifications can be used to reduce the immunogenicity of the antibody. For example, one or more framework region residues are "back mutated" to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody was derived. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody was derived.

[0102] Another type of framework region modification includes mutating one or more residues in the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the immunogenicity that the antibody may cause. This method is also called "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043.

[0103] In addition, or as another option outside of the modification of the framework region or CDR region, the antibody of the present invention can be subjected to Fc modification, which is generally used to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cellular toxicity. In addition, the antibody of the present invention can be chemically modified (for example, one or more chemical functional groups can be attached to the antibody), or modified to change its glycosylation, to change one or more functional properties of the antibody.

[0104] In one embodiment, C H1 -Hinge region modification, thereby changing (e.g., increasing or decreasing) the number of cysteine ​​residues in the hinge region. This method is described in detail in U.S. Patent No. 5,677,425. Changing C H1 - The number of cysteine ​​residues in the hinge region, which can, for example, facilitate the assembly of the heavy and light chains, or increase / decrease the stability of the antibody.

[0105] In another embodiment, the Fc-hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 The domain interface region results in an antibody with reduced Staphylococcus protein A (SpA) binding relative to the native Fc-hinge domain. This approach is described in more detail in U.S. Patent No. 6,165,745.

[0106] In another embodiment, the glycosylation of the antibody is altered. For example, a deglycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. Altering glycosylation can, for example, increase the affinity of the antibody for the antigen. Such glycosylation modification can be achieved, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate a glycosylation site in the framework region of one or more variable regions, thereby eliminating glycosylation at the site. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0107] In addition, or, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucose residues, or antibodies with increased bisecting GlcNac structures. It has been shown that changes in this glycosylation form can increase or decrease the ADCC activity of the antibody. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms are known in the art and can be used as host cells for expressing the recombinant antibodies of the present invention to prepare antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase gene), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose in their sugars. Ms704, Ms705 and Ms709 FUT8- / - cell lines were prepared by using two replacement vectors to target the disruption of the FUT8 gene in CHO / DG44 cells (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87: 614-22). As another example, EP1,176,195 describes a cell line in which the function of the FUT8 gene is disrupted, which encodes a fucosyltransferase, so that the antibody expressed in the cell line exhibits low fucosylation due to the reduction or elimination of the enzyme related to the α-1,6 bond. EP1,176,195 also describes a cell line that has low or lacks the enzyme activity of adding fucose to N-acetylglucosamine that binds to the Fc region of an antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO03 / 035835 describes a CHO variant cell line, Lec13 cells, which have a reduced ability to add fucose to Asn(297)-linked sugars, resulting in hypofucosylated antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with altered glycosylation profiles can also be produced in eggs, as described in WO06 / 089231. Alternatively, antibodies with altered glycosylation profiles can be produced in plant cells, such as Lemna. Methods for producing antibodies in plant systems are disclosed in U.S. patent application corresponding to Alston & Bird LLP attorney docket number 040989 / 314911 filed on August 11, 2006. Fucosidase can be used to cleave the fucose residues of the antibody, for example, α-L-fucosidase is used to remove the fucose residues of the antibody (Tarentino et al., (1975) Biochem. 14:5516-23).

[0108] Another modification of the antibodies of the present invention is polyethylene glycolization (PEGylation). The antibody is PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate the antibody, the antibody or its fragment is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derive other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the PEGylated antibody is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be used for the antibodies of the present invention. See, for example, EP0154316 and EP0401384.

[0109] Physical properties of antibodies

[0110] The antibodies of the invention can be characterized by their various physical properties in order to detect and / or distinguish their species.

[0111] For example, an antibody may contain one or more glycosylation sites in the light chain variable region or the heavy chain variable region. These glycosylation sites may lead to an increase in antibody immunogenicity or a change in the antibody pK value due to changes in antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al (1985) Nature 316: 452-7; Mimura et al., (2000) Mol Immunol 37: 697-706). It is known that glycosylation occurs in a motif containing an NXS / T sequence. In some cases, it is preferred that the variable region of the anti-IL4Rα antibody does not contain glycosylation. This can be achieved by selecting an antibody whose variable region does not contain a glycosylation motif or by mutating residues in the glycosylation region.

[0112] In a preferred embodiment, the antibody does not contain an asparagine isomerization site. Deamidation of asparagine may occur in NG or DG sequences and result in the generation of an isoaspartic acid residue, which introduces a kink into the polypeptide backbone and reduces its stability (isoaspartate effect).

[0113] Each antibody has a unique isoelectric point (pI), which generally falls within the pH range of 6-9.5. The pI of IgG1 antibodies generally falls within the pH range of 7-9.5, while the pI of IgG4 antibodies generally falls within the pH range of 6-8. It is speculated that antibodies with pI outside the normal range may undergo some unfolding and instability in vivo. Therefore, anti-IL4Rα antibodies with pI values ​​falling within the normal range are preferred. This can be achieved by selecting antibodies with pI values ​​within the normal range or by mutating charged surface residues.

[0114] Nucleic acid molecules encoding the antibodies of the present invention

[0115] On the other hand, the present invention provides nucleic acid molecules encoding the heavy chain variable region and / or light chain variable region or CDR of the antibody of the present invention. The nucleic acid can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. After the nucleic acid is purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins by standard techniques, the nucleic acid is "isolated" or "substantially pure". The nucleic acid of the present invention can be DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0116] The nucleic acid of the present invention can be obtained by standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes as described below), cDNAs encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from gene libraries.

[0117] Preferably, the nucleic acid molecule of the present invention comprises a V encoding an IL4Rα monoclonal antibody. H and V L Once the sequences or CDRs encoding V H and V L These DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. H or V L A DNA fragment encoding a protein is operably linked to a DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" used in this case means that the two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments are in the reading frame.

[0118] The code VH DNA encoding the heavy chain constant region (C H1 , C H2 and C H3 ) can be operably linked to another DNA molecule encoding V H The isolated DNA of the heavy chain region is converted into a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art, and a DNA fragment comprising the human heavy chain constant region gene can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, the V H The DNA of the heavy chain C H1 Another DNA molecule with a constant region.

[0119] The code V L DNA encoding the light chain constant region C L Another DNA molecule can be operably linked to the V L The isolated DNA of the region is converted into a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art, and a DNA fragment comprising the human light chain constant region gene can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region is a kappa or lambda constant region.

[0120] To prepare scFv genes, the V H and V L The DNA fragment of V is operably linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3, so that V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The regions are connected by this flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0121] Preparation of monoclonal antibodies of the present invention

[0122] The monoclonal antibodies (mAbs) of the present invention can be prepared using the somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495, which is well known in the art. Other embodiments for preparing 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. See, for example, U.S. Patent Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the entire contents of which are expressly incorporated herein by reference.

