Anti-IL4R antibody and application thereof

Abstract

Relates to an anti-IL4R antibody. Specifically, the invention provides an antibody capable of specifically binding to IL4R or an antigen binding fragment thereof, and the antibody or the antigen binding fragment thereof can specifically bind to IL4R with high affinity and can be used as a drug for treating IL4R-related diseases.

Description

Anti-IL4R antibodies and their applications

[0001] The present invention claims priority to Chinese patent application No. 202310960138.5, filed on August 1, 2023, entitled “Anti-IL4R Antibodies and Applications Thereof”, the entire contents of which, including the appendices, are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of antibodies, and in particular, to anti-IL4R antibodies. Background Art

[0003] The Th2 cytokines interleukin-4 (IL-4) and IL-13, and their interacting heterodimeric IL-4 receptor (IL-4R) complex, play key roles in the pathogenesis of allergic diseases.

[0004] Interleukin-4 (IL-4) is a cytokine produced primarily by activated T cells, monocytes, basophils, mast cells, and eosinophils. The biological effects of IL-4 include stimulating the proliferation of activated B and T cells and the differentiation of CD4+ T cells into type II helper T cells. It also plays a key role in regulating humoral and adaptive immunity. IL-4 induces B cell antibody class switching to IgE and upregulates the production of type II major histocompatibility complexes. Studies have shown that IL-4 plays multiple roles in immune responses to infectious diseases, autoimmune diseases, tumors, and other conditions.

[0005] The IL-4R is a type I transmembrane protein that binds interleukin-4 and interleukin-13 to regulate IgE antibody production in B cells. In T cells, the encoded protein also binds interleukin-4 to promote Th2 cell differentiation. Human IL-4R is a heterodimer composed of a common subunit, IL-4Rα, which pairs with different accessory subunits to mediate the effects of IL-4 and IL-13 in different tissues. IL-4Rα pairs with the γc chain to form the IL-4R type I complex, which is expressed on hematopoietic cells and specifically binds IL-4. It then binds to the low-affinity IL-13 receptor, IL-13Rα1, to form a type II heterodimeric complex that binds IL-13 and IL-4 and is expressed on both hematopoietic and non-hematopoietic cells. Once IL-4 or IL-13 binds to the receptor, it triggers transphosphorylation and activation of the receptor subunit-associated Janus family protein kinases (JAKs). Several companies have conducted research on IL-4Rα, and studies have found that human monoclonal antibodies can effectively relieve and treat symptoms such as asthma, eczema, and atopic dermatitis.

[0006] IL-13 is a cytokine produced by Th2 cells, CD4 cells, natural killer T cells, mast cells, basophils, and eosinophils. IL-13 is a central regulator of IgE synthesis, goblet cell proliferation, mucus hypersecretion, airway hyperresponsiveness, fibrosis, and chitinase upregulation. It is a mediator of allergic inflammation and various diseases, including asthma. IL-13 expression has been shown to be increased in bronchoalveolar lavage (BAL) fluid and cells from patients with atopic mild asthma after allergen challenge. Genome-wide association studies have identified multiple polymorphisms in IL-13 and the gene encoding the IL-13 receptor that are associated with asthma susceptibility, bronchial hyperresponsiveness, and elevated IgE levels. Although no studies have directly linked IL-13 to disease control in humans, numerous polymorphisms in the IL-13 gene have been shown to increase the risk of atopic respiratory diseases such as asthma.

[0007] Atopic dermatitis is a chronic, recurrent, inflammatory skin disease that often occurs on the face, neck, elbows, and other areas. It is a special type of eczema characterized by dry skin, chronic eczematous lesions, and significant itching. Scratching can also cause redness, exudation, and thickening of the skin. In addition to skin manifestations, some AD patients also often have other atopic diseases such as allergic asthma, allergic rhinitis, and allergic conjunctivitis. Studies have shown that cytokines such as IL-4 and IL-13 are involved in the pathogenesis of the disease.

[0008] Asthma is a chronic airway inflammation involving multiple inflammatory cells, including eosinophils (Eos), mast cells (Mc), and T lymphocytes. It is characterized by elevated IgE levels, airway infiltration of Mc, Eos, and T cells, and airway hyperresponsiveness. IL4 is a key cytokine in the pathogenesis of asthma and allergies. It transmits signals to the cell nucleus via the IL4 receptor on the cell membrane, producing biological effects.

[0009] In view of the role and function of IL-4Rα in various related diseases, the goal of the present invention is to provide an immunotherapeutic agent that can effectively inhibit IL-4Rα-mediated and treat or improve diseases such as atopic dermatitis and asthma.

[0010] Summary of the Invention

[0011] The present invention provides anti-IL4R antibodies, nucleic acids encoding the same, methods for preparing the antibodies, pharmaceutical compositions containing the antibodies, and related uses of the pharmaceutical compositions for treating immune diseases.

[0012] In a first aspect, the present invention provides an antibody or antigen-binding fragment that specifically binds to IL4R, comprising:

[0013] (1) a heavy chain variable region (VH), comprising HCDR1, HCDR2, and HCDR3 selected from the group consisting of a VH represented by any one of SEQ ID NOs: 10-15, 135-136, 141-143, 147-148, 154-155, 161, and 164-165;

[0014] (2) a light chain variable region (VL), comprising LCDR1, LCDR2, and LCDR3 selected from the group consisting of the VL represented by any one of SEQ ID NOs: 16-21, 134, 140, 145-146, 153, 158-160, and 163.

[0015] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the VH set forth in any one of SEQ ID NOs: 10, 135, and 136 have the sequences set forth in SEQ ID NOs: 22-24, SEQ ID NOs: 25-27, or SEQ ID NOs: 28-30 according to the Kabat, IMGT, or Chothia numbering systems;

[0016] The HCDR1, HCDR2, and HCDR3 of the VH set forth in SEQ ID NOs: 11 and 141-143 have the sequences set forth in SEQ ID NOs: 31-33, SEQ ID NOs: 34-36, or SEQ ID NOs: 37-39 according to the Kabat, IMGT, or Chothia numbering systems;

[0017] The HCDR1, HCDR2, and HCDR3 of the VH set forth in SEQ ID NO: 12 have the sequences set forth in SEQ ID NOs: 40-42, 43-45, or 46-48 according to the Kabat, IMGT, or Chothia numbering systems;

[0018] The HCDR1, HCDR2, and HCDR3 of the VH represented by SEQ ID NOs: 13, 154, and 155 have the sequences represented by SEQ ID NOs: 49-51, 52-54, or 55-57 according to the Kabat, IMGT, or Chothia numbering systems;

[0019] The HCDR1, HCDR2, and HCDR3 of the VH set forth in SEQ ID NOs: 14 and 161 have the sequences set forth in SEQ ID NOs: 58-60, SEQ ID NOs: 61-63, or SEQ ID NOs: 64-66 according to the Kabat, IMGT, or Chothia numbering systems;

[0020] The HCDR1, HCDR2, and HCDR3 of the VH set forth in SEQ ID NOs: 15, 164, and 165 have the sequences set forth in SEQ ID NOs: 67-69, SEQ ID NOs: 70-72, or SEQ ID NOs: 73-75 according to the Kabat, IMGT, or Chothia numbering systems;

[0021] The LCDR1, LCDR2, and LCDR3 of the VL set forth in SEQ ID NOs: 16 and 134 have the sequences set forth in SEQ ID NOs: 76-78, SEQ ID NOs: 79-81, or SEQ ID NOs: 82-84 according to the Kabat, IMGT, or Chothia numbering systems;

[0022] The LCDR1, LCDR2, and LCDR3 of the VL set forth in SEQ ID NOs: 17 and 140 have the sequences set forth in SEQ ID NOs: 85-87, 88-90, or 91-93 according to the Kabat, IMGT, or Chothia numbering systems;

[0023] The LCDR1, LCDR2, and LCDR3 of the VL represented by SEQ ID NOs: 18 and 145 have the sequences represented by SEQ ID NOs: 94-96, 97-99, or 101-102 according to the Kabat, IMGT, or Chothia numbering systems;

[0024] The LCDR1, LCDR2, and LCDR3 of the VL set forth in SEQ ID NOs: 19 and 153 have the sequences set forth in SEQ ID NOs: 103-105, 106-108, or 109-111 according to the Kabat, IMGT, or Chothia numbering systems;

[0025] The LCDR1, LCDR2, and LCDR3 of the VL set forth in SEQ ID NOs: 20, 158, or 159 have the sequences set forth in SEQ ID NOs: 112-114, 115-117, or 118-120 according to the Kabat, IMGT, or Chothia numbering systems;

[0026] The LCDR1, LCDR2, and LCDR3 of the VL set forth in SEQ ID NOs: 21 and 163 have the sequences set forth in SEQ ID NOs: 121-123, 124-126, or 127-129 according to the Kabat, IMGT, or Chothia numbering systems;

[0027] The HCDR1, HCDR2, and HCDR3 of the VH represented by SEQ ID NOs: 147 and 148 have the sequences represented by SEQ ID NOs: 40, 149, and 42 according to the Kabat numbering system;

[0028] The LCDR1, LCDR2 and LCDR3 of the VL shown in SEQ ID NO: 146 have the sequences shown in SEQ ID NOs: 94, 150 and 96 according to the Kabat numbering system.

