Anti-IL-4R antibody or its antigen-binding fragment complex and pharmaceutical applications

Abstract

The present invention provides conjugates of anti-IL-4R antibodies or antigen-binding fragments thereof and pharmaceutical uses thereof. Specifically, the present invention provides conjugates of antibodies or antigen-binding fragments thereof that specifically bind to IL-4R covalently linked to toxins, pharmaceutical compositions containing the conjugates, and uses thereof in the preparation of medicaments for treating IL-4R-mediated diseases or conditions, particularly in the preparation of anti-cancer agents.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 202011529235.1, filed on 22 December 2020.

[0002] This application relates to a complex of an anti-IL-4R antibody or its antigen-binding fragment, and its use as an anticancer agent. [Background technology]

[0003] Malignant tumors (cancer) are the second leading cause of death worldwide, after heart disease. Glioblastoma (GBM) is the most common primary malignant brain tumor. Patients with glioblastoma treated with standard therapies have a median survival time of less than 15 months. Although research in tumor biology is progressing, it has not translated into products or therapies that benefit patients. Therefore, the development of effective methods for treating glioblastoma is urgently needed.

[0004] IL-4R is an interleukin-4 receptor expressed in glioblastoma tumor cells and invasive immunosuppressive cells (e.g., tumor-associated macrophages (TAMs), myeloid-derived immunosuppressive cells (MDSCs)). Therapies targeting IL-4R can not only kill tumor cells but also improve the tumor microenvironment. In preclinical animal models and early clinical trials of glioblastoma, IL-4R-targeted therapies have consistently shown superior efficacy compared to conventional therapies.

[0005] MDNA55, developed by Medicenna, is a fusion protein formed from IL-4 and the bacterial toxin Pseudomonas aeruginosa exotoxin A (PE38KDEL), and has shown positive therapeutic effects in clinical development trials (see WO9527732, Biochem. J. (1995) 307, 29-37, CANCER RESEARCH 55, 3357-3363, August 1, 1995).

[0006] However, first-generation immunotoxin therapies have problems such as insufficient affinity to their targets. Improving the targeting and affinity of immunotoxins has become one of the focus areas and challenges in research on tumor immunotoxin therapy (see "China Clinical Oncology Yearbook 2009," edited by Zhao Ping, China Union Medical College Press, July 2010, p. 61).

[0007] Antibody-drug conjugates (ADCs) are intended to combine the selectivity of monoclonal antibodies (mAbs) with the cytotoxic potential of chemotherapeutic drugs. An antibody-drug conjugate consists of three main components: an antibody, a linker, and a drug molecule. Compared to conventional fully or partially humanized antibodies or antibody fragments, antibody-drug conjugates theoretically offer higher therapeutic efficacy because they can release highly active cytotoxins in tumor tissue, and they exhibit higher resistance or lower side effects compared to fusion proteins. The affinity of an antibody-drug conjugate to its target antigen influences the process by which it penetrates tumor tissue and binds to the target antigen, and directly affects the binding efficiency of the antibody-drug conjugate (see "Biotechnology Drug Discovery," edited by Feng Meiqing, China Pharmaceutical and Technology Press, January 2016, pp. 209-211).

[0008] When designing antibody-drug conjugates, antibody selection is one of the most important factors, and high specificity for the antigen is crucial. Antibodies that do not have high specificity and cross-react with other antigens cannot perform the intended action, for example, causing off-target toxicity by interacting with healthy tissue or being cleared by the body prematurely before reaching the tumor site (see "Introduction to Antibody-Drug Conjugates (ADCs)," Ilona Pysz, Paul JM Jackson and David E. Thurston, CHAPTER 1: Introduction to Antibody-Drug Conjugates (ADCs), in Cytotoxic Payloads for Antibody-Drug Conjugates, 2019).

[0009] Currently, numerous pharmaceutical companies in various countries are working on the development of monoclonal antibodies against IL-4R, and related patent applications include, for example, WO2010053751, WO2001092340, WO2008054606, and WO2014031610. The inventor's prior application, WO2020038454, also relates to a novel anti-IL-4R antibody. However, antibody-drug conjugates targeting IL-4R have rarely been clearly reported. For example, WO2015188934, WO2014124227, and WO2018217227 all generally mention that the antibody in the antibody-drug conjugate may selectively target IL-4R, but do not disclose specific anti-IL-4R antibody-drug conjugates.