[0123] Generation of transfectomas for producing monoclonal antibodies of the invention

[0124] The antibodies of the present invention can also be prepared in a host cell transfectoma using, for example, recombinant DNA technology well known in the art in combination with gene transfection methods (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors so that the genes are operably linked to transcription and translation regulatory sequences. In this case, the term "operably linked" means that the antibody gene is linked to the vector so that the transcription and translation regulatory sequences in the vector perform their established functions of regulating antibody gene transcription and translation.

[0125] The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (such as adenovirus major late promoter (AdMLP)) and polyoma virus. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters may be used. In addition, the regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat sequence of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472). The expression vector and expression regulatory sequence compatible with the expression host cell used are selected.

[0126] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is inserted into an expression vector that already encodes the heavy chain constant region and the light chain constant region of the desired isotype to construct a full-length antibody gene, so that V H C H Operably connected, V L C L Operably connected. In addition, or, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0127] In order to express the light chain and the heavy chain, the expression vectors encoding the heavy chain and the light chain are transfected into the host cell by standard techniques. Different forms of "transfection" include a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is theoretically feasible to express the antibodies of the present invention in prokaryotic or eukaryotic host cells, the antibodies are preferably expressed in eukaryotic cells, most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely to assemble antibodies and secrete properly folded and immunologically active antibodies than prokaryotic cells.

[0128] Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells used with the DHFR selectable marker described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, and the DHFR selectable marker described, for example, in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. Particularly when NSO myeloma cells are used, another preferred expression system is the GS gene expression system described in WO87 / 04462, WO89 / 01036, and EP338,841. After the recombinant expression vector encoding the antibody gene is introduced into the mammalian host cells, the antibody is prepared by culturing the host cells for a period of time sufficient to allow the antibody to be expressed in the host cells, or preferably sufficient to allow the antibody to be secreted into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0129] Bispecific molecules

[0130] On the other hand, the present invention provides a bispecific molecule comprising one or more antibodies of the present invention, which are linked to at least one other functional molecule such as another peptide or protein (e.g., another antibody or receptor ligand) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Therefore, the "bispecific molecule" used in the present invention includes molecules with three or more specificities.

[0131] In one embodiment, the bispecific molecule has a third specificity in addition to the Fc binding specificity and the IL4Rα binding specificity.

[0132] Bispecific molecules can appear in a variety of forms 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 of the size spectrum, bispecific molecules are composed of two single-chain antibody fragments (scFv) connected by peptide chains, the so-called Bs(scFv)2 construct. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic modification, somatic cell hybridization or chemical methods. See, for example, See, eg, Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), and the references cited therein.

[0133] Immunoconjugates

[0134] The antibody of the present invention can be coupled with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADC, the antibody and therapeutic agent are preferably coupled by a cleavable linker, such as a peptide linker, a disulfide linker or a hydrazone linker. A preferred linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02 / 096910; WO07 / 038,658; WO07 / 051,081; WO07 / 059,404; WO08 / 083,312; and WO08 / 103,693; U.S. Patent Publication Nos. 20060024317; 20060004081; and 20060247295, the disclosures of which are incorporated herein by reference.

[0135] Oncolytic viruses encoding or carrying antibodies

[0136] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the present invention can be used together with oncolytic viruses. Alternatively, an oncolytic virus encoding the antibodies of the present invention can be introduced into the human body.

[0137] Chimeric Antigen Receptor

[0138] The present invention also provides a chimeric antigen receptor (CAR) comprising an anti-IL4Rα scFv, wherein the anti-IL4Rα scFv comprises the CDR and heavy / light chain variable regions of the present invention.

[0139] 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.

[0140] CAR can include a signal peptide at the N-terminus of the extracellular antigen binding domain to guide the nascent receptor to enter the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen binding domain that makes the receptor easier to bind. Preferably, the intracellular signaling domain of CAR includes a primary intracellular signaling domain and one or more costimulatory signaling domains. The most commonly used and most effective primary intracellular signaling domain is the CD3-zeta cytoplasmic domain, which includes ITAMs, and phosphorylation of ITAMs leads to T cell activation. The costimulatory signaling domain can be derived from costimulatory proteins, such as CD28, CD137, and OX40.

[0141] CARs can be further supplemented with elements that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines and co-stimulatory ligands.

[0142] The present invention also provides engineered immune effector cells, which include CAR provided by the present invention. In some embodiments, immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells or embryonic stem cells. In some embodiments, immune effector cells are T cells.

[0143] Pharmaceutical composition

[0144] On the other hand, the present invention provides a pharmaceutical composition comprising one or more antibodies (or antigen-binding portions of antibodies, bispecific molecules, CAR-T cells, oncolytic viruses or immunoconjugates) of the present invention formulated with a pharmaceutically acceptable carrier. When the composition comprises more than one antibody (or antigen-binding portion of an antibody, bispecific molecules, CAR-T cells, oncolytic viruses or immunoconjugates), the antibodies (or antigen-binding portions of antibodies, bispecific molecules, CAR-T cells, oncolytic viruses or immunoconjugates) may be administered separately. The pharmaceutical composition may optionally include one or more other pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug or an antiallergic agent.

[0145] The pharmaceutical composition may include any number of excipients. Available excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersions or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0146] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient can be coated in a material to protect it from acid and other natural conditions that may inactivate it. The phrase "parenteral administration" used in the present invention refers to a mode of administration other than enteral and topical administration, typically using injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibodies of the present invention can be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual or topical administration.

[0147] The pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0148] The amount of active ingredient prepared into a single dose together with the carrier material varies according to the subject to be treated and the specific mode of administration, and is generally the amount of the composition that produces a therapeutic effect. Usually, in terms of percentage, the amount is about 0.01% to about 99%, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0149] The dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the severity of the therapeutic situation. It is particularly advantageous to formulate parenteral 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 for a single dose of a subject to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect together with the desired pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained release formulation, in which case the required frequency of administration is reduced.

[0150] For administration of the composition, the dosage may be about 0.0001 to 100 mg / kg of the host body weight, more typically 0.01 to 5 mg / kg of the host body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight, or within the range of 1-10 mg / kg body weight. Exemplary treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. A preferred dosage regimen for the anti-IL4Rα antibody of the present invention includes intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight, the administration of which uses one of the following dosing regimens: (i) once every four weeks for a total of 6 times, then once every three months; (ii) once every three weeks; (iii) 3 mg / kg body weight once, then 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, about 25-300 μg / ml.

[0151] A "therapeutically effective amount" of the anti-IL4Rα antibody of the present invention, or its antigen-binding portion, bispecific molecule, CAR-T cell, oncolytic virus or immunoconjugate, preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of the disease's asymptomatic period, or prevents damage or disability caused by the disease. For example, for the treatment of tumor subjects, a "therapeutically effective amount" is preferably an inhibition of at least about 20%, preferably an inhibition of at least about 40%, more preferably an inhibition of at least about 60%, and more preferably an inhibition of at least about 80% of tumor growth compared to untreated subjects. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or alleviate symptoms in a subject, who is typically a human or other mammal.