[0029] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequences:

[0030] (1) SEQ ID NO: 22, 23, 24, 76, 77, 78, respectively;

[0031] (2) SEQ ID NOs: 25, 26, 27, 79, 80, and 81, respectively;

[0032] (3) SEQ ID NOs: 28, 29, 30, 82, 83, and 84, respectively;

[0033] (4) SEQ ID NOs: 31, 32, 33, 85, 86, and 87, respectively;

[0034] (5) SEQ ID NO: 34, 35, 36, 88, 89, 90, respectively;

[0035] (6) SEQ ID NO: 37, 38, 39, 91, 92, 93, respectively;

[0036] (7) SEQ ID NO: 40, 41, 42, 94, 95, 96, respectively;

[0037] (8) SEQ ID NO: 43, 44, 45, 97, 98, 99, respectively;

[0038] (9) SEQ ID NO: 46, 47, 48, 100, 101, 102, respectively;

[0039] (10) SEQ ID NO: 49, 50, 51, 103, 104, 105, respectively;

[0040] (11) SEQ ID NO: 52, 53, 54, 106, 107, 108, respectively;

[0041] (12) SEQ ID NO: 55, 56, 57, 109, 110, 111, respectively;

[0042] (13) SEQ ID NO: 58, 59, 60, 112, 113, 114, respectively;

[0043] (14) SEQ ID NO: 61, 62, 63, 115, 116, 117, respectively;

[0044] (15) SEQ ID NO: 64, 65, 66, 118, 119, 120, respectively;

[0045] (16) SEQ ID NOs: 67, 68, 69, 121, 122, and 123, respectively;

[0046] (17) SEQ ID NO: 70, 71, 72, 124, 125, 126, respectively;

[0047] (18) SEQ ID NOs: 73, 74, 75, 127, 128, and 129, respectively;

[0048] (19) SEQ ID NOs: 40, 149, 42, 94, 95, and 96, respectively;

[0049] (20) SEQ ID NOs: 40, 149, 42, 94, 150, and 96, respectively;

[0050] (21) A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in any one of (1) to (20) above, or having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

[0051] In some embodiments, the antibody or antigen-binding fragment comprises:

[0052] a heavy chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NOs: 10-15, 135-136, 141-143, 147-148, 154-155, 161, 164-165; or / and a light chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NOs: 16-21, 134, 140, 145-146, 153, 158-160, 163;

[0053] Preferably, (1) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 10, and the light chain variable region comprises the sequence shown in SEQ ID NO: 16;

[0054] (2) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 11, and the light chain variable region comprises the sequence shown in SEQ ID NO: 17;

[0055] (3) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 12, and the light chain variable region comprises the sequence shown in SEQ ID NO: 18;

[0056] (4) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 13, and the light chain variable region comprises the sequence shown in SEQ ID NO: 19;

[0057] (5) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 14, and the light chain variable region comprises the sequence shown in SEQ ID NO: 20;

[0058] (6) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the sequence shown in SEQ ID NO: 21;

[0059] (7) the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 135 and 136, and the light chain variable region comprises the sequence shown in SEQ ID NO: 134;

[0060] (8) the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 141-143, and the light chain variable region comprises the sequence shown in SEQ ID NO: 140;

[0061] (9) the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 147 and 148, and the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 145 and 146;

[0062] (10) the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 154 and 155, and the light chain variable region comprises the sequence shown in SEQ ID NO: 153;

[0063] (11) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 161, and the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 158-160;

[0064] (12) the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 164-165, and the light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 163;

[0065] (13) The heavy chain variable region and / or the light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in groups (1) to (12); or, a sequence that has at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the sequence shown in groups (1) or (2); the mutations may be selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids.

[0066] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region;

[0067] Preferably, the heavy chain constant region is selected from IgG, such as IgG1, IgG2, IgG3 or IgG4, and the IgG can be selected from human IgG, such as human IgG4; alternatively, the heavy chain constant region can be selected from the Fc region, the CH3 region or the complete heavy chain constant region, alternatively, the heavy chain constant region is a human Fc region; the light chain constant region is selected from the κ chain or the λ chain, preferably the κ chain.

[0068] In some embodiments, the antibody or antigen-binding fragment specifically binds to human or monkey IL4R protein; preferably, the KD of the antibody or antigen-binding fragment is better than 1.00E-9M.

[0069] In some embodiments, the antibody or antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof.

[0070] In some embodiments, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a whole antibody, a fragment of a whole antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, a diabody, or a single domain antibody.

[0071] In some embodiments, the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

[0072] In another aspect, the present invention provides a multispecific molecule comprising any one of the antibodies or antigen-binding fragments described above; preferably, the multispecific molecule further comprises an antibody or antigen-binding fragment that specifically binds to an antigen other than IL4R or binds to an IL4R epitope different from any one of the antibodies or antigen-binding fragments described above;

[0073] Preferably, the antigen other than IL4R is selected from the following groups: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) target for recruiting and / or activating immune cells.

[0074] In another aspect, the present invention provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises any one of the above antibodies or antigen-binding fragments.

[0075] On the other hand, the present invention provides an immune effector cell expressing the chimeric antigen receptor, or comprising a nucleic acid fragment encoding the chimeric antigen receptor; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

[0076] In another aspect, the present invention provides an isolated nucleic acid fragment encoding any one of the above antibodies or antigen-binding fragments, or the multispecific molecule, or the chimeric antigen receptor.

[0077] In another aspect, the present invention provides a vector comprising the nucleic acid fragment.

[0078] In another aspect, the present invention provides a host cell comprising the vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0079] In another aspect, the present invention provides a method for preparing any one of the above antibodies or antigen-binding fragments or the multispecific molecules, which comprises culturing the host cells, and isolating the antibody or antigen-binding fragment expressed by the cells, or isolating the multispecific molecules expressed by the cells.

[0080] On the other hand, the present invention provides a method for preparing the immune effector cell, which comprises introducing a nucleic acid fragment encoding the CAR into the immune effector cell, and optionally, further comprises initiating the immune effector cell to express the CAR.

[0081] In another aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned antibodies or antigen-binding fragments, the multispecific molecule, the immune effector cell, the nucleic acid fragment, the vector, the host cell, or a product prepared by any one of the methods; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

[0082] On the other hand, the present invention provides any one of the above antibodies or antigen-binding fragments, the multispecific molecule, the immune effector cell, the nucleic acid fragment, the vector, the host cell, a product prepared by any one of the methods; or the use of the pharmaceutical composition in the preparation of a medicament for preventing and / or treating immune diseases; the immune diseases include dermatitis, asthma, nasal polyposis, eosinophilic esophagitis, urticaria, prurigo, or other diseases related to IL4R.

[0083] In another aspect, the present invention provides a method for preventing and / or treating an immune disease, comprising administering to a patient in need thereof an effective amount of any one of the above-mentioned antibodies or antigen-binding fragments, the multispecific molecule, the immune effector cell, the nucleic acid fragment, the vector, the host cell, the product obtained by any one of the methods, or the pharmaceutical composition; wherein the immune disease includes dermatitis, asthma, nasal polyposis, eosinophilic esophagitis, urticaria, prurigo, or other diseases related to IL4R.

[0084] On the other hand, the present invention provides any one of the above antibodies or antigen-binding fragments, the multispecific molecule, the immune effector cell, the nucleic acid fragment, the vector, the host cell, the product obtained by any one of the methods, or the pharmaceutical composition, for preventing and / or treating immune diseases; wherein the immune diseases include dermatitis, asthma, nasal polyposis, eosinophilic esophagitis, urticaria, prurigo, or other diseases related to IL4R.

[0085] On the other hand, the present invention provides a kit comprising any one of the above antibodies or antigen-binding fragments, the multispecific molecule, the immune effector cell, the nucleic acid fragment, the vector, the host cell, the product prepared by any one of the methods, or the pharmaceutical composition.

[0086] Beneficial Effects: The present invention obtains a high-affinity, specific antibody against IL-4R through hybridoma antibody screening, and a humanized antibody is obtained based on this antibody. The antibody of the present invention can bind to IL-4R with high specificity and block the binding of IL-4 to IL-4R. It has low in vivo toxicity and a good safety profile, and can be used to treat immune diseases such as dermatitis and asthma.

[0087] Definitions and Explanations of Terms

[0088] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0089] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0090] The terms "comprising," "including," and "having" are used interchangeably herein to denote the inclusiveness of a solution, meaning that the solution may contain additional elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also encompasses "consisting of" solutions. For example, "a composition comprising A and B" should be understood to encompass the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0091] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the term."

[0092] The term "IL4R" herein refers to a cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4Rα. IL-4R is intended to encompass various forms of IL-4R molecules at various stages in the body, such as, but not limited to, molecules produced during IL-4R gene amplification, replication, transcription, splicing, processing, translation, and modification, such as precursor IL-4R, mature IL-4R, naturally occurring IL-4R splice variants, modified IL-4R, or fragments thereof.

[0093] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an affinity of approximately 1 × 10 -6 M or less, 1×10 -7 M or less, about 1×10 -8 M or less, about 1×10 -9 M or lower or about 1×10 -10 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, see Example 10 herein for methods for obtaining KD values.

[0094] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0095] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004) (the contents of which are incorporated herein by reference). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0096] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0097] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0098] "Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0099] The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or produced during the production of the preparation, such variants typically being present in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.

[0100] The term "monospecific" as used herein is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0101] The term "multispecific" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0102] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0103]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

[0104] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0105] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, along with the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the light chain VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites (the two Fab fragments) and a portion of the Fc region.

[0106] The term "Fd" herein refers to an antibody composed of a VH and CH1 domain. The term "Fv" herein refers to an antibody fragment composed of a single-arm VL and VH domain. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0107] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv). (The foregoing is incorporated herein by reference).

[0108] The term "diabody" herein refers to an antibody whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)) (the contents of which are incorporated herein by reference).

[0109] The term "naked antibody" herein refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term "conjugated antibody" refers to an antibody that is conjugated to a therapeutic agent or tracer.

[0110] The term "chimeric antibody" herein refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)) (the contents of which are incorporated herein by reference). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0111] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0112] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted onto human framework sequences.

[0113] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007) (the contents of which are incorporated herein by reference). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0114] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways. (The foregoing is incorporated herein by reference).

[0115] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) (the contents of which are incorporated herein by reference).

[0116] The term "IMGT numbering system" herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003 (the contents of which are incorporated herein by reference).

[0117] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) (the contents of which are incorporated herein by reference).

[0118] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from the CH1, Fc, or CH3 domains.

[0119] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0120] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can be, for example, removed during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and therefore, the Fc region may or may not include Lys447.

[0121] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0122] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0123] 1) Alanine (A), serine (S), threonine (T);

[0124] 2) Aspartic acid (D), glutamic acid (E);

[0125] 3) Asparagine (N), glutamine (Q);

[0126] 4) Arginine (R), Lysine (K), Histidine (H);

[0127] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0128] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0129] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0130] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0131] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix (incorporated herein by reference) with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0132] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (Version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4 (the aforementioned contents are incorporated herein by reference).

[0133] Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present invention can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov (the aforementioned contents are incorporated herein by reference).

[0134] The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, comprising at least (1) an extracellular antigen binding domain, such as a variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0135] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone bonding or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into the subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1, the contents of which are incorporated herein by reference). "Isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0136] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0137] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0138] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0139] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject of treatment, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0140] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (e.g., monkeys), or non-primate mammals being treated for a disease or condition.

[0141] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0142] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the terms "tumor" or "neoplasm" refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0143] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] FIG1A shows that the non-reducing band (NR) of the hFc-tagged protein in the extracellular region of human IL-4Rα is approximately 130 KDa, and the reducing band (R) is approximately 60 KDa.

[0145] FIG1B shows that the non-reducing band of the extracellular region of human IL-4Rα protein with an mFc tag is around 130 KDa, and the reducing band is around 60 KDa.

[0146] Figure 1C shows that the sizes of the non-reducing band and the reducing band of the his-tagged protein in the extracellular region of human IL-4Rα are both around 40 kDa.