[0010] This disclosure describes the preparation of a second-generation immunotoxin drug that targets and delivers drugs using a high-affinity anti-IL-4R antibody, in order to achieve the objective of potently killing cancer cells and to meet the demands for cancer treatment and delaying cancer progression. [Overview of the Initiative]

[0011] This disclosure relates to a complex of an anti-IL-4R antibody or its antigen-binding fragment, a method for preparing it, and its pharmaceutical applications. compound

[0012] This disclosure is, An anti-IL-4R antibody or its antigen-binding fragment, and one or more toxin molecules, The present invention provides a complex of an anti-IL-4R antibody or its antigen-binding fragment, which includes the above.

[0013] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the complex includes a heavy chain variable region and a light chain variable region, of which the heavy chain variable region is (I) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or (II) comprising HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. and / or, the antibody light chain variable region is (I) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs. 6, 7 and 8, respectively, or (II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or (III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively, or (IV) comprising LCDR1, LCDR2, and LCDR3, whose amino acid sequences are shown in SEQ ID NO: 42, SEQ ID NO: 39, and SEQ ID NO: 8, respectively. Of these, the anti-IL-4R antibody or its antigen-binding fragment covalently or noncovalently binds to the toxin.

[0014] [Table 1]

[0015] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment is (I) to (IV) below, i.e., (I) Heavy chain variable regions including HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NOs. 3, 4, and 5, respectively, and Light chain variable regions including LCDR1, LCDR2, and LCDR3, whose amino acid sequences are shown in SEQ ID NOs. 6, 7, and 8, respectively. (II) Heavy chain variable regions including HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and Light chain variable regions including LCDR1, LCDR2, and LCDR3, whose amino acid sequences are shown in SEQ ID NOs. 14, 15, and 16, respectively. (III) A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 whose amino acid sequences are represented by SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and A light chain variable region comprising LCDR1, LCDR2, and LCDR3 whose amino acid sequences are represented by SEQ ID NO: 38, SEQ ID NO: 7, and SEQ ID NO: 40, respectively, (IV) A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 whose amino acid sequences are represented by SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and A light chain variable region comprising LCDR1, LCDR2, and LCDR3 whose amino acid sequences are represented by SEQ ID NO: 42, SEQ ID NO: 39, and SEQ ID NO: 8, respectively, comprises any one selected from the following.

[0016] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (I) a sequence represented by SEQ ID NO: 1, or a sequence having at least 70%, 80%, 90%, 95%, 98%, 99% identity with SEQ ID NO: 1, or (II) a sequence represented by SEQ ID NO: 9, or a sequence having at least 70%, 80%, 90%, 95%, 98%, 99% identity with SEQ ID NO: 9, or <x (III) a sequence represented by SEQ ID NO: 43, or a sequence having at least 70%, 80%, 90%, 95%, 98%, 99% identity with SEQ ID NO: 43, and / or and / or the light chain variable region (I) a sequence represented by SEQ ID NO: 2, or a sequence having at least 70%, 80%, 90%, 95%, 98%, 99% identity with SEQ ID NO: 2, or (II) a sequence represented by SEQ ID NO: 10, or a sequence having at least 70%, 80%, 90%, 95%, 98%, 99% identity with SEQ ID NO: 10, or (III) a sequence represented by SEQ ID NO: 37, or a sequence having at least 70%, 80%, 90%, 95%, 98%, 99% identity with SEQ ID NO: 37, or (IV) A sequence represented by sequence number 41, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 41.

[0017] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0018] In at least one embodiment, the anti-IL-4R antibody or antigen-binding fragment is: The sequence of the heavy chain variable region is shown in sequence number 1, and the sequence of the light chain variable region is shown in sequence number 2, or The sequence of the heavy chain variable region is shown in sequence number 9, and the sequence of the light chain variable region is shown in sequence number 10, or The sequence of the heavy chain variable region is shown in sequence number 43, and the sequence of the light chain variable region is shown in sequence number 37, or The sequence of the heavy chain variable region is shown in sequence number 43, and the sequence of the light chain variable region is shown in sequence number 41, or The sequence of the heavy chain variable region is shown in sequence number 47, and the sequence of the light chain variable region is shown in sequence number 48.

[0019] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, of which, The heavy chain variable region is, (I) A sequence represented by one of sequence numbers 25 to 27, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 25 to 27, (II) A sequence that is represented by one of sequence numbers 31 to 33, or has at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 31 to 33, and / or, the light chain variable region is (I) A sequence represented by one of sequence numbers 28 to 30, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 28 to 30, (II) A sequence that is represented by one of sequence numbers 34 to 36, or has at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 34 to 36.