[0152] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0153] The pharmaceutical composition can be administered by the following medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal delivery devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0154] In certain embodiments, the monoclonal antibodies of the present invention can be formulated to ensure appropriate in vivo distribution. For example, to ensure that the therapeutic antibodies of the present invention cross the blood-brain barrier, the antibodies can be formulated in liposomes, and the liposomes containing the antibodies can also additionally contain a group with a targeting function to enhance the selective delivery to specific cells or organs. See, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; VV Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090; Keinanen et al., (1994) J. Biol. Chem. 269:9090; and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0155] Uses and methods of the invention

[0156] The compositions of the invention comprising antibodies or antigen-binding portions thereof, bispecific molecules, CAR-T cells, oncolytic viruses, or immunoconjugates have a variety of in vitro and in vivo uses, involving, for example, the treatment of allergic diseases associated with excessive IL4 and / or IL13 signaling.

[0157] In view of the ability of the anti-IL4Rα antibody of the present invention to block the binding of IL4Rα to IL4 or IL13-IL13Rα1 to reduce type 2 immunity, the present invention provides a method for treating type 2 immunity-related allergic diseases, 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.

[0158] On the other hand, since IL4 or IL13 signaling can activate STAT6, and it has been found that STAT6 inhibitors can inhibit cancer cell growth, the present invention provides a method for inhibiting the growth of tumor cells in a subject, the method comprising administering a composition of the present invention to the subject, thereby inhibiting the growth of the tumor in the subject. Non-limiting examples of tumors that can be treated by the antibodies of the present invention include, but are not limited to, melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, and urothelial cancer.

[0159] On the other hand, the present invention provides a method for reducing or inhibiting cell activation in response to IL-4 or IL-13. In some embodiments, inhibiting activation includes inhibiting the production or secretion of cytokines. In some embodiments, inhibiting activation includes inhibiting proliferation. Cells that respond to IL-4 by activating the hybrid IL-4Rα / γC receptor include, but are not limited to, B cells, eosinophils, and macrophages. Cells that respond to IL-13 by activating the hybrid IL-4Rα / IL-3Rα1 receptor include, but are not limited to, fibroblasts and smooth muscle cells. Therefore, in one embodiment, 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.

[0160] On the other hand, the present invention provides diagnostic methods, compositions and kits. In one embodiment, the antibodies of the present invention are used to determine the presence and expression of IL4Rα in cells or tissues. In one embodiment, the diagnosis can indicate prognosis and / or guide treatment and / or follow-up. For example, it has been found that overexpression of IL4Rα in human bladder cancer is associated with the pathological grade and stage of the disease. In one embodiment, the antibodies of the present invention are used to diagnose the grade and stage of bladder cancer. It has been found that high expression of IL-4Rα is associated with an increase in the occurrence or recurrence of oral cancer. In one embodiment, the antibodies of the present invention can be used in oral cancer diagnostic kits or diagnostic methods to determine prognosis and appropriate treatment and follow-up. Tumor IL-4Rα expression is negatively correlated with the survival rate of patients with surgical resection of epithelial malignant pleural mesothelioma (MPM). In one embodiment, the antibodies of the present invention can be used in diagnostic kits or diagnostic methods to determine the prognosis of MPM and appropriate treatment and / or follow-up.

[0161] Combination therapy

[0162] In one aspect, the present invention provides a combination therapy, wherein the combination therapy is to co-administer the anti-IL4Rα antibody or antigen-binding portion thereof, bispecific molecule or oncolytic virus of the present invention with one or more other drugs that can effectively alleviate type 2 immune-related allergic diseases. The drug can be an antihistamine (targeting H 1 Histamine receptors), or corticosteroids, beta-adrenergic receptor agonists and drugs targeting cyc-LTs used clinically for asthma treatment. Omalizumab, an anti-IgE antibody, can also be used in the treatment of allergic diseases with the antibodies or antigen-binding portions thereof, bispecific molecules or oncolytic viruses of the present invention. In certain embodiments, the subject is a human.

[0163] On the other hand, the present invention provides a method of combined therapy, the method is to co-administer the anti-IL4Rα antibody of the present invention or its antigen binding portion, bispecific molecule, CAR-T cell, oncolytic virus or immunoconjugate with one or more other antibodies that can effectively inhibit tumor growth in a subject. In one embodiment, the present invention provides a method of inhibiting tumor growth in a subject, comprising administering an anti-IL4Rα antibody (or its antigen binding portion, bispecific molecule, oncolytic virus, CAR-T cell or immunoconjugate) and one or more other antibodies, such as anti-OX40 antibodies, anti-TIM-3 antibodies, anti-CD137 antibodies, anti-GITR antibodies, anti-LAG-3 antibodies, anti-PD-L1 antibodies and anti-PD-1 antibodies to the subject. In certain embodiments, the subject is human. IL4Rα pathway inhibitors can also be further combined with standard treatments for cancer. For example, IL4Rα pathway inhibitors can be combined with LAG-3 and / or PD-1 inhibitors and chemotherapy regimens. For example, a chemotherapeutic drug can be administered together with an anti-IL4Rα antibody, and the chemotherapeutic drug can be a cytotoxic drug. For example, epirubicin, oxaliplatin and 5-fluorouracil can be administered to patients who are receiving anti-IL4Rα treatment. Optionally, the combination of anti-IL4Rα and one or more other antibodies (e.g., anti-LAG-3 and / or anti-PD-1 antibodies) can be further combined with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and sugar molecules) and cells transfected with genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol. 173: 4919-28). Non-limiting examples of available tumor vaccines include melanoma antigen peptides such as gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. Other therapies that can be combined with anti-IL4Rα antibodies include, but are not limited to, administration of interleukin 2 (IL-2), radiation, surgery or hormone blockade.

[0164] The combination or combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, each of which is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0165] Furthermore, if multiple administrations of the combination therapy are performed sequentially, the order of sequential administration at each time point may be reversed or remain the same, sequential administration may be combined with simultaneous administration or any combination thereof.