[0147] FIG1D shows that the sizes of the non-reducing band and the reducing band of the his-tagged protein in the extracellular region of monkey IL-4Rα are both around 40 KDa.

[0148] FIG2A shows the generation of monoclonal 293T cells stably expressing human IL-4Rα.

[0149] FIG2B shows that a monkey CHO cell line stably expressing IL4Rα-Flpin was obtained.

[0150] FIG2C shows the obtained TF1-Luc pool cell line.

[0151] FIG3A shows the protein binding of humanized antibodies to hIL-4Rα.

[0152] FIG3B shows the binding of humanized antibodies to cyno IL-4Rα protein.

[0153] Figure 4 shows that humanized antibodies block the binding of hIL-4Rα to IL-4.

[0154] FIG5A shows the binding of humanized antibodies to the stably transfected cell line human IL4Rα-293T expressing hIL-4Rα.

[0155] FIG5B shows the binding of the humanized antibody to the stable cell line monkey IL4Rα-FlpinCHO expressing cynoIL-4Rα.

[0156] Figure 6A shows that the humanized antibody can inhibit IL-4-stimulated IL-4IL-13Reporter 293 cell signal transduction.

[0157] Figure 6B shows that the humanized antibody can inhibit IL-13-stimulated IL-4IL-13Reporter 293 cell signal transduction.

[0158] FIG7A shows that the humanized antibody can inhibit IL-4-stimulated proliferation of TF1-luc cells.

[0159] FIG7B shows that the humanized antibody can inhibit IL-13-stimulated proliferation of TF1-luc cells. DETAILED DESCRIPTION

[0160] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0161] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0162] Example 1 Preparation of IL4Rα Antigen and Stable Cell Lines

[0163] 1.1 Preparation of IL4Rα extracellular domain antigen

[0164] The nucleotides encoding the full-length amino acid sequence of hIL4Rα (NCBI: NP_000409.1) were codon-optimized according to the codon usage preference of Homo sapiens, and the N-terminal amino acid fragment 26-232 was gene synthesized. The hFc fragment, mFc fragment and His tag (HHHHHH) were inserted into the C-terminus of the hIL4Rα-ECD(26-232) fragment and cloned into the pTT5 vector (provided by General Bio (Anhui) Co., Ltd.) to obtain pTT5-hIL4Rα-ECD(26-232)-hFc, pTT5-hIL4Rα-ECD(26-232)-mFc and pTT5-hIL4Rα-ECD(26-232)-His, respectively. The plasmid pTT5-cyno IL4Rα-ECD(26-232)-His encoding cynoIL4Rα (NCBI: EHH60265.1) was obtained using the same method. The plasmid and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293 cells (Thermofisher, Catalog No. A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. The day after transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma, Catalog No. G7528) were added. On the sixth day after transfection, cell supernatants were collected and purified to obtain hIL4Rα-ECD(26-232)-hFc (SEQ ID NO: 1), hIL4Rα-ECD(26-232)-mFc (SEQ ID NO: 2), hIL4Rα-ECD(26-232)-His (SEQ ID NO: 3), and cyno IL4Rα-ECD(26-232)-His (SEQ ID NO: 4) proteins. The results are shown in Figure 1.

[0165] Fusion protein of hIL4Rα extracellular domain and hFc: hIL4Rα-ECD(26-232)-hFc

[0166] Fusion protein of hIL4Rα extracellular domain and mFc: hIL4Rα-ECD(26-232)-mFc

[0167] Fusion protein of the extracellular domain of hIL4Rα and His tag: hIL4Rα-ECD(26-232)-His

[0168] cynoIL4Rα extracellular domain and His tag fusion protein: cyno IL4Rα-ECD(26-232)-His

[0169] 1.2 Preparation of IL-4Rα stably transfected cell lines

[0170] A. Obtaining a stable human IL4Rα-293T cell line

[0171] The nucleotide sequence encoding the full-length amino acid sequence of hIL4Rα (NCBI: NP_000409.1) was cloned into the pLVX vector (purchased from Clontech) and a plasmid was prepared. A 293T cell line (purchased from the Chinese Academy of Sciences) was transfected with plasmids to produce lentivirus (PEI MAX, purchased from Polyscience, Catalog No. 24765-1). The virus was harvested and then reinfected into 293T cells. The cells were selectively cultured for 2 weeks in DMEM medium containing 10% (w / w) fetal bovine serum and 5 μg / ml puromycin. Positive monoclonal cells were then sorted using a FACSAria II flow cytometer (purchased from BD Biosciences) using an IL4Ra antibody (dupilumab, self-produced, VH and VL sequences as shown in SEQ ID NOs: 6-7, the same below) and a donkey anti-human IgG h+l antibody (Jackson, Catalog No. 109605088). The cells were then transferred to a 96-well plate and incubated at 37°C in 5% (v / v) CO2. After approximately 2 weeks, a portion of the monoclonal wells were selected for expansion. The amplified clones were screened by flow cytometry. The results are shown in Figure 2A.

[0172] Full-length hIL4Rα (NCBI: NP_000409.1): used to construct the human IL4Rα overexpression cell line human IL4Rα-293T

[0173] Dupilumab-VH:

[0174] Dupilumab-VL:

[0175] B. Obtaining monkey IL4Rα-FlpinCHO cell line

[0176] The nucleotide sequence encoding the full-length amino acid sequence of cynoIL4Rα (NCBI: EHH60265.1) was cloned into the pCDNA5 vector (purchased from ThermoFisher) to prepare the plasmid. The Flpin CHO cell line (purchased from ThermoFisher) was transfected with the plasmid (Lopofect300-, purchased from ThermoFisher) and selectively cultured for two weeks in F12 medium containing 10% (w / w) fetal bovine serum (FBS) and 800 μg / ml hygromycine. Positive monoclonal cells were then isolated using a FACSAria II (purchased from BD Biosciences) flow cytometer using an IL4Ra antibody (dupilumab, in-house) and a donkey anti-human IgG h+l antibody (Jackson, Cat. No. 109605088). The cells were then placed into 96-well plates and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a subset of monoclonal wells were selected for expansion. The expanded clones were screened by flow cytometry. The results are shown in Figure 2B.

[0177] Full-length cynoIL4Rα (NCBI: EHH60265.1): used to construct the cynomolgus monkey IL4Rα overexpression cell line monkey IL4Rα-FlpinCHO

[0178] C. Obtaining the TF1-Luc pool

[0179] The nucleotide sequence encoding the full-length amino acid sequence of nanoLuc (SEQ ID NO: 9) (kindly provided by Shengzhao Bio) was cloned into the pLVX vector (purchased from Clontech) to prepare a plasmid. Lentivirus (PEI MAX, purchased from Polyscience, Cat. No. 24765-1) was produced by transfecting the plasmid into 293T cells (purchased from the Chinese Academy of Sciences). The virus was harvested and then used to infect TF-1 cells (Nanjing Kebai). The cells were selectively cultured for two weeks in 1640 medium supplemented with 10% (w / w) fetal bovine serum (FBS) and 1 μg / ml puromycin. Luciferase expression levels were determined using the nanoLuc detection kit (purchased from Promega). The cells were further expanded and stored in liquid nitrogen. The results are shown in Figure 2C.

[0180] Full-length nanoLuc: used to construct TF1-Luc with low IL4R expression

[0181] Example 2 Preparation of anti-hIL-4Rα hybridoma antibodies

[0182] A. Mouse Immunization and Serum Titer Detection

[0183] The animal immunization experiment was divided into seven groups. The experimental animals were 6-8 week old female Balb / c, SJL, and C57 BL / 6J mice (purchased from Shanghai Slake Co., Ltd.). The animals were housed in an SPF environment. Orbital blood was collected from the mice before immunization to serve as negative serum.

[0184] The first immunization group consisted of five Balb / c mice. The first and second immunogens were Human Recombinant IL-4R / CD124-his protein (purchased from Sino Biological Inc., Catalog No. 10402-H08H), and the third, fifth, and fifth immunogens were hIL4Ra-ECD(26-232)-his. For the first immunization, 0.1 ml of the protein was emulsified with Alum (purchased from Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally. The protein was then emulsified with TiterMax (purchased from Sigma, Catalog No. T2684) and CpG and injected subcutaneously and at multiple sites in the paws, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were administered every two weeks. For booster immunizations, 25 μg of the immunogen was injected subcutaneously at multiple sites in the paws, for a total of four booster immunizations.

[0185] The second immunization group consisted of five Balb / c mice. The first and second immunogens were Human Recombinant IL-4R / CD124-hFc protein (purchased from Sino Biological Inc., Catalog No. 10402-H02H), and the third, fifth, and fifth immunogens were hIL4Ra-ECD(26-232)-hFc. For the first immunization, 0.1 ml of the protein was emulsified with Alum (purchased from Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally. The protein was then emulsified with TiterMax (purchased from Sigma, Catalog No. T2684) and CpG and injected subcutaneously and at multiple sites in the paws, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were administered every two weeks. For booster immunizations, 25 μg of the immunogen was injected subcutaneously at multiple sites in the paws, for a total of four booster immunizations.

[0186] The third immunization group consisted of five C57 BL / 6J mice. The second and fourth immunogens were cyno IL4Ra-ECD(26-232)-hFc, and the remaining immunogens were hIL4Ra-ECD(26-232)-hFc proteins. For the initial immunization, 0.1 ml of the protein was emulsified with Alum (Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally. Furthermore, the protein was emulsified with TiterMax (Sigma, Catalog No. T2684) and CpG and injected subcutaneously and at multiple sites in the paws, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were administered weekly. For each booster immunization, 25 μg of the immunogen was injected subcutaneously at multiple sites, using either Alum / CpG or TiterMax / CpG emulsified. For a total of six booster immunizations, each mouse received a total of 25 μg of the immunogen.

[0187] The fourth immunization group consisted of five SJL mice. The second and fourth immunogens were cyno IL4Ra-ECD(26-232)-hFc, and the remaining immunogens were hIL4Ra-ECD(26-232)-hFc proteins. For the initial immunization, the protein was emulsified with Alum (purchased from Thermo Fisher Scientific, catalog number 77161) and CpG (synthesized by Sangon, catalog number ODN1826) and injected intraperitoneally at 0.1 ml. The protein was emulsified with TiterMax (purchased from Sigma, catalog number T2684) and CpG and injected subcutaneously and at multiple sites in the soles of the feet, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were performed weekly. For the booster immunizations, the protein was emulsified with Alum / CpG or TiterMax / CpG and injected subcutaneously at multiple sites, for a total of six booster immunizations.