[0020] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0021] In some specific embodiments, the arrangement of the heavy chain variable region is shown in one of sequence numbers 25 to 27, and the arrangement of the light chain variable region is shown in one of sequence numbers 28 to 30, or The sequence of the heavy chain variable region is shown by one of sequence numbers 31 to 33, and the sequence of the light chain variable region is shown by one of sequence numbers 34 to 36.

[0022] In some embodiments, the amino acids at position 44 (VH-44) in the heavy chain variable region and position 100 (VL-100) in the light chain variable region are selectively mutated to cysteine.

[0023] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises a heavy chain and a light chain, of which the heavy chain is (I) A sequence shown in sequence number 17, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 17, (II) A sequence shown in sequence number 19, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 19, (III) A sequence represented by sequence number 44, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 44, and / or, the light chain, (I) A sequence shown in sequence number 18, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 18, (II) A sequence shown in sequence number 20, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 20, (III) A sequence shown in SEQ ID NO: 45, or a sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 45, (IV) A sequence represented by sequence number 46, or a sequence having at least 90%, 95%, 98%, or 99% identity with sequence number 46.

[0024] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0025] In at least one embodiment, the heavy chain sequence is shown in SEQ ID NO: 17, and the light chain sequence is shown in SEQ ID NO: 18, or The heavy chain sequence is shown in SEQ ID NO: 19, and the light chain sequence is shown in SEQ ID NO: 20, or The heavy chain sequence is shown in SEQ ID NO: 44, and the light chain sequence is shown in SEQ ID NO: 45, or The heavy chain sequence is shown in sequence number 44, and the light chain sequence is shown in sequence number 46.

[0026] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment is a mouse antibody, a chimeric antibody, a fully human antibody, a humanized antibody, or a fragment thereof. In some specific embodiments, the anti-IL-4R antibody or antigen-binding fragment is humanized.

[0027] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises an FR region sequence derived from the human germline light chain template IGKV3-11*01 (SEQ ID NO: 22, for antibody 25G7) or a revertant mutation sequence having at least 95% identity thereto. In some specific embodiments, the revertant mutation is selected from one or more of 46P, 47W, and 71Y.

[0028] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises an FR region sequence derived from the human germline heavy chain template IGHV3-48*01 (SEQ ID NO: 21, for antibody 25G7) or a revertant mutation sequence having at least 95% identity thereto. In some specific embodiments, the revertant mutation is selected from one or more of 94A, 67S, and 93T.

[0029] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises an FR region sequence derived from the human germline light chain template IGKV2D-29*01 (SEQ ID NO: 24, for antibody 7B10) or a revertant mutation sequence having at least 95% identity thereto. In some specific embodiments, the revertant mutation is selected from 4L and / or 58I.

[0030] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises an FR region sequence derived from the human germline heavy chain template IGHV1-2*02 (SEQ ID NO: 23, for antibody 7B10) or a revertant mutation sequence having at least 95% identity thereto. In some specific embodiments, the revertant mutation is selected from one or more of 69L, 71I, 73K, and 94K.

[0031] [ka] [ka] [ka]

[0032] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises a heavy chain constant region or a variant thereof selected from human IgG1, IgG2, IgG3, and IgG4. In some specific forms, it comprises the heavy chain constant region or a variant thereof of human IgG1. In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises the constant regions of human κ, λ chains or variants thereof.

[0033] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment is a humanized antibody, the heavy chain sequence is shown in SEQ ID NO: 17 or has at least 85% sequence identity thereto, and the light chain sequence is shown in SEQ ID NO: 18 or has at least 85% sequence identity thereto.

[0034] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment is such that the antibody is a humanized antibody, the heavy chain sequence is shown in SEQ ID NO: 19 or has at least 85% sequence identity thereto, and the light chain sequence is shown in SEQ ID NO: 20 or has at least 85% sequence identity thereto.

[0035] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment is such that the antibody is a humanized antibody, the heavy chain sequence is represented by SEQ ID NO: 44 or has at least 85% sequence identity thereto, and the light chain sequence is represented by SEQ ID NO: 45 or has at least 85% sequence identity thereto.

[0036] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment is a humanized antibody, the heavy chain sequence is represented by SEQ ID NO: 44 or has at least 85% sequence identity thereto, and the light chain sequence is represented by SEQ ID NO: 46 or has at least 85% sequence identity thereto.

[0037] In some embodiments, isolated anti-IL-4R antibodies or antigen-binding fragments thereof are provided, characterized in that they bind to human IL-4R or its epitope in competition with any one of the above-described anti-IL-4R antibodies or its antigen-binding fragment.