[0166] The present invention is further described by the following examples, which should not be interpreted as limiting. All drawings of the present application and all references, Genebank sequences, patents and published patent applications cited in the present application are expressly incorporated herein by reference. Example

[0168] Example 1: Production of mouse anti-IL4Rα monoclonal antibodies using hybridoma technology

[0169] Immunization

[0170] Mice were immunized according to the method described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. Recombinant human IL4Rα-his protein (Sino biological inc., product catalog 10402-H08H) was used as an immunogen, and the in-house prepared human IL4Rα-his protein (amino acid sequence as shown in SEQ ID NO: 57) was used to determine the titer of antiserum and screen hybridomas secreting antigen-specific antibodies. The immunization dose for the primary immunization and booster immunization was 20 μg of human IL4Rα-his protein per mouse per injection. In order to increase the immune response, complete Freund's adjuvant and incomplete Freund's adjuvant (Sigma, St. Louis, Mo., USA) were used for the primary immunization and booster immunization, respectively. Briefly, the adjuvant-immunogen mixture was prepared as follows: first, the adjuvant was gently mixed in a vial by vortexing; the required amount of adjuvant was transferred to an autoclaved 1.5 mL microcentrifuge tube; the immunogen was diluted with PBS or saline to a concentration range of 0.2-0.3 mg / mL; then the calculated amount of immunogen was added to the microcentrifuge tube together with the adjuvant and gently vortexed for 2 minutes to allow the mixture to form an oil-in-water emulsion. The adjuvant-immunogen emulsion was aspirated with a suitable syringe for animal injection. A total of 20 μg of immunogen was injected in a volume of 150-200 μL. After each animal was immunized, it was boosted 2 to 3 times according to the antiserum titer. Before cell fusion, the animals with better titers were given a final booster immunization by intraperitoneal injection.

[0171] Hybridoma fusion and screening

[0172] Prior to cell fusion, cells of the mouse myeloma cell line (SP2 / 0-Ag14, ATCC#CRL-1581) were cultured to the logarithmic phase. According to the method described in Kohler G, and Milstein C, "Continuous cultures of fused cellssecreting antibody of predefined specificity," Nature, 256:495-497 (1975), spleen cells from immunized mice were aseptically removed and fused with myeloma cells. The fused "hybrid cells" in DMEM / 20% FCS / HAT medium were then added to a 96-well plate. Seven to ten days after fusion, surviving hybridoma cell colonies were observed under a microscope. Two weeks later, the supernatant of each well was tested by ELISA using recombinant human IL4Rα-his protein. In brief, human IL4Rα-his protein (2.0 μg / mL) dissolved in PBS was coated on an ELISA plate at 60 μL / well and incubated overnight at 4°C. The plate was washed 4 times with PBST, and then 200 μL of blocking buffer (PBST containing 5% w / v skim milk) was added to each well for blocking. 60 μL of diluted hybridoma supernatant was added to each well and incubated at 37°C for 40 minutes. The plate was then washed 4 times, detected using HRP-goat anti-mouse IgG (Jackson Immuno research, product catalog 115-036-071), and the OD value of binding was detected at 450nm. Positive hybridomas that secrete antibodies that bind to human IL4Rα-his protein were screened and transferred to 24-well plates. Hybridoma clones that produce antibodies with high specific binding activity to IL4Rα and IL4Rα-IL4 or IL4Rα-13Rα1-IL13 blocking activity were subcloned by limiting dilution to ensure the clonality of the cell line, and then the monoclonal antibodies were purified. In brief, protein A agarose column (bestchrom (Shanghai) Biosciences, product catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. Cell supernatant was passed through the protein A agarose column, and then the column was washed with PBS buffer until the absorbance of the protein reached baseline. The column was eluted with elution buffer (0.1M glycine-HCl, pH 2.7), and the eluate was immediately collected in a 1.5mL tube and neutralized with neutralization buffer (1M Tris-HCl, pH 9.0). The immunoglobulin-containing parts were merged and dialyzed overnight at 4°C in PBS. Subsequently, the in vitro functional activity of the purified monoclonal antibody was characterized according to the method described below.

[0173] Example 2: Affinity measurement of mouse anti-IL4Rα monoclonal antibody using BIACORE surface plasmon resonance Certainly

[0174] The binding affinity and binding kinetics of the anti-IL4Rα mouse monoclonal antibody (mAb) purified in Example 1 were characterized by Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

[0175] Briefly, goat anti-mouse IgG (GE healthcare, catalog BR100838, MouseAntibody Capture Kit) was covalently linked to a CM5 chip (carboxymethylated dextran coated chip) via primary amine groups using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA). Unreacted portions on the biosensor surface were blocked with ethanolamine. Then, the purified anti-IL4Rα antibody of the present invention at a concentration of 66.67 nM and the anti-IL4Rα antibody reference ( Also referred to as BM) flowed through the chip at a flow rate of 10 μL / min. Then, recombinant human IL4Rα-his (prepared in-house, the amino acid sequence is shown in SEQ ID NO: 57), cynomolgus monkey IL4Rα-his protein (Sino biological inc., product catalog 90897-C08H), or marmoset IL4Rα-his protein (Sino biological inc. custom product, also referred to as cal-IL4Rα-his, the amino acid sequence is shown in SEQ ID NO: 58) diluted in a gradient manner with HBS EP buffer (provided by Biacore) flowed through the chip at a flow rate of 30 μL / min. The antigen-antibody binding kinetics were tracked for 2 minutes, and the dissociation kinetics were tracked for 10 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIA evaluation software. Determination of K D , K a and K d The values ​​are summarized in Table 2 below. Table 2. Binding affinity of mouse anti-IL4Rα antibodies *Not tested.

[0176] All mouse antibodies of the present invention specifically bind to human IL4Rα, and most of them have comparable or higher binding affinity than the reference product.

[0177] Example 3: Binding activity of mouse anti-IL4Rα monoclonal antibody to IL4Rα

[0178] 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.

[0179] 3.1 Capture ELISA

[0180] Briefly, goat anti-mouse IgG (Fcγ fragment specific) (Jackson Immuno Research, catalog 115-005-008) at a concentration of 2 μg / mL in PBS was coated in 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 at 37°C for 2 hours. The plate was washed again, and 100 μL of serial dilutions (starting at 66.7 nM, 5-fold serial dilutions in PBST containing 2.5% w / v skim milk) of the anti-IL4Rα antibody of the present invention, reference or negative control hIgG (human immunoglobulin for intravenous injection (pH 4, Hualan Biological Engineering Inc.) was added to each well, incubated at 37°C for 40 minutes, and then the plate was washed 4 times. 100 μL / well of biotinylated human IL4Rα-his protein (SEQ ID NO: 57, prepared in-house, dissolved in PBST containing 2.5% w / v skim milk, concentration of 0.14 nM) was added to the 96-well plate containing the capture anti-IL4Rα antibody and incubated at 37°C for 40 minutes. The plate was washed 4 times, and then 100 μL / well of HRP-labeled streptavidin (diluted 1:10000 in PBST, Jackson ImmunoResearch, catalog 016-030-084) was added and incubated at 37°C for 40 minutes. After the last wash, 100 μL / well of ELISA substrate TMB (Innoreagents, catalog TMB-S-002) was added for incubation. After 10 minutes, 50 μL / well of 1M H 2 SO 4 The reaction was terminated and the absorbance was read at 450 nm. The data were analyzed using Graphpad Prism software and the EC 50 value.