[0188] The fifth immunization group consisted of five Balb / c mice, and the immunogen was hIL4Ra-ECD(26-232)-hFc protein. For the initial immunization, 0.1 ml of the protein was emulsified with Alum (Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally. Furthermore, the protein was emulsified with TiterMax (Sigma, Catalog No. T2684) and CpG and injected subcutaneously and at multiple sites in the plantar follicles, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were administered weekly, with 25 μg of the immunogen injected subcutaneously at multiple sites using either Alum / CpG or TiterMax / CpG, for a total of eight booster immunizations.

[0189] The sixth immunization group consisted of five SJL mice. The second, fourth, and sixth immunogens were the stably transfected 293T cell line hIL4Rα, and the remaining immunogens were hIL4Ra-ECD(26-232)-hFc protein. For the initial immunization, the protein was emulsified with Alum (Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally at 0.1 ml. The protein was then emulsified with TiterMax (Sigma, Catalog No. T2684) and CpG and injected subcutaneously and at multiple sites in the paws, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were performed weekly for a total of eight booster immunizations. For the cell-based boost immunization, 0.1 ml of the cell and CpG suspension was injected intraperitoneally, with 1E7 cells per mouse. For the protein-based boost immunization, the protein was emulsified with TiterMax / CpG and injected subcutaneously at multiple sites, for a total of 25 μg of immunogen per mouse.

[0190] The seventh immunization group consisted of five SJL mice, and the immunogen was hIL4Ra-ECD(26-232)-hFc protein. For the initial immunization, 0.1 ml of the protein was emulsified with Alum (Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally. The protein was then emulsified with TiterMax (Sigma, Catalog No. T2684) and CpG and injected subcutaneously and at multiple sites in the soles of the feet, for a total of 50 μg of immunogen per mouse. Thereafter, booster immunizations were administered every two weeks. For booster immunizations, 25 μg of the immunogen was injected subcutaneously at multiple sites using TiterMax / CpG emulsification, for a total of four booster immunizations.

[0191] After booster immunization in the above immune groups, blood was collected from the mouse orbits, and the binding titers of antibodies in the mouse serum and human IL4Ra-His protein were detected by ELISA.

[0192] After booster immunization, mice with high antibody titers in serum were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was performed, and 50 μg / mouse of immunogen solution prepared with physiological saline was injected subcutaneously, in the plantar and intraperitoneally.

[0193] B. Spleen Cell Fusion and Hybridoma Screening

[0194] The spleen and lymph nodes were sterilely removed, ground and filtered with a 40 μm cell strainer (purchased from BD Falcon), and 5 ml of ACK Lysing Buffer (purchased from Gibco, catalog number: A1049201) was added to lyse the red blood cells to obtain a cell suspension. The cells were washed twice with DMEM (purchased from Gibco, catalog number: 12800017) basal medium at 1500 rpm, and then mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC) at a ratio of 2:1 in terms of the number of viable cells. Cell fusion was performed using the BTX ECM2001+ high-efficiency electrofusion method (see ECM2001+ELECTROFUSION PROTOCOL). The fused cells were diluted into DMEM culture medium containing 20% ​​fetal bovine serum (purchased from ExCell Bio, catalog number: FND500) and 1×HAT (purchased from Sigma, catalog number: H0262-10VL). The percentages are volume percentages. The fused cells were plated at 5×10 4 200 μl of the solution was added to each well of a 96-well cell culture plate and incubated in a 5% CO2, 37°C incubator. After 7 days, the supernatant of the cell fusion plate was assayed. Hybridoma cells from qualified fusion plate wells were subjected to limiting dilution in DMEM (Gibco, Catalog No. 12800017). Cells were observed under a microscope and the number of viable cells was counted. Approximately 200 cells were added to 2 ml of Medium D (STEMCELL, Catalog No. 03810), mixed well, and plated into a 6-well cell culture plate. The plates were incubated at 37°C, 5% CO2. After 7 days, single clones were selected and cultured in DMEM supplemented with 10% FBS and 1×HT (Sigma, Catalog No. H0137-10VL) at 37°C, 5% CO2 for 2 days. Initial screening was performed using ELISA. Positive single clones were selected and expanded to 24-well cell culture plates for further culture. After 3 days, the supernatant was assayed for activity.

[0195] Based on the test results of the 24-well plate samples, the best clone was selected and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2. After 7 days, the supernatant was collected and purified with protein A to obtain purified hybridoma monoclonal antibodies.

[0196] The hybridoma antibodies identified above were identified by ELISA, FACS, blocking experiments, etc., and 6 candidate antibodies with good binding and blocking activities were obtained, namely IL4R-SFM-04, IL4R-SFM-07, IL4R-SFM-13, IL4R-SFM-14, IL4R-SFM-17, and IL4R-SFM-18.

[0197] Example 3 Construction and preparation of anti-IL-4Rα chimeric antibody

[0198] Using molecular biological techniques, DNA encoding heavy and light chain immunoglobulins was obtained from the above six hybridoma antibodies, and chimeric antibodies were constructed after sequencing.

[0199] The nucleic acids encoding the amino acid sequences of the heavy and light chain variable regions (VH) of the hybridoma antibodies were codon-optimized according to the codon usage preference of Homo sapiens, followed by full sequence synthesis. The heavy chain variable region was cloned into the vector PTT5-huIgG4CH1-CH2-CH3, containing the human heavy chain constant region and regulatory elements, to express the complete IgG heavy chain in mammalian cells. Similarly, the light chain variable region was cloned into the vector PTT5-huIgGLC(Kappa), containing the human light chain constant region and regulatory elements, to express the complete IgG light chain in mammalian cells. After sequencing, the sequences were transfected into Expi-293 mammalian cells, where IgG was expressed and secreted into the culture medium. The supernatants were pooled, filtered, and purified. IgG was purified by Protein A chromatography. The culture supernatant was loaded onto an appropriately sized Protein A column and eluted with 3–5 column volumes of a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4). The protein was eluted with 50 mM NaAc-HAc, pH 3.5. The protein was concentrated by ultrafiltration using a Millipore concentrator, and the OD280 was measured. The IgG concentration was determined by spectrophotometry. The purified IgG was analyzed for aggregation or degradation using SDS-PAGE.

[0200] Table 1 shows the VH / VL sequences of the chimeric antibody, Table 2 shows the CDR analysis results of the chimeric antibody VH, and Table 3 shows the CDR analysis results of the chimeric antibody VL.

[0201] Table 1 Anti-IL-4Rα chimeric antibody VH / VL sequences

[0202] Table 2 CDR analysis results of anti-IL-4Rα chimeric antibody VH

[0203] Table 3 CDR analysis results of anti-IL-4Rα chimeric antibody VL

[0204] Example 4 Identification of anti-IL-4Rα chimeric antibodies

[0205] 4.1 ELISA detection of the binding level of chimeric antibody to hIL4Rα-ECD(26-232)-His protein

[0206] A. Detection of the binding level of chimeric antibody to hIL4Rα-ECD(26-232)-His protein

[0207] The hIL4Rα-ECD(26-232)-His prepared in Example 1.1 was labeled with biotin. According to the instructions of the Biotin Labeling kit (Cat. No. LK03, purchased from DO JINDO), 100 μL WS buffer and 200 μg hIL4Rα-ECD(26-232)-His protein were added to the Filtration tube, mixed, and centrifuged at 8000g for 10 minutes; 10 μL DMSO was added to the NH2-Reactive Biotin Tube and mixed for later use; after centrifugation, 100 μL Reaction Buffer and 8 μL NH2-Reactive Biotin solution were added to the Filtration tube, mixed, and placed in a 37°C constant temperature incubator for 10 minutes, followed by 100 μL WS Buffer and centrifugation at 8000g for 10 minutes. The filtrate was discarded and 200 μL WS Buffer was added and centrifuged at 8000g for 10 minutes. The above steps were repeated once, and 100 μL WS Buffer was added to recover the labeled hIL4Rα-ECD(26-232)-His into a new centrifuge tube. The concentration was measured and set aside.

[0208] First, dilute streptavidin (SA, Catalog No. S4762, purchased from Sigma) with PBS to a final concentration of 5 μg / mL. Then, add 50 μL per well to a 96-well ELISA plate and incubate overnight at 4°C. The next day, discard the supernatant and add blocking buffer (PBS buffer containing 5% (w / w) skim milk powder (purchased from Sangon, Catalog No. A600669-0250)) at 37°C for 2 hours. Discard the blocking buffer and wash the plate three times with PBST. Add 0.5 μg / mL biotin-labeled hIL4Rα-ECD(26-232)-His to each well at 50 μL / well, incubate at 37°C for 1 hour, and wash three times with PBST. Add 50 μL of the purified antibody from Example 4 to each well, starting at 20 nM and serially diluting 5-fold. After incubation at 37°C for 1 hour, wash the plate three times with PBST. 50 μL of a 1:5000 dilution of horseradish peroxidase (HRP)-labeled secondary antibody (Jackson Immuno, Catalog No. 109-035-098) was added to each well in PBS containing 1% (w / w) BSA (Sangon, Catalog No. A500023-0100). After incubation at room temperature for 1 hour, the plate was washed five times with PBST. 50 μL of TMB substrate was added to each well and incubated at room temperature for 5 minutes. 50 μL of stop solution (1.0 N HCl) was added to each well. The plate was read using a microplate reader (PowerWave HT, Biotek) at A450 nm. The results are shown in Table 4. Compared to the control antibody (the control antibody used in Example 4 was dupilumab, produced in-house, with VH and VL sequences as shown in SEQ ID NOs: 6-7, the same below), the anti-IL4Rα chimeric antibody effectively bound to hIL4Rα-ECD(26-232)-His.

[0209] B. Detection of the binding level of chimeric antibody to cyno IL4Rα-ECD(26-232)-His protein

[0210] Referring to the method of Example 4.1(A), cyno IL4Rα-ECD(26-232)-His was labeled with biotin and detected by ELISA. The results are shown in Table 4. Compared with the control antibody, the anti-IL4Rα chimeric antibody can effectively bind to cyno IL4Rα-ECD(26-232)-His.