[0038] In some embodiments, a bispecific or multispecific antibody is provided that includes any one of the above-described anti-IL-4R antibodies or its antigen-binding fragment, comprising a light chain variable region and / or a heavy chain variable region.

[0039] In some other embodiments, a single-chain antibody is provided comprising any one of the above-described anti-IL-4R antibodies or its antigen-binding fragment, or a light chain variable region and / or heavy chain variable region.

[0040] In some embodiments, the humanized anti-IL-4R antibody or its antigen-binding fragment further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In at least one embodiment, the human IgG2 or IgG4 heavy chain constant region is included because IgG2 or IgG4 is not ADCC toxic. In another embodiment, IgG1 that is not ADCC (antibody-dependent cell-mediated cytotoxicity) toxic after amino acid mutation is used. In at least one embodiment, the variant comprises heavy chain constant region mutations in which ADCC effector function is reduced or deleted, such as IgG1 297A, 234A, and 235A, for example, but not limited to these.

[0041] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment may be an antibody variant having 1 to 10 amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) in the light chain and / or 1 to 10 amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) in the heavy chain.

[0042] In some embodiments, the above-mentioned variants have biological functions or effects similar to or similar to those of the parental anti-IL-4R antibody or its fragments.

[0043] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment may be a known one, for example, the anti-IL-4R antibody or its antigen-binding fragment described in WO2010053751, WO2001092340, WO2008054606, WO2014031610, and WO2020038454 (each incorporated herein by reference).

[0044] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment includes, but is not limited to, Dupixent, PRS-060, AK-120, 63IgG1, CBP201, AMG-317, or its antigen-binding fragment.

[0045] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment is an anti-human IL-4R antibody or its antigen-binding fragment.

[0046] In some embodiments, the antigen-binding fragments include, but are not limited to, Fab, Fab', Fv, F(ab')2, linear antibodies, scFv (single-chain Fv antibody), tandem di-scFv, tandem tri-scFv, double-chain antibodies (diabody), triple-chain antibodies (triabody), quadruple-chain antibodies (tetrabody), sdAb (single-domain antibody or nano-antibody), sdFv, peptide antibodies (peptibody), domain antibodies, multispecific antibodies (e.g., bispecific, triplicate, or quadruplicate antibodies), dsFv (disulfide-bond-stabilized Fv), and ScdsFv (disulfide-bond-stabilized single-chain Fv antibody).

[0047] In some embodiments, an anti-IL-4R antibody or antigen-binding fragment is subjected to mutations that stabilize the antibody structure, for example, several amino acid residues are mutated to cysteine ​​residues. In some specific embodiments, the mutation occurs in the framework region; in some specific embodiments, the mutation occurs in the heavy chain variable region and / or the light chain variable region; and in some specific embodiments, the mutation occurs in the heavy chain or light chain variable region and the framework region.

[0048] In some embodiments, an amino acid residue at a position selected from one or more of the following groups: VL100 and VH44, VL101 and VH44, VL34 and VH100, VL43 and VH91, VL43 and VH103, VL49 and H100, VL87 and VH45, VL91 and VH98, VL91 and VH99, VL96 and VH47, and VL98 and VH45, is mutated to a cysteine ​​residue.

[0049] In some specific embodiments, an amino acid residue at a position selected from one or more of the following groups: VL100 and VH44, VL43 and VH91, VL43 and VH103, and VL98 and VH45, is mutated into a cysteine ​​residue.

[0050] In one specific embodiment, the amino acids at positions VH44 and VL100 are mutated to cysteine.

[0051] Unless otherwise specified, the amino acid sequences of the anti-IL-4R antibodies or their antigen-binding proteins relating to this disclosure are encoded by Kabat.

[0052] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment is scFv or ScdsFv, and the heavy chain variable region (VH) and the light chain variable region (VL) are linked by a linking peptide L2.

[0053] In some embodiments, the linker peptide L2 is greater than and less than the length of 12 amino acid residues and is rich in nonpolar amino acid residues, polar amino acid residues and / or hydrophilic amino acid residues, such as glycine, serine and threonine.

[0054] In some embodiments, the linker peptide L2 may be selected from a plurality of linkers known in the prior art, and may include a "GS" linker, for example, (GxS)n, (SxG)n, (GGGGS)n, (G)n, where x is an integer from 1 to 6 and n is an integer from 1 to 30.

[0055] In some embodiments, non-limiting examples of linker peptide L2 include GKSSGSGSESKS (SEQ ID NO: 54), EGKSSGSGSESKEF (SEQ ID NO: 55), GTSTGSGKSSEGKG (SEQ ID NO: 56), GTSTGSGKSSEGSGSTKG (SEQ ID NO: 57), GTSTGSGKPGSGEGSTKG (SEQ ID NO: 58), SRSSG (SEQ ID NO: 59), and SGSSC (SEQ ID NO: 60).