[0181] 3.2 Cell-based binding FACS

[0182] The binding activity of mouse anti-IL4Rα antibodies to IL4Rα expressed on the surface of 293F-IL4Rα cells was detected by flow cytometry (FACS). Briefly, 293F cells (Thermofisher Inc., catalog 11625019) were transfected with a pCMV-TP plasmid construct, which had nucleotides encoding human IL4Rα (amino acid residues 1-825 of uniprot#P24394-1) between EcoRI and XbaI, and a stable cell pool (named 293F-IL4Rα) was selected for subsequent cell-based binding FACS and cell-based ligand blocking FACS analysis. 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (phosphate buffered saline (PBS) containing 2% v / v fetal bovine serum). Then, in a flask containing 2×10 5 100 μL / well of the anti-IL4Rα antibody or control diluted in FACS buffer (starting at 80 nM, 4-fold serial dilution) was added to the 96-well plate with 10 cells / well and placed on ice for 40 minutes. After washing the cells twice with FACS buffer, 100 μL / well of R-phycoerythrin-labeled affinity purified F(ab') 2 Fragmented goat anti-mouse IgG (H+L) (diluted 1:1000 in FACS buffer, Jackson Immunoresearch, catalog 115-116-146). After incubation at 4°C in the dark for 40 minutes, the cells were washed three times and resuspended in FACS buffer. Fluorescence values ​​were measured using a Becton Dickinson FACSCanto II-HTS. Data were analyzed using Graphpad Prism software and EC values ​​were obtained. 50 value.

[0183] 3.3 Indirect ELISA

[0184] Cross-reaction of anti-IL4Rα antibody with cynomolgus IL4Rα protein or cal-IL4Rα-his protein was detected. Briefly, 2 μg / mL cynomolgus IL4Rα-his protein (Sinobiological inc., catalog 90897-C08H) dissolved in carbonate / bicarbonate buffer (pH 9.6) or 0.2 μg / mL cal-IL4Rα-his protein (Sino biological inc. custom product, catalog BAX2) dissolved in carbonate / bicarbonate buffer (pH 9.6) was coated in 96-well plates at 100 μL / well and incubated at 37°C for 2 hours. 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 at 37°C for 2 hours. Wash the plate again, add 100 μL of the anti-IL4Rα antibody of the present invention or the control substance (0.004-66.7 nM, starting at 66.7 nM, 5-fold gradient dilution in PBST containing 2.5% w / v skim milk) to each well, and incubate at 37°C for 40 minutes. Wash the plate 4 times, then add 100 μL / well of peroxidase-labeled affinity-purified goat anti-mouse IgG (Fcγ fragment specific) (diluted at a ratio of 1:5000 in PBST buffer, Jackson Immunoresearch, product catalog 115-036-071), and incubate at 37°C for 40 minutes. After the last wash, add 100 μL / well of TMB (Innoreagents) for incubation. After 3-10 minutes, add 50 μL / well of 1M H 2 SO 4 The reaction was terminated and the absorbance was read at 450 nm. The data were analyzed using Graphpad Prism software and the EC 50 value.

[0185] The results of the three determinations are shown in Table 3 and Figures 1A-1C , 2A-2D, and 3.

[0186] The results showed that the mouse anti-IL4Rα antibodies of the present invention can specifically bind to human IL4Rα with high binding affinity, and some of the antibodies have higher binding activity to monkey IL4Rα protein than the reference. Table 3. Binding activity of mouse anti-IL4Rα antibodies *Not tested.

[0187] Example 4: Blocking activity of mouse anti-IL4Rα antibody on IL4Rα-reference or IL4Rα-IL4 interaction

[0188] 4.1 Ligand blocking ELISA

[0189] The ability of the anti-IL4Rα antibodies of the present invention to block IL4-IL4Rα interactions was determined by competitive ELISA. Briefly, human IL4Rα-his protein (SEQ ID NO: 57, prepared in-house) dissolved in PBS at a concentration of 2 μg / mL was coated in 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 PBST containing 5% w / v skim milk was added and blocked at 37°C for 2 hours. Then, the plate was washed again with wash buffer.

[0190] Anti-IL4Rα antibody or control was diluted with PBST buffer containing 2.5% w / v skim milk (starting at 80nM, 4-fold gradient dilution), and the gradient diluted anti-IL4Rα antibody or control was added to the plate coated with IL4Rα at 100μL / well, and incubated with human IL4Rα-his protein at 37°C for 40 minutes. The plate was washed 4 times with washing buffer, and then 100μL of biotin-labeled human IL4 protein (Sino biological inc., product catalog 11846-HNAE) at a concentration of 0.56nM was added to each well and incubated at 37°C for 40 minutes. The plate was washed again with washing buffer. Then 100μL / well of HRP-labeled streptavidin (diluted at a ratio of 1:10000 in PBST buffer, Jackson Immunoresearch, product catalog 016-030-084) was added and incubated at 37°C for 40 minutes. Wash the plate again with wash buffer. Finally, add TMB and wash with 1MH 2 SO 4 The reaction was terminated and the absorbance was read at 450 nm. The data were analyzed using Graphpad Prism software and the IC 50 value.

[0191] 4.2 Reference Blocking ELISA

[0192] The ability of the anti-IL4Rα antibodies of the present invention to block the binding of the reference substance to human IL4Rα was determined by competitive ELISA. Briefly, the reference substance dissolved in PBS at a concentration of 2 μg / mL was coated in a 96-well plate at 100 μL / well and incubated overnight at 4°C. The next day, the plate was washed with washing buffer (PBS+0.05% w / v Tween-20, PBST), and PBST containing 5% w / v skim milk was added and blocked at 37°C for 2 hours. During the blocking of the 96-well plate, the anti-IL4Rα antibody of the present invention or the reference substance (starting at 100 nM, 4-fold serial dilution) was diluted with biotin-labeled human IL4Rα-his protein (SEQ ID NO: 57, prepared in-house, dissolved in PBST containing 2.5% w / v skim milk, concentration of 0.55 nM) and incubated at 25°C for 40 minutes. After washing the plate, add 100 μL / well of the antibody / IL4Rα-his mixture to the plate coated with the reference. Incubate at 37°C for 40 minutes and wash the plate with wash buffer. Then add 100 μL / well of HRP-labeled streptavidin to the plate and incubate at 37°C for 40 minutes to detect biotinylated human IL4Rα-his bound to the plate. Wash the plate again with wash buffer. Finally, add TMB and wash with 1M H 2 SO 4 The reaction was terminated and the absorbance was read at 450 nm. The data were analyzed using Graphpad Prism software and the IC 50 value.

[0193] 4.3 Cell-based ligand blocking FACS

[0194] Using the 293F-IL4Rα cells prepared as described above, flow cytometry (FACS) was used to evaluate the activity of anti-IL4Rα antibodies in blocking the binding of IL4 protein to cell surface IL4Rα.