[0211] 4.2 Enzyme-linked immunosorbent assay (ELISA) detection of chimeric antibodies blocking the binding of human IL4 to IL4Rα-ECD protein

[0212] Anti-mFc (Jackson, Catalog No. 115-006-071) was diluted in PBS to a final concentration of 4 μg / mL. 50 μL of anti-mFc was then added to each well of a 96-well ELISA plate. The plate was sealed with plastic wrap and incubated overnight at 4°C. The next day, the supernatant was discarded and blocking solution (PBS buffer containing 5% (w / w) skim milk powder (Sangon, Catalog No. A600669-0250)) was added. The plate was blocked at 37°C for 2 hours. The blocking solution was discarded and the plate was washed three times with PBST. 50 μL of 0.2 μg / mL hIL4R-ECD(26-232)-mFc was added to each well. The plate was incubated at 37°C for 1 hour and washed three times with PBST. 25 μL of human IL4-Avi-His-biotin (Acro, Catalog No. IL4-H82E0) protein was added to each well at a final concentration of 0.1 μg / mL. Then, 25 μL of purified antibody was added, starting at 100 nM and serially diluted 5-fold. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. 50 μL of horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma, Catalog No. S2438) was added to each well at a 1:5000 dilution in PBS containing 1% (w / w) BSA (Sangon, Catalog No. A500023-0100). After incubation at room temperature for 1 hour, the plate was washed five times with PBST. 50 μL of TMB substrate was added to each well, incubated at room temperature for 5 minutes, and 50 μL of stop solution (1.0 N HCl) was added to each well. The A450 nm reading was read using a microplate reader (PowerWaveHT, Biotek). The results are shown in Table 4. Compared with the control antibody, the anti-IL4Rα chimeric antibody can effectively block the binding of human IL4 to IL4Rα-ECD protein.

[0213] 4.3 Flow cytometry (FACS) assay to detect the binding activity of chimeric antibodies to cells

[0214] A. Detection of the binding level of chimeric antibodies to human IL4Rα-293T cells

[0215] Human IL4Rα-293T cells were expanded and cultured in T-175 cell culture flasks to 90% confluence. The culture medium was removed, washed once with PBS buffer (purchased from Hyclone, product number SH30256.01), and digested with 3 ml of 0.25% trypsin (purchased from Gibico, product number 25200-072) for 2 minutes. The cells were resuspended in DMEM (purchased from Gibco, product number 11995-040) containing 10% (v / v) fetal bovine serum. After cell counting, centrifugation was performed at 1000 rpm for 5 minutes at room temperature and the culture medium was discarded. 1×10 550 μL of the purified antibody to be tested was added to each well of a U-bottom 96-well FACS plate (Corning, Cat. No. 3795) and stored at 4°C or on ice until ready to use. The purified antibody to be tested was diluted in PBS containing 2% (w / w) fetal bovine serum and 50 μL was added to the cells per well, mixed thoroughly, and incubated on ice for 1 hour. The plate was washed twice with 200 μL of PBS buffer per well and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and 100 μL of diluted fluorescently labeled secondary antibody (Jackson ImmunoResearch, Cat. No. 109-605-098) was added to each well and incubated on ice for 1 hour. The plate was washed twice with FACS buffer by centrifugation. The results were detected and analyzed using FACS (FACS Canto II, BD Biosciences). As shown in Table 4, the anti-IL4Rα chimeric antibody effectively bound to human IL4Rα-293T cells compared to the control antibody.

[0216] B. Detection of the binding level of chimeric antibodies to monkey IL4Rα-FlpinCHO cells

[0217] Monkey IL4Rα-Flpin CHO cells were cultured as described in Example 4.3(A), and the cells were resuspended in DMEM / F12 (Gibco, Catalog No. 11330-032) containing 10% (v / v) fetal bovine serum. As shown in Table 4, the anti-IL4Rα chimeric antibody effectively bound to monkey IL4Rα-Flpin CHO cells compared to the control antibody.

[0218] 4.4 Detection of chimeric antibodies blocking the binding of IL4 to IL-4IL-13 Reporter 293 cells

[0219] IL-4IL-13 Reporter 293 cells (purchased from Jiman Bio, product number GM-C01511) were expanded and cultured in T-175 cell culture flasks to 90% confluence. The culture medium was removed, washed once with PBS buffer (purchased from Hyclone, product number SH30256.01), and digested with 3 ml of 0.25% trypsin (purchased from Gibico, product number 25200-072) for 2 minutes. The cells were resuspended in DMEM (purchased from Gibco, product number 11995-040) containing 2% (v / v) fetal bovine serum. After counting the cells, centrifuge at 1000 rpm for 5 minutes at room temperature and discard the culture medium. 2×10 450 μL of the test antibody was added to each well of a flat-bottom 96-well plate (purchased from Kawei Biosciences, Catalog No. 062096) and placed in an incubator until ready to use. The test antibody was diluted in DMEM containing 2% (w / w) fetal bovine serum to a starting concentration of 400 nM. After a 10-fold dilution, 25 μL of the test antibody was added to each well and mixed thoroughly. IL4 (purchased from SinoBiological, Catalog No. 11846-HNAE) was diluted in DMEM containing 2% (w / w) fetal bovine serum and added to the cells at a rate of 25 μL per well, mixing thoroughly to a final IL4 concentration of 0.15 ng / mL. The cells were incubated in a cell culture incubator for 5 hours. 50 μL of Bright lite (purchased from Novozymes, Catalog No. DD1204) was added to each well and shaken at room temperature for 10 minutes. The results were detected and analyzed using PE-Ensight (purchased from PerkinElmer). The results are shown in Table 4. Compared with the control antibody, the anti-IL4Rα chimeric antibody can effectively block the binding of IL4 to IL-4IL-13Reporter 293 cells.

[0220] 4.5 Detection of chimeric antibodies inhibiting the proliferation of TF1-Luc cells

[0221] TF1-Luc cells were cultured in T-175 cell culture flasks. After cell counting, the cells were centrifuged at 1000 rpm for 5 minutes at room temperature and the culture medium was discarded. The cells were resuspended in RPMI-1640 (Gibco, Cat. No. 22400-089) containing 2% (v / v) fetal bovine serum. After cell counting, 4 × 10 cells were plated per well. 4 50 μL of the antibody to be tested was added to each well of a flat-bottom 96-well plate (Cave Bio, Catalog No. 062096) and placed in an incubator until ready to use. The purified antibody to be tested was diluted in RPMI-1640 containing 2% (w / w) fetal bovine serum to a starting concentration of 400 nM. The antibody was diluted 10-fold and added to the cells at a rate of 25 μL per well, mixing thoroughly. IL4 (SinoBiological, Catalog No. 11846-HNAE) was diluted in RPMI-1640 containing 2% (w / w) fetal bovine serum and added to the cells at a rate of 25 μL per well, mixing thoroughly, to a final IL4 concentration of 0.4 ng / mL. The cells were incubated in a cell culture incubator for 5 hours. 50 μL of Brightlite (Novozymes, Catalog No. DD1204) was added to each well and shaken at room temperature for 10 minutes. The results were detected and analyzed using PE-Ensight (PerkinElmer). The results are shown in Table 4. Compared with the control antibody, the anti-IL4Rα chimeric antibody can effectively inhibit the proliferation of TF1-Luc cells.

[0222] 4.6 Flow cytometry (FACS) assay to detect the internalization efficiency of chimeric antibodies in human IL4Rα-293T cells

[0223] Human IL4Rα-293T cells were expanded and cultured in T-175 cell culture flasks to 90% confluence. The culture medium was removed, washed once with PBS buffer (purchased from Hyclone, product number SH30256.01), and digested with 3 ml of 0.25% trypsin (purchased from Gibico, product number 25200-072) for 2 minutes. The cells were resuspended in DMEM (purchased from Gibco, product number 11995-040) containing 10% (v / v) fetal bovine serum. After counting the cells, centrifuge at 1000 rpm for 5 minutes at room temperature and discard the culture medium. Resuspend with pre-cooled PBS and plate at 2×10 per well. 5 100 μL of the cells were added to each well of a U-bottom 96-well plate (Corning, Cat. No. 3795). The plate was centrifuged at 1000 rpm for 5 minutes at 4°C, and the supernatant was discarded. The purified antibody to be tested was diluted to 20 nM in PBS containing 2% (w / w) fetal bovine serum. 200 μL of the antibody was added to each well of the plate, mixed thoroughly, and incubated on ice for 1 hour. 100 μL of the sample was transferred from each well of the 96-well plate to a new 96-well plate, washed twice at 4°C, and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, 100 μL of pre-chilled complete medium was added to each well. One plate was placed at 4°C, and the other in a cell culture incubator. The plates were incubated for 2 hours, and both plates were placed on ice for 5 minutes to terminate endocytosis. The plates were washed twice at 4°C, and centrifuged at 1500 rpm for 5 minutes. A diluted fluorescently conjugated secondary antibody (Jackson ImmunoResearch, Cat. No. 109-605-098) was added and incubated on ice for 1 hour. The cells were washed twice by centrifugation with pre-cooled PBS buffer. The results were detected and analyzed using FACS (FACS Canto II, purchased from BD Biosciences). The results are shown in Table 4. Compared with the control antibody, the anti-IL4Rα chimeric antibody had a better internalization efficiency.

[0224] 4.7 Biacore Affinity Detection of Chimeric Antibodies

[0225] A BIAcore 8K instrument was used to detect antibody-antigen binding strength using the Protein A capture method. Multi-cycle kinetics was used to determine antibody-antigen affinity. In each cycle, the test antibody was first captured with Protein A. A single concentration of hIL4Rα-ECD(26-232)-His (prepared in Example 1.1) was then injected. The binding and dissociation processes between the antibody and antigen were recorded. Finally, the chip was regenerated using Glycine pH 1.5. The mobile phase consisted of HBS-EP pH 7.4 (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The flow rate was 30 μL / min, the association time was 240 seconds, the dissociation time was 600 seconds, the regeneration time was 30 seconds, and the detection temperature was 25°C. Finally, the data were analyzed according to a 1:1 binding model, and the antibody-antigen binding kinetic parameters, including the association rate constant ka, dissociation rate constant kd, equilibrium dissociation constant KD, and maximum binding signal Rmax, were fitted. The results are shown in Table 4. Compared with the control antibody, the anti-IL4Rα chimeric antibody has a good affinity for hIL4Rα-ECD(26-232)-his.

[0226] Table 4 Identification results of anti-IL4Rα chimeric antibodies

[0227] Example 5 Humanization of anti-IL-4Rα chimeric antibody

[0228] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to mouse antibodies as templates. The mouse antibody CDRs were then transplanted into the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the antibody's three-dimensional structure, we backmutated buried residues, residues that directly interact with the CDR regions, and residues in the framework regions that have a significant impact on VL and VH conformation, resulting in humanized monoclonal antibodies.

[0229] 5.1 Humanization of IL4R-CHI-04

[0230] The humanized light chain templates of the chimeric antibody IL4R-CHI-04 are IGKV1-9*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV4-38-2*02 and IGHJ6*01. The CDRs of IL4R-CHI-04 were transplanted into their human templates to obtain the corresponding humanized versions.