[0195] Briefly, 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (PBS containing 2% v / v fetal bovine serum). 5100 μL / well of anti-IL4Rα antibody or control dissolved in FACS buffer in a gradient dilution (starting at 80 nM, 4-fold gradient dilution) was added to a 96-well plate with 10 cells / well, and ice-bathed for 40 minutes. After washing the plate twice with FACS buffer, 100 μL of 1.67 nM biotin-labeled human IL4 protein (Sino biological inc., product catalog 11846-HNAE) was added to each well and incubated at 4°C in the dark for 40 minutes. After washing the plate twice with FACS buffer, 100 μL of R-phycoerythrin-labeled streptavidin (diluted 1:500 with FACS buffer, Jackson Immunoresearch, product catalog 016-110-084) was added to each well and incubated at 4°C in the dark for 40 minutes. After washing the cells twice, resuspend them in FACS buffer. Fluorescence values ​​were measured using Becton Dickinson FACS Canto II-HTS. The data were analyzed using Graphpad Prism software, and the IC 50 value.

[0196] The results of the three determinations are shown in Table 4 and Figures 4A-4B , 5A-5B, 6A-6C.

[0197] As shown in Table 4 and Figures 4A-4B As shown, all anti-IL4Rα antibodies of the present invention can block the human IL4-human IL4Rα interaction with a blocking activity comparable to that of the reference product.

[0198] Figure 5A and 5B It was shown that some antibodies of the present invention were able to block the binding of human IL4Rα-reference, indicating that the epitope they bind to may be the same as or similar to the epitope bound by the reference.

[0199] In addition, as shown in Table 4 and Figures 6A-6C As shown in Figure 2, all anti-IL4Rα antibodies were able to block the binding of IL4 to cell surface IL4Rα, and their blocking abilities were very close to those of the reference (although their IC 50 The value is slightly higher than that of the reference product). Table 4. Blocking activity of anti-IL4Rα antibodies against reference-IL4Rα or IL4-IL4Rα binding

[0200] Example 5: Cell-based functional assay of mouse anti-IL4Rα antibody

[0201] IL4 and IL13 can bind to IL4Rα and induce STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. STAT6 phosphorylation is crucial in the IL4 / IL13 signaling pathway.

[0202] Briefly, HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells were prepared in-house by stably transfecting HEK293T cells (ATCC CRL-11268) naturally expressing IL13Rα1 with a pcDNA3.1-Puro (YouBio biological inc., catalog VT9222) plasmid construct (with nucleotides encoding human IL4Rα between BamHI and XhoI), a STAT6 plasmid (Sino biologicalinc., catalog HG13190-NH) (with nucleotides encoding human STAT6 between KpnI and XbaI), and a STAT6 luciferase reporter plasmid STAT6-Luc (Yeasen biological inc., catalog 11588ES03). Single cell clone LB2 was then selected for all subsequent functional assays.

[0203] The inhibitory effect of the anti-IL4Rα antibody of the present invention on STAT6 phosphorylation induced by IL4 and IL13 was detected.

[0204] Briefly, HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells in the logarithmic phase were resuspended in culture medium (RPMI1640 + 10% FBS) and seeded into 96-well plates at 100 μL / well, with 5 × 10 5cells. Then, 50 μL of gradient dilutions of anti-IL4Rα antibody or control (including anti-CD22 antibody prepared in-house) was added to each well (with a starting concentration of 100 nM, 5-fold gradient dilution) and incubated at 37° C. for 30 minutes. Then 50 μL of IL4 protein (600 pg / mL, Sinobiological inc., product catalog 11846-HNAE) or IL13 protein (50 ng / mL, Sino biological inc., product catalog 10369-HNAC) was added to each well and incubated at 37° C. for 20 minutes. The plate was centrifuged and washed twice with staining buffer (prepared in-house, DPBS+0.5% w / v BSA+2mM EDTA), and then 50 μL of fixation buffer (BD biosciences inc., product catalog 5545655) was added to each well and incubated at 4° C. for 30 minutes. The cells were washed twice, 200 μL of permeabilization buffer (BD biosciences inc., catalog number 558050) was added to each well, and ice bathed for 30 minutes. The plate was washed 3 times with staining buffer. Then anti-pSTAT6 antibody (pSTAT6 stock solution diluted 20 times, BD biosciences inc., product catalog number 562079) was added and allowed to stand on ice for 60 minutes. Finally, the plate was washed twice and resuspended in staining buffer. Fluorescence values ​​were measured using Becton Dickinson FACSCanto II-HTS. Data were analyzed using Graphpad Prism software and IC 50 value.

[0205] The results are shown in Table 5 and Figure 7 , 8 shown.

[0206] The results showed that all anti-IL4Rα antibodies could block IL4- or IL13-induced STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells with comparable or higher blocking activity than the reference product. Table 5. Functional assay results of anti-IL4Rα antibodies

[0207] Example 6: Production and characterization of chimeric antibodies

[0208] The heavy chain variable region and light chain variable region of the anti-IL4Rα mouse monoclonal antibody were sequenced, and the sequence IDs are summarized in Table 1.

[0209] The heavy chain variable region and light chain variable region of anti-IL4Rα mouse monoclonal antibodies C2C1A1A1 and B8G11F2B7G5E8 were cloned into a vector containing a human IgG4 heavy chain constant region (SEQ ID NO: 55) and a vector containing a human κ light chain constant region (SEQ ID NO: 56), respectively, wherein the C-terminus of the variable region was connected to the N-terminus of the corresponding constant region.

[0210] A vector comprising nucleotides encoding a heavy chain variable region linked to a human IgG4 heavy chain constant region, and a vector comprising nucleotides encoding a light chain variable region linked to a human kappa light chain constant region, were transiently transfected into 50 mL of 293F suspension cells with 1 mg / mL PEI at a ratio of 60%:40% light chain construct:heavy chain construct.

[0211] After culturing for six days in a shake flask, the cell supernatant was harvested, the cells in the supernatant were centrifuged, and filtered through a 0.22 μm filter to separate immunoglobulins. The chimeric antibody was purified by protein A affinity chromatography. In short, the protein A agarose column (bestchrom (Shanghai) Biosciences, product catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was passed through the protein A agarose column, and then the column was washed with PBS buffer until the absorbance of the protein reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and the eluate was immediately collected in a 1.5 mL tube and neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0). The immunoglobulin-containing parts were merged and dialyzed overnight at 4 ° C in PBS.

[0212] Purified antibodies were tested by capture ELISA, competition ELISA, BIAcore affinity assay, cell-based binding FACS assay, and cell-based functional assay following the protocols in the previous examples with minor modifications as described below.

[0213] For capture ELISA, goat anti-mouse IgG (Fcγ fragment specific) was replaced with 2 μg / mL goat anti-human IgG (affinity purified goat anti-human IgG, Fcγ fragment specific, Jackson Immunoresearch, catalog 109-005-098), 100 μL / well.

[0214] For indirect ELISA, affinity purified F(ab') 2Fragmented goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-036-098) replaced peroxidase-labeled affinity purified goat anti-mouse IgG (Fcγ fragment specific), 100 μL / well.

[0215] For BIAcore, goat anti-mouse IgG was replaced with goat anti-human IgG (GE healthcare, catalog BR100839, Human Antibody Capture Kit) and covalently linked to a CM5 chip.

[0216] For cell-based binding FACS, R-phycoerythrin-labeled affinity purified goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-115-098) was used instead of R-phycoerythrin-labeled affinity purified F(ab') 2 Fragmented goat anti-mouse IgG (H+L) was diluted 1:1000 in FACS buffer, 100 μL / well.

[0217] The results are shown in Table 6 and Figure 9-13 The data showed that the chimeric antibody had similar binding affinity / potency and blocking activity as its parental mouse antibody. Table 6. Binding and functional activities of chimeric antibodies

[0218] Example 7: Humanization of anti-IL4Rα monoclonal antibodies B8G11F2B7G5E8 and C2C1A1A1

[0219] Mouse anti-IL4Rα antibodies B8G11F2B7G5E8 and C2C1A1A1 were humanized and further characterized. Humanization of mouse antibodies was performed using an established CDR grafting approach as described below.

[0220] In order to select the acceptor framework for the humanization of mouse antibodies B8G11F2B7G5E8 and C2C1A1A1, the sequences of the light chain variable region and the heavy chain variable region of each mouse antibody were BLASTed with the human immunoglobulin gene database. The human germline antibody with the highest homology was selected as the acceptor framework for humanization. The mouse antibody heavy / light chain variable region CDRs were inserted into the selected framework, and the residues in the framework were further back-mutated to obtain more candidate heavy / light chain variable regions. A total of 13 exemplary humanized B8G11F2B7G5E8 antibodies, namely huB8G11F2B7G5E8-V1 to huB8G11F2B7G5E8-V11, huB8G11F2B7G5E8-V13 and huB8G11F2B7G5E8-V14, and 16 exemplary humanized C2C1A1A1 antibodies, namely huC2C1A1A1-V1 to huC2C1A1A1-V16, were obtained, and their heavy / light chain variable region sequence IDs are shown in Table 1.

[0221] A vector comprising nucleotides encoding a humanized heavy chain variable region linked to a human IgG4 heavy chain constant region (SEQ ID NO: 55), and a vector comprising 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 of 293F suspension cells with 1 mg / mL PEI at a ratio of 60%:40% light chain construct:heavy chain construct.

[0222] After culturing for six days in a shake flask, the cell supernatant was harvested, the cells in the supernatant were centrifuged, and filtered through a 0.22 μm filter to separate immunoglobulins. The antibody was purified by protein A affinity chromatography. In short, the protein A agarose column (bestchrom (Shanghai) Biosciences, product catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was flowed through the protein A agarose column, and then the column was washed with PBS buffer until the absorbance of the protein reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and the eluate was immediately collected in a 1.5 mL tube and neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0). The immunoglobulin-containing parts were merged and dialyzed overnight at 4 ° C in PBS.

[0223] Example 8: Characterization of humanized antibodies Table 7. Binding affinity of humanized B8G11F2B7G5E8 monoclonal antibody

[0224] According to the protocol in the previous example, the binding affinity of the humanized antibody to human IL4Rα was evaluated by BIAcore technology. a , K d and K D values ​​and are summarized in Tables 7 and 8. Table 8. Binding affinity of humanized C2C1A1A1 monoclonal antibodies Dissociation % = (End (RU) - Start (RU)) / Start (RU)

[0225] The results showed that the humanized antibodies had similar human IL4Rα binding affinities as the chimeric antibodies, and all humanized huC2C1A1A1 antibodies showed higher human IL4Rα binding affinities compared with the reference.

[0226] Humanized antibodies huB8G11F2B7G5E8-V2, huB8G11F2B7G5E8-V4, huB8G11F2B7G5E8-V14, huC2C1A1A1-V14 and huC2C1A1A1-V15 were further tested by Biacore, capture ELISA, indirect ELISA, cell-based binding FACS, competition ELISA and cell-based functional assays according to the protocols in the previous examples (with minor modifications as described below).

[0227] For capture ELISA, goat anti-mouse IgG (Fcγ fragment specific) was replaced with 2 μg / mL goat anti-human IgG (affinity purified goat anti-human IgG, Fcγ fragment specific, Jackson Immunoresearch, catalog 109-005-098), 100 μL / well.

[0228] For indirect ELISA, affinity purified F(ab') 2 Fragmented goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-036-098) replaced peroxidase-labeled affinity purified goat anti-mouse IgG (Fcγ fragment specific), 100 μl / well.

[0229] For BIAcore, goat anti-mouse IgG was replaced with goat anti-human IgG (GE healthcare, catalog BR100839, Human Antibody Capture Kit) and covalently linked to a CM5 chip.

[0230] For cell-based binding FACS, R-phycoerythrin-labeled affinity purified goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-115-098) was used instead of R-phycoerythrin-labeled affinity purified F(ab') 2 Fragmented goat anti-mouse IgG (H+L) was diluted 1:1000 in FACS buffer, 100 μL / well.

[0231] The thermal stability of humanized antibodies huB8G11F2B7G5E8-V14 and huC2C1A1A1-V15 was also tested. TM Thermal Shift Protein Stability Kit (Biotium, catalog 33022-T, lot number 181214) was used to determine the Tm (melting temperature) of proteins by thermal shift assay. TM The dye was thawed to room temperature. The vial containing the dye was vortexed and centrifuged. Then, 5 μL of 200× dye was added to 95 μL PBS to prepare 10× dye. 2 μL of 10× dye and 10 μg of humanized antibody were added to the reaction system, and PBS was added to a total reaction volume of 20 μL. The centrifuge tube containing the dye and antibody was briefly centrifuged and placed in a real-time PCR thermal cycler (Roche, LightCycler480II), and the Melt Curve program in the thermal cycler was set using the parameters in Table 9. Table 9. Parameters of Melt Curve program Profile Steps temperature Heating rate Duration Initial hold 25℃ NA 30s Melt curve 25-99℃ 0.1℃ / s NA

[0232] The results are shown in Tables 10-1 to 10-3 and Figures 14A-14B As shown in 22. Table 10-1. Binding activity and functional activity of humanized monoclonal antibodies *Not tested. Table 10-2. Binding activity and functional activity of humanized monoclonal antibodies *Not tested. Table 10-3. Binding activity and functional activity of humanized monoclonal antibodies *Not tested.