[0231] The amino acid sequence of the humanized light chain template IGKV1-9*01 is shown in SEQ ID NO: 130:

[0232] The amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 131:

[0233] The amino acid sequence of the humanized heavy chain template IGHV4-38-2*02 is shown in SEQ ID NO: 132:

[0234] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 133:

[0235] As needed, key amino acids in the FR region sequence of the humanized antibody were backmutated to maintain the original affinity. The specific design is shown in Table 5 (backmutations are in natural numbering order).

[0236] Table 5 Back mutation design of humanized antibody IL4R-CHI-04

[0237] Note: Graft represents the implantation of mouse antibody CDR into human germline template FR sequence; L45P represents the mutation of L at position 45 of Graft to P, and so on.

[0238] The amino acid sequences of the humanized heavy and light chain variable regions are shown in Table 6:

[0239] Table 6 Variable region amino acid sequences of the humanized antibody IL4R-CHI-04

[0240] From the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned humanized anti-IL4Rα antibody, different light chain and heavy chain sequences were selected for cross-combination, and finally two humanized anti-IL4Rα antibodies were obtained. The specific combinations are shown in Table 7.

[0241] Table 7 IL4R-CHI-04 humanized antibody combination

[0242] The analysis results of the VH and VL sequences of the above two humanized antibodies according to the Kabat numbering system are shown in Table 8.

[0243] Table 8 Kabat analysis results of the VH and VL sequences of the second round of humanized antibody IL4R-CHI-04

[0244] 5.2 Humanization of IL4R-CHI-07

[0245] The humanized light chain templates of the chimeric antibody IL4R-CHI-07 are IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The CDRs of IL4R-CHI-07 were transplanted into their human templates to obtain the corresponding humanized versions.

[0246] The amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 137:

[0247] The amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 138:

[0248] The amino acid sequence of the humanized heavy chain template IGHV1-69*02 is shown in SEQ ID NO: 139:

[0249] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 133:

[0250] If necessary, key amino acids in the FR region sequence of the humanized antibody were backmutated to maintain the original affinity. The specific design is shown in Table 9 (backmutations are in natural numbering order).

[0251] Table 9 Back mutation design of humanized antibody IL4R-CHI-07

[0252] The amino acid sequences of the humanized heavy and light chain variable regions are shown in Table 10:

[0253] Table 10 Variable region amino acid sequences of the humanized antibody IL4R-CHI-07

[0254] From the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned humanized anti-IL4Rα antibody, different light chain and heavy chain sequences were selected for cross-combination, and finally three anti-IL4Rα humanized antibodies were obtained. The specific combinations are shown in Table 11.

[0255] Table 11 IL4R-CHI-07 humanized antibody combination

[0256] The analysis results of the VH and VL sequences of the above three humanized antibodies according to the Kabat numbering system are shown in Table 12.

[0257] Table 12 Kabat analysis results of IL4R-CHI-07 humanized antibody VH and VL sequences

[0258] 5.3 Humanization of IL4R-CHI-13

[0259] The humanized light chain templates of the chimeric antibody IL4R-CHI-13 are IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of IL4R-CHI-13 were transplanted into their human templates to obtain the corresponding humanized versions.

[0260] The amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 137:

[0261] The amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 138:

[0262] The amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 144:

[0263] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 133:

[0264] As needed, key amino acids in the FR region of the humanized antibody were backmutated to maintain original affinity. Antibodies contain sites susceptible to chemical modification, and point mutations were performed to eliminate these modifications. Furthermore, to reduce the predicted immunogenicity risk, empirically based point mutations were performed on L2. Detailed design is shown in Table 13 (backmutations are numbered in natural order).

[0265] Table 13 Design of humanized antibody back mutation of IL4R-CHI-13

[0266] The amino acid sequences of the heavy and light chain variable regions after humanization and immunogenicity reduction are shown in Table 14:

[0267] Table 14 Variable region amino acid sequences of humanized IL4R-CHI-13 and reduced immunogenicity antibodies

[0268] From the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned humanized anti-IL4Rα antibody, different light chain and heavy chain sequences were selected for cross-combination, and finally 4 anti-IL4Rα humanized antibodies were obtained. The specific combinations are shown in Table 15.

[0269] Table 15 IL4R-CHI-13 Second Round Humanized Antibody Combinations

[0270] The analysis results of the VH and VL sequences of the above four humanized antibodies according to the Kabat numbering system are shown in Table 16.

[0271] Table 16 Kabat analysis results of IL4R-CHI-13 humanized antibody VH and VL sequences

[0272] 5.4 Humanization of IL4R-CHI-14

[0273] The humanized light chain templates of the chimeric antibody IL4R-CHI-14 are IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV3-7*01 and IGHJ6*01. The CDRs of IL4R-CHI-14 were transplanted into their human templates to obtain the corresponding humanized versions.

[0274] The amino acid sequence of the humanized light chain template IGKV1-39*01 is shown in SEQ ID NO: 151:

[0275] The amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 138:

[0276] The amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown in SEQ ID NO: 152:

[0277] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 133:

[0278] If necessary, key amino acids in the FR region sequence of the humanized antibody were backmutated to maintain the original affinity. The specific design is shown in Table 17 (backmutations are in natural numbering order).

[0279] Table 17 Humanized antibody backmutation design of IL4R-CHI-14

[0280] The amino acid sequences of the humanized heavy and light chain variable regions are shown in Table 18:

[0281] Table 18 Variable region amino acid sequences of humanized antibodies against IL4R-CHI-14

[0282] From the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned humanized anti-IL4Rα antibody, different light chain and heavy chain sequences were selected for cross-combination, and finally two humanized anti-IL4Rα antibodies were obtained. The specific combinations are shown in Table 19.

[0283] Table 19 IL4R-CHI-14 humanized antibody combination

[0284] The analysis results of the VH and VL sequences of the above two humanized antibodies according to the Kabat numbering system are shown in Table 20.

[0285] Table 20 Kabat analysis results of IL4R-CHI-14 humanized antibody VH and VL sequences

[0286] 5.5 Humanization of IL4R-CHI-17

[0287] The humanized light chain templates of the chimeric antibody IL4R-CHI-17 are IGKV1-39*01 / IGKV2-28*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ1*01. The CDRs are transplanted into their human templates to obtain the corresponding humanized versions.

[0288] The amino acid sequence of the humanized light chain template IGKV1-39*01 is shown in SEQ ID NO: 151:

[0289] The amino acid sequence of the humanized light chain template IGKV2-28*01 is shown in SEQ ID NO: 156:

[0290] The amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 138:

[0291] The amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 144:

[0292] The amino acid sequence of the humanized heavy chain template IGHJ1*01 is shown in SEQ ID NO: 157:

[0293] As needed, key amino acids in the FR region of the humanized antibody were backmutated to maintain original affinity. Antibodies contain sites susceptible to chemical modification, and point mutations were performed at these sites to eliminate modification risks. Detailed design is shown in Table 21 (backmutations are numbered in natural order).

[0294] Table 21 Humanized antibody back mutation design of IL4R-CHI-17

[0295] The amino acid sequences of the humanized heavy and light chain variable regions are shown in Table 22:

[0296] Table 22 Variable region amino acid sequences of humanized antibodies against IL4R-CHI-17

[0297] From the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned humanized anti-IL4Rα antibody, different light chain and heavy chain sequences were selected for cross-combination, and finally three anti-IL4Rα humanized antibodies were obtained. The specific combinations are shown in Table 23.

[0298] Table 23 IL4R-CHI-17 humanized antibody combination

[0299] The analysis results of the VH and VL sequences of the above three humanized antibodies according to the Kabat numbering system are shown in Table 24.

[0300] Table 24 Kabat analysis results of IL4R-CHI-17 humanized antibody VH and VL sequences

[0301] 5.6 Humanization of IL4R-CHI-18

[0302] The humanized light chain templates of the chimeric antibody IL4R-CHI-18 are IGKV1-39*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV2-26*01 and IGHJ6*01. The CDRs of IL4R-CHI-18 were transplanted into their human templates to obtain the corresponding humanized versions.

[0303] The amino acid sequence of the humanized light chain template IGKV1-39*01 is shown in SEQ ID NO: 151:

[0304] The amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 131:

[0305] The amino acid sequence of the humanized heavy chain template IGHV2-26*01 is shown in SEQ ID NO: 162:

[0306] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 133:

[0307] If necessary, key amino acids in the FR region sequence of the humanized antibody were backmutated to maintain the original affinity. The specific design is shown in Table 25 (backmutations are in natural numbering order).

[0308] Table 25 Humanized antibody back mutation design of IL4R-CHI-18

[0309] The amino acid sequences of the humanized heavy and light chain variable regions are shown in Table 26:

[0310] Table 26 Variable region amino acid sequences of humanized antibodies against IL4R-CHI-18

[0311] From the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned humanized anti-IL4Rα antibody, different light chain and heavy chain sequences were selected for cross-combination, and finally two anti-IL4Rα humanized antibodies were obtained. The specific combinations are shown in Table 27.

[0312] Table 27 IL4R-CHI-18 humanized antibody combination

[0313] The analysis results of the VH and VL sequences of the above two humanized antibodies according to the Kabat numbering system are shown in Table 28.

[0314] Table 28 Kabat analysis results of IL4R-CHI-18 humanized antibody VH and VL sequences

[0315] 5.7 Construction, Expression, and Purification of Humanized Anti-IL4Rα Antibodies

[0316] The light and heavy chain derivatives obtained above were synthesized separately and cloned into the pTT5 vector containing the constant region of the antibody kappa chain or the CH1-CH3 constant region of human IgG4. The plasmids were paired and transfected into Expi293 cells for expression for 6 days. The culture supernatant was collected and purified using AKTA Pure for Protein A affinity and molecular sieve purification. The resulting antibodies were quantitatively and qualitatively analyzed by SDS-PAGE, SEC-HPLC, and CE-SDS. The specific purification and analysis methods are as follows.

[0317] 1. Use Protein A column (Mabselect SuRe TM , purchased from Cytiva) was initially purified. The Protein A column was first equilibrated with 3–5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and then the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading, the column was eluted with a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3–5 column volumes. Protein bound to the Protein A column was eluted with an eluent (50 mM NaAc-HAc, pH 3.5), and protein elution was monitored by the A280 UV absorbance peak. The eluted protein was collected and neutralized to pH 5.0–6.0 by adding 1 M Tris-HCl, pH 8.0.

[0318] 2. Purify with molecular sieves (Ezload 16 / 60 Chromdex 200 pg purchased from Bogelon or Superdex 200 Increase 10 / 300 GL purchased from Cytiva) and collect the target sample. After concentration, dialyze into 559 buffer (10 mM NaAc-HAc, 9% sucrose, pH 5.5), sterile filter with a 0.22 μm filter, and store aseptically to obtain the purified corresponding antibody.