[0233] The data showed that compared with the reference, the humanized C2C1A1A1 antibody showed comparable (if not better) human IL4Rα binding affinity / activity and blocking ability for IL4Rα-IL4 / IL13, while the humanized B8G11F2B7G5E8 antibody had significantly better blocking ability for IL4 / IL13-IL13Rα1-IL4Rα interaction.

[0234] Although the present invention has been described in conjunction with one or more embodiments, it should be understood that the present invention not only covers these embodiments, but also is intended to cover all alternatives, modifications and equivalents included in the spirit and scope of the appended claims. All documents cited herein are fully incorporated herein by reference.

[0235] The sequence information of this application is summarized in the table below.

Claims

1. An isolated monoclonal antibody or antigen-binding portion thereof, which binds to interleukin-4 receptor alpha subunit (IL4Rα), comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise: (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 sequence of SEQ ID NOs: 1, 5 and 10, respectively; (2) 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 sequence of SEQ ID NOs: 1, 5 and 10, respectively; 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 1, 6 and 11; (3) amino acid sequences that are 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: 2, 7 and 12, respectively; (4) amino acid sequences that are 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: 3, 7 and 13, respectively; 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 3, 8 and 13; (5) 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: 4, 8 and 13, respectively; or (6) 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: 5, 8 and 13, respectively. The amino acid sequences shown in NOs: 3, 9 and 14 have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

2. The isolated monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence 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=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), or amino acid sequences shown in 43, 44, 47, 49, 51 or 53 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

3. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a light chain variable region comprising CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise: (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 sequence set forth in SEQ ID NOs: 15, 22 and 26, respectively; (2) 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 sequence set forth in SEQ ID NOs: 16, 22 and 27, respectively; (3) 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 sequence set forth in SEQ ID NOs: 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; (4) amino acid sequences that are 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: 18, 24 and 29, respectively; (5) amino acid sequences that are 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: 18, 24 and 29, respectively; 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 19, 24 and 30; (6) amino acid sequences that are 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: 20, 25 and 31, respectively; or (7) amino acid sequences that are 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: 21, 23 and 34, respectively. The amino acid sequences shown in IDNOs: 21, 25 and 31 have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

4. The isolated monoclonal antibody or antigen-binding portion thereof of claim 3, wherein the light chain variable region comprises 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 sequence shown in SEQ ID NOs: 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 isolated monoclonal antibody or antigen-binding portion thereof of claim 3, wherein the heavy chain variable region and the light chain variable region comprise: (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 sequence set forth in SEQ ID NOs: 1, 5, 10, 15, 22 and 26, respectively; (2) 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 sequence set forth in SEQ ID NOs: 1, 6, 11, 16, 22 and 27, respectively; (3) 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 sequence set forth in SEQ ID NOs: 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 2, 7, 12, 17, 23 and 28; (4) amino acid sequences that are 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: 3, 8, 13, 18, 24 and 29, respectively; (5) amino acid sequences that are 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: 3, 8, 13, 18, 24 and 29, respectively; 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 4, 8, 13, 19, 24 and 30; (6) amino acid sequences that are 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: 3, 9, 14, 20, 25 and 31, respectively; or (7) amino acid sequences that are 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: 3, 9, 14, 20, 25 and 31, respectively. The amino acid sequences shown in NOs: 3, 9, 14, 21, 25 and 31 have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

6. The isolated monoclonal antibody or antigen-binding portion thereof of claim 5, wherein the heavy chain variable region and the light chain variable region comprise: (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 sequence of SEQ ID NOs: 32 and 35, respectively; (2) 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 sequence of SEQ ID NOs: 32 and 35, respectively; NOs:33 (X1=W, X2=S) and 36 (X1=L, X2=I; X1=F, X2=V; X1=F, X2=I) 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 36 (X1=L, X2=I; X1=F, X2=V; X1=F, X2=I); (3) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:33 (X1=W, X2=S) and 37, respectively; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: NOs:34 and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) 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) 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:34 and 37, respectively; (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:34 and 37, respectively; NOs:33 (X1 = L, X2 = A) and 36 (X1 = L, X2 = I; X1 = F, X2 = V; X1 = F, X2 = I) 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); (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:33 (X1 = L, X2 = A) and 37, respectively; (8) 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: NOs: 33 (X1=W, X2=A) and 36 (X1=L, X2=I; X1=F, X2=V;91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 33 (X1 = W, X2 = A) and 37, respectively; (10) amino acid sequences set forth in SEQ ID NOs: 34 (X1 = W, X2 = A) and 38 (X1 = F, X2 = I) that are 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: 35 (X1 = W, X2 = A) and 39, respectively; (11) amino acid sequences set forth in SEQ ID NOs: 37 (X1 = W, X2 = A) and 38, respectively. NOs:38 and 39 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:38 and 39; (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; 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 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; (13) respectively with 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 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; (14) respectively with SEQ ID The amino acid sequences set forth in NOs: 43 and 46 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;(15) amino acid sequences that are 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: 44 and 46, respectively; (16) amino acid sequences that are 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: 47 and 48, respectively; (17) amino acid sequences that are 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: 48 and 49, respectively. 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences set forth in SEQ ID NOs:49 and 50; (18) 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:51 and 52, respectively; or (19) 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:51 and 52, respectively. The amino acid sequences set forth in NOs: 53 and 54 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; Optionally, it comprises a heavy chain constant region connected to the heavy chain variable region, and a light chain constant region connected to the light chain variable region, the heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 55, and the light chain constant region having the amino acid sequence shown in SEQ ID NO:

56.

7. 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 monkey IL4Rα; (c) blocks IL4Rα-IL4 interaction; and (d) blocks IL4Rα-IL13-IL13Rα1 interaction; Optionally, the monoclonal antibody or antigen-binding portion thereof is a mouse, human, chimeric or humanized antibody; Optionally, the monoclonal antibody or antigen binding portion thereof is of IgG1, IgG2 or IgG4 isotype.

8. A nucleotide encoding the isolated monoclonal antibody or antigen-binding portion thereof of claim 1.

9. An expression vector comprising the nucleotide according to claim 8. 10 . A host cell comprising the nucleotide of claim 8 or the expression vector of claim 9 .

11. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof of claim 1, the nucleotide of claim 8, the expression vector of claim 9 or the host cell of claim 10, and a pharmaceutically acceptable carrier; Optionally, the pharmaceutical composition further comprises an antiallergic agent or an antitumor agent; Optionally, the anti-allergic agent is an antihistamine, a corticosteroid, a β-adrenergic receptor agonist, a drug targeting cyc-LTs or a drug targeting IgE.

12. A method for treating an allergic disease associated with excessive IL4 and / or IL13 signaling, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 11; Optionally, the allergic disease is atopic dermatitis, anaphylaxis, allergic rhinitis or allergic asthma.

13. 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 11; Optionally, the tumor is a solid tumor.

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