[0319] 3. Protein purity was analyzed by SDS-PAGE using SurePAGE (Bis-Tris 4%-12% from GenScript) and a PowerPAC BASIC (Bio-Rad) electrophoresis system. Fast Protein Stain (Tanon) was used for staining, and imaging was performed using a Gel Doc EZ (Bio-Rad) gel imaging system. The results showed clear target bands with no smearing, indicating high purity.

[0320] Example 6 Identification of humanized anti-IL-4Rα antibodies

[0321] 6.1 Enzyme-linked immunosorbent assay (ELISA) to detect the binding level of humanized antibodies to hIL4Rα-ECD(26-232)-His and cyno IL4Rα-ECD(26-232)-His proteins

[0322] The binding levels of humanized antibodies to hIL4Rα-ECD(26-232)-His and cyno IL4Rα-ECD(26-232)-His proteins were measured and analyzed using the method of Example 4.1. As shown in Figures 3A and 3B , all humanized antibodies of the present invention effectively bound to the hIL4Rα-ECD(26-232)-His protein. IL4R-hum-18H5L1, IL4R-hum-13H1aL2a, IL4R-hum-14L2H2, and IL4R-hum-04H7L1 also effectively bound to the cyno IL4Rα-ECD(26-232)-His protein.

[0323] 6.2 Enzyme-linked immunosorbent assay (ELISA) to detect the blocking effect of humanized antibodies on the binding of human IL4 to IL4Rα-ECD protein

[0324] The humanized antibodies were used to detect the level of blocking effect on the binding of human IL4 to the IL4Rα-ECD protein, and the data were analyzed using the method described in Example 4.2. The results are shown in Figure 4 and Table 29, indicating that the humanized antibodies of the present invention can effectively block the binding of human IL4 to the IL4Rα-ECD protein.

[0325] Table 29 Humanized antibodies block IL-4 binding to human IL4Rα-ECD protein

[0326] 6.3 Flow cytometry (FACS) assay for binding activity of humanized antibodies to human IL4Rα-293T cells and cyno IL4R-FlpinCHO

[0327] The binding activity of the humanized antibodies to human IL4Rα-293T cells and cyno IL4R-FlpinCHO was assayed and the data analyzed using the method described in Example 4.3. As shown in Figures 5A and 5B , all humanized antibodies of the present invention effectively bound to human IL4Rα-293T cells, while IL4R-hum-18H5L1 and IL4R-hum-13H1aL2a effectively bound to cyno IL4R-FlpinCHO cells.

[0328] 6.4 Detection of humanized antibodies blocking the binding of IL4 to IL-4IL-13 reporter 293 cells

[0329] A. Detection of humanized antibodies blocking the binding of IL4 to IL-4IL-13 Reporter 293 cells

[0330] The humanized antibodies were tested for blocking IL4 binding to IL-4IL-13Reporter 293 cells using the same method as in Example 4.4, and the data were analyzed. As shown in Figure 6A , the humanized antibodies of the present invention were able to effectively block IL4 binding to IL-4IL-13Reporter 293 cells compared to the control antibody.

[0331] B. Detection of humanized antibodies blocking the binding of IL13 to IL-4IL-13 Reporter 293 cells

[0332] IL-4IL-13 Reporter 293 cells were expanded and cultured in T-175 cell culture flasks to 90% confluence. The culture medium was removed, washed once with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and digested with 3 ml of 0.25% trypsin (purchased from Gibico, catalog number SH30256.01) for 2 minutes. The cells were resuspended in DMEM (purchased from Gibco, catalog number 11995-040) containing 2% (v / v) fetal bovine serum. After cell counting, the cells were centrifuged at 1000 rpm for 5 minutes at room temperature and the culture medium was discarded. 2×10 cells were plated per well. 4 50 μL of the test antibody was added to each well of a flat-bottom 96-well plate (purchased from Kawei Biosciences, Catalog No. 062096) and placed in an incubator until ready to use. The test antibody was diluted in DMEM containing 2% (w / w) fetal bovine serum to a starting concentration of 400 nM. After a 10-fold dilution, 25 μL of the test antibody was added to each well and mixed thoroughly. IL13 (purchased from SinoBiological, Catalog No. 10369-HNAC) was diluted in DMEM containing 2% (w / w) fetal bovine serum and 25 μL of the test antibody was added to each well of the plate to give a final IL13 concentration of 1 ng / mL. The cells were incubated in a cell culture incubator for 5 hours. 50 μL of Bright lite (purchased from Novozymes, Catalog No. DD1204) was added to each well and shaken at room temperature for 10 minutes. The results were detected and analyzed using PE-Ensight (purchased from PerkinElmer). The results are shown in FIG6B , showing that the humanized antibodies of the present invention can effectively block the binding of IL13 to IL-4IL-13 Reporter 293 cells.

[0333] 6.5 Detection of humanized antibodies inhibiting the proliferation of TF1-Luc cells

[0334] A. Detection of humanized antibodies to inhibit IL4-promoted TF1-Luc cell proliferation

[0335] The humanized antibody was tested for its ability to inhibit IL4-stimulated TF1-Luc cell proliferation, and data analysis was performed using the same method as in Example 4.5. As shown in Figure 7A , the humanized antibody of the present invention effectively inhibited IL4-stimulated TF1-Luc cell proliferation compared to the control antibody.

[0336] B. Detection of humanized antibodies to inhibit IL13-promoted TF1-Luc cell proliferation

[0337] TF1-Luc cells were cultured in T-175 cell culture flasks. After counting the cells, the cells were centrifuged at 1000 rpm for 5 minutes at room temperature and the culture medium was discarded. The cells were resuspended in RPMI-1640 (Gibco, Cat. No. 22400-089) containing 2% (v / v) fetal bovine serum. After counting the cells, 4 × 10 cells were plated per well. 4 50 μL of the purified antibody from the hybridoma to be tested was added to each well of a flat-bottom 96-well plate (Cave Bio, Catalog No. 062096) and placed in an incubator until ready to use. Dilute the purified antibody from the hybridoma to be tested in RPMI-1640 containing 2% (w / w) fetal bovine serum to a starting concentration of 400 nM, dilute it 10-fold, and add 25 μL of it to the cells per well, mixing thoroughly. IL-13 (Sino Biological, Catalog No. 10369-HNAC) was diluted in RPMI-1640 containing 2% (w / w) fetal bovine serum and added to the cells at a final concentration of 2 ng / mL. Incubate in a cell culture incubator for 5 hours. Add 50 μL of Brightlite (Novozymes, Catalog No. DD1204) to each well and shake at room temperature for 10 minutes. Analyze and analyze the results using PE-Ensight (PerkinElmer). The results are shown in FIG7B . Compared with the control antibody, the humanized antibody of the present invention can effectively inhibit the proliferation of TF1-Luc cells promoted by IL13.

[0338] 6.6 Flow cytometry (FACS) assay to detect the internalization efficiency of humanized antibodies in cells

[0339] A. Detection of the endocytosis efficiency of humanized antibodies and human IL4Rα-293T cells

[0340] The endocytosis efficiency of the humanized antibody and human IL4Rα-293T cells was detected and the data were analyzed according to the method of Example 4.6. The results are shown in Table 30. Compared with the control antibody, the humanized antibody of the present invention has a good endocytosis efficiency.

[0341] Table 30 Internalization of humanized antibodies on human IL4Rα-293T cells

[0342] B. Detection of the internalization efficiency of humanized antibodies on Romas cells

[0343] Romas cells were cultured in T-175 cell culture flasks. After counting the cells, the cells were centrifuged at 1000 rpm for 5 minutes at room temperature and the culture medium was discarded. The cells were resuspended in pre-cooled PBS and the culture medium was 2 × 10 cells per well. 5 For each cell, 100 μL was added to each well of a U-bottom 96-well plate (Corning, Cat. No. 3795). Centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant. Dilute the purified antibody to be tested to 100 nM in PBS containing 2% (w / w) fetal bovine serum. Add 200 μL per well to the cells, mix thoroughly, and incubate on ice for 1 hour. Transfer 100 μL of the sample from the 96-well plate to a new 96-well plate. Wash the duplicate 96-well plates twice at 4°C and centrifuge at 1500 rpm for 5 minutes. After discarding the supernatant, add 100 μL of pre-chilled complete medium to each well. Place one plate at 4°C and the other in a cell culture incubator. Incubate for 2 hours, then place both plates on ice for 5 minutes to terminate endocytosis. Wash twice at 4°C and centrifuge at 1500 rpm for 5 minutes. Diluted fluorescently labeled secondary antibody (purchased from Jackson ImmunoResearch, Cat. No. 109-605-098) was incubated on ice for 1 hour. The cells were washed twice with pre-chilled PBS by centrifugation. Results were detected and analyzed using FACS (FACS Canto II, purchased from BD Biosciences). The results are shown in Table 31.

[0344] Table 31 Endocytosis in Romas cells

[0345] 6.7 Biacore Affinity Detection of Humanized Antibodies

[0346] The affinity of the humanized antibody was tested according to the method of Example 4.7. The results are shown in Table 32.

[0347] Table 32 Biacore detection of affinity of humanized antibodies to hIL4Rα-ECD(26-232)-his

Claims

1. An antibody or antigen-binding fragment that specifically binds to IL4R, characterized in that: The antibody or antigen-binding fragment comprises: (1) a heavy chain variable region (VH), comprising HCDR1, HCDR2 and HCDR3 selected from the group consisting of VH shown in any one of SEQ ID NOs: 10-15, 135-136, 141-143, 147-148, 154-155, 161, and 164-165; (2) a light chain variable region (VL), comprising LCDR1, LCDR2 and LCDR3 selected from the group consisting of the VL shown in any one of SEQ ID NOs: 16-21, 134, 140, 145-146, 153, 158-160, 163.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that The HCDR1, HCDR2 and HCDR3 of the VH set forth in any one of SEQ ID NOs: 10, 135, 136 have the sequences set forth in SEQ ID NOs: 22-24, SEQ ID NOs: 25-27 or SEQ ID NOs: 28-30 according to the Kabat, IMGT or Chothia numbering system; The HCDR1, HCDR2 and HCDR3 of the VH set forth in SEQ ID NOs: 11, 141-143 have the sequences set forth in SEQ ID NOs: 31-33, SEQ ID NOs: 34-36 or SEQ ID NOs: 37-39 according to the Kabat, IMGT or Chothia numbering system; The HCDR1, HCDR2 and HCDR3 of the VH set forth in SEQ ID NO:12 have the sequences set forth in SEQ ID NOs:40-42, SEQ ID NOs:43-45 or SEQ ID NOs:46-48 according to the Kabat, IMGT or Chothia numbering system; The HCDR1, HCDR2 and HCDR3 of the VH set forth in SEQ ID NOs: 13, 154, 155 have the sequences set forth in SEQ ID NOs: 49-51, SEQ ID NOs: 52-54 or SEQ ID NOs: 55-57 according to the Kabat, IMGT or Chothia numbering system; The HCDR1, HCDR2 and HCDR3 of the VH set forth in SEQ ID NOs: 14 and 161 have the sequences set forth in SEQ ID NOs: 58-60, SEQ ID NOs: 61-63 or SEQ ID NOs: 64-66 according to the Kabat, IMGT or Chothia numbering system; The HCDR1, HCDR2 and HCDR3 of the VH set forth in SEQ ID NOs: 15, 164, 165 have the sequences set forth in SEQ ID NOs: 67-69, SEQ ID NOs: 70-72 or SEQ ID NOs: 73-75 according to the Kabat, IMGT or Chothia numbering system; LCDR1, LCDR2 and LCDR3 of the VL set forth in SEQ ID NOs: 16 and 134 have the sequences set forth in SEQ ID NOs: 76-78, SEQ ID NOs: 79-81 or SEQ ID NOs: 82-84 according to the Kabat, IMGT or Chothia numbering system; LCDR1, LCDR2 and LCDR3 of the VL set forth in SEQ ID NOs: 17 and 140 have the sequences set forth in SEQ ID NOs: 85-87, SEQ ID NOs: 88-90 or SEQ ID NOs: 91-93 according to the Kabat, IMGT or Chothia numbering system; LCDR1, LCDR2 and LCDR3 of the VL set forth in SEQ ID NOs: 18 and 145 have the sequences set forth in SEQ ID NOs: 94-96, SEQ ID NOs: 97-99 or SEQ ID NOs: 101-102 according to the Kabat, IMGT or Chothia numbering system; LCDR1, LCDR2 and LCDR3 of the VL set forth in SEQ ID NOs: 19 and 153 have the sequences set forth in SEQ ID NOs: 103-105, SEQ ID NOs: 106-108 or SEQ ID NOs: 109-111 according to the Kabat, IMGT or Chothia numbering system; The LCDR1, LCDR2 and LCDR3 of the VL set forth in SEQ ID NOs: 20, 158, 159 have the sequences set forth in SEQ ID NOs: 112-114, SEQ ID NOs: 115-117 or SEQ ID NOs: 118-120 according to the Kabat, IMGT or Chothia numbering system; LCDR1, LCDR2 and LCDR3 of VL shown in SEQ ID NO: 21, 163 are according to Kabat, IMGT or Chothia numbering system, having a sequence as shown in SEQ ID NO: 121-123, SEQ ID NO: 124-126 or SEQ ID NO: 127-129; The HCDR1, HCDR2 and HCDR3 of the VH set forth in SEQ ID NOs: 147 and 148 have the sequences set forth in SEQ ID NOs: 40, 149 and 42 according to the Kabat numbering system; The LCDR1, LCDR2 and LCDR3 of the VL shown in SEQ ID NO: 146 have the sequences shown in SEQ ID NOs: 94, 150 and 96 according to the Kabat numbering system.

3. The antibody or antigen-binding fragment according to any one of claims 1 to 2, characterized in that: The antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequence: (1) SEQ ID NO: 22, 23, 24, 76, 77, 78, respectively; (2) SEQ ID NO: 25, 26, 27, 79, 80, 81, respectively; (3) SEQ ID NO: 28, 29, 30, 82, 83, 84, respectively; (4) SEQ ID NO: 31, 32, 33, 85, 86, 87, respectively; (5) SEQ ID NO: 34, 35, 36, 88, 89, 90, respectively; (6) SEQ ID NO: 37, 38, 39, 91, 92, 93, respectively; (7) SEQ ID NO: 40, 41, 42, 94, 95, 96, respectively; (8) SEQ ID NO: 43, 44, 45, 97, 98, 99, respectively; (9) SEQ ID NO: 46, 47, 48, 100, 101, 102, respectively; (10) SEQ ID NO: 49, 50, 51, 103, 104, 105, respectively; (11) SEQ ID NO: 52, 53, 54, 106, 107, 108, respectively; (12) SEQ ID NO: 55, 56, 57, 109, 110, 111, respectively; (13) SEQ ID NO: 58, 59, 60, 112, 113, 114, respectively; (14) are SEQ ID NOs: 61, 62, 63, 115, 116, 117, respectively; (15) are SEQ ID NOs: 64, 65, 66, 118, 119, 120, respectively; (16) SEQ ID NO: 67, 68, 69, 121, 122, 123, respectively; (17) SEQ ID NO: 70, 71, 72, 124, 125, 126, respectively; (18) SEQ ID NO: 73, 74, 75, 127, 128, 129, respectively; (19) are SEQ ID NOs: 40, 149, 42, 94, 95, 96, respectively; (20) SEQ ID NO: 40, 149, 42, 94, 150, 96, respectively; (21) A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in any one of (1) to (20) above, or having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NOs: 10-15, 135-136, 141-143, 147-148, 154-155, 161, 164-165; or / and a light chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NOs: 16-21, 134, 140, 145-146, 153, 158-160, 163; Preferably, (1) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 10, and the light chain variable region comprises the sequence shown in SEQ ID NO: 16; (2) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 11, and the light chain variable region comprises the sequence shown in SEQ ID NO: 17; (3) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 12, and the light chain variable region comprises the sequence shown in SEQ ID NO: 18; (4) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 13, and the light chain variable region comprises the sequence shown in SEQ ID NO: 19; (5) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 14, and the light chain variable region comprises the sequence shown in SEQ ID NO: 20; (6) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the sequence shown in SEQ ID NO: 21; (7) the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 135 and 136, and the light chain variable region comprises the sequence shown in SEQ ID NO: 134; (8) the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 141-143, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 140; (9) the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 147 and 148, and the light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 145 and 146; (10) the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 154 and 155, and the light chain variable region comprises the sequence shown in SEQ ID NO: 153; (11) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 161, and the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 158-160; (12) the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 164-165, and the light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 163; (13) The heavy chain variable region and / or the light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in groups (1) to (12); or, a sequence that has at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the sequence shown in groups (1) or (2); the mutations may be selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is selected from IgG, such as IgG1, IgG2, IgG3 or IgG4, and the IgG can be selected from human IgG, such as human IgG4; alternatively, the heavy chain constant region can be selected from the Fc region, the CH3 region or the complete heavy chain constant region, alternatively, the heavy chain constant region is a human Fc region; the light chain constant region is selected from the κ chain or the λ chain, preferably the κ chain.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, characterized in that: The antibody or antigen-binding fragment specifically binds to human or monkey IL4R protein; preferably, the KD of the antibody or antigen-binding fragment binding to human IL4R protein is better than 1.00E-9M.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, characterized in that: The antibody or antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a complete antibody, a fragment of a complete antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, a diabody, or a single domain antibody.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, further coupled to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from drugs, toxins, radioisotopes, chemotherapeutic drugs or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.

10. A multispecific molecule comprising the antibody or antigen-binding fragment of any one of claims 1 to 9; preferably, the multispecific molecule further comprises an antibody or antigen-binding fragment that specifically binds to an antigen other than IL4R or binds to an IL4R epitope different from the antibody or antigen-binding fragment of any one of claims 1 to 9; Preferably, the antigen other than IL4R is selected from the following groups: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) target for recruiting and / or activating immune cells.

11. A chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen binding fragment described in any one of claims 1-9.

12. An immune effector cell, which expresses the chimeric antigen receptor according to claim 11, or comprises a nucleic acid fragment encoding the chimeric antigen receptor according to claim 11; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

13. An isolated nucleic acid fragment encoding the antibody or antigen-binding fragment of any one of claims 1 to 9, or the multispecific molecule of claim 10, or the chimeric antigen receptor of claim 11. A vector comprising the nucleic acid fragment according to claim 13.

15. A host cell comprising the vector of claim 14; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

16. A method for preparing the antibody or antigen-binding fragment of any one of claims 1 to 9 or the multispecific molecule of claim 10, comprising culturing the cell of claim 15, and isolating the antibody or antigen-binding fragment expressed by the cell, or isolating the multispecific molecule expressed by the cell.

17. A method for preparing the immune effector cell of claim 12, comprising introducing a nucleic acid fragment encoding the CAR of claim 11 into the immune effector cell, and optionally, further comprising starting the immune effector cell to express the CAR of claim 11.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-9, or the multispecific molecule of claim 10, or the immune effector cell of claim 12, or the nucleic acid fragment of claim 13, or the vector of claim 14, or the host cell of claim 15, or a product prepared by the method of any one of claims 16-17; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

19. The antibody or antigen-binding fragment of any one of claims 1-9, or the multispecific molecule of claim 10, or the immune effector cell of claim 12, or the nucleic acid fragment of claim 13, or the vector of claim 14, or the host cell of claim 15, or the product obtained by the method of any one of claims 16-17; or the use of the pharmaceutical composition of claim 18 in the preparation of a medicament for preventing and / or treating an immune disease; the immune disease comprises dermatitis, asthma, nasal polyposis, eosinophilic esophagitis, urticaria, prurigo, or other diseases associated with IL4R.

20. A method for preventing and / or treating an immune disease, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment of any one of claims 1-9, or the multispecific molecule of claim 10, or the immune effector cell of claim 12, or the nucleic acid fragment of claim 13, or the vector of claim 14, or the host cell of claim 15, or the product prepared by the method of any one of claims 16-17; or the pharmaceutical composition of claim 18; wherein the immune disease comprises dermatitis, asthma, nasal polyposis, eosinophilic esophagitis, urticaria, prurigo, or other diseases associated with IL4R.

21. The antibody or antigen-binding fragment of any one of claims 1-9, or the multispecific molecule of claim 10, or the immune effector cell of claim 12, or the nucleic acid fragment of claim 13, or the vector of claim 14, or the host cell of claim 15, or the product prepared by the method of any one of claims 16-17; or the pharmaceutical composition of claim 18, for preventing and / or treating immune diseases; wherein the immune diseases include dermatitis, asthma, nasal polyposis, eosinophilic esophagitis, urticaria, prurigo, or other diseases associated with IL4R.

22. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 9, or the multispecific molecule of claim 10, or the immune effector cell of claim 12, or the nucleic acid fragment of claim 13, or the vector of claim 14, or the host cell of claim 15, or the product prepared by the method of any one of claims 16 to 17, or the pharmaceutical composition of claim 18.

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