[0056] In some embodiments, the linker peptide L2 is composed of a repeating GGGGS amino acid sequence (SEQ ID NO: 61) or a variant thereof, for example, (GGGGS)n, where n may be 0, 1, 2, 3, 4, 5 or more, for example, n is 2, 3, 4, or for example, n is 3.

[0057] In some embodiments, the structure of ScFv is VH-L2-VL, where L2 is connected to the C-terminus of VH and also to the N-terminus of VL. In some embodiments, the structure of ScFv is VL-L2-VH, where L2 is connected to the C-terminus of VL and also to the N-terminus of VH. In some embodiments, the structure of ScdsFv is VH-L2-VL, where L2 is connected to the C-terminus of VH and also to the N-terminus of VL. In some embodiments, the structure of ScdsFv is VL-L2-VH, where L2 is connected to the C-terminus of VL and also to the N-terminus of VH.

[0058] In some embodiments, the toxin is selected from bacterial toxins, animal toxins, or plant toxins.

[0059] In some embodiments, the toxin comprises a cleavage toxin or a toxin variant, the toxin variant comprising a deletion, insertion, substitution, or combination thereof of an amino acid.

[0060] Some toxins include pore-forming toxins.

[0061] In some embodiments, the pore-forming toxin comprises aerolysin or proaerolysin.

[0062] In some embodiments, the toxin includes bouganin, ricin, Pseudomonas exotoxin (PE), cholera toxin, or diphtheria toxin.

[0063] In some embodiments, the toxin includes, but is not limited to, cytotoxic fragments or cytotoxic variants of Pseudomonas aeruginosa exotoxins. For example, one or more of the following types of PE-LR, PE-LO10R456A, PE-T20, PE-T20-KDEL, PE4E, PE40, PE38, PE24, PE25, PE38QQR, PE38KDEL, and PE35 as described in U.S. Patents 4,892,827, 5,512,658, 5,602,095, 5,608,039, 5,821,238, 5,854,044, 8,871,906, 8,907,060, 8,936,792, 9,346,859, 9,206,240, and 9,388,222 (each incorporated herein by reference).

[0064] In some embodiments, the toxin includes PE38KDEL, shown in SEQ ID NO: 49, and variants such as PE38DKEL, PE38RDEL, and PE38KNEL, which have similar functions, may also be used.

[0065] The anti-IL-4R antibody or the antigen-binding fragment complex relating to this disclosure further selectively comprises linker L1.

[0066] Linker L1 links the toxin to the anti-IL-4R antibody or its antigen-binding fragment.

[0067] A suitable linker L1 generally folds each component of the disclosure into a three-dimensional structure, such a three-dimensional structure being very similar to the structure formed when each component is absent from any linker or other components.

[0068] In some embodiments, linker L1 is stable extracellularly, allowing ADCs to remain intact when present in the extracellular environment, but becoming cleaved when internalized in cells such as cancer cells.

[0069] In some embodiments, a suitable linker L1 may include, for example, a protease-sensitive, environmental redox potential-sensitive, pH-sensitive, acid-cleavable, photocleavable, and / or high-temperature-sensitive linker.

[0070] In some embodiments, linker L1 may be non-proteinaceous, for example, a chemical linker. Examples of non-proteinaceous chemical linkers include N-succinimidyl(4-iodoacetyl)-aminobenzoic acid, S-(N-succinimidyl)thioacetic acid (SATA), N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridinyldithio)toluene (SMPT), N-succinimidyl 4-(2-pyridinyldithio)-pentanoic acid (SPP), succinimidyl 4-(N-maleimidemethyl) (L)Cyclohexanecarboxylic acid ester (SMCC or MCC), sulfosuccinimidyl (4-iodoacetyl)-aminobenzoic acid ester, 4-succinimidyl-oxycarbonyl-α-(2-pyridinyldithio)toluene, sulfosuccinimidyl-6-(α-methyl-α-(pyridinyldithio)-tolueneamide)caproic acid ester, N-succinimidyl-3-(-2-pyridinyldithio)-propionic acid ester (SPDP), succinimidyl 6 (3(-(-2-pyridinyldithio)-propionamide) caproate, sulfosuccinimidyl 6 (3(-(-2-pyridinyldithio)-propionamide) caproate, maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), 3-maleimidobenzoate N-hydroxysuccinimid (MB This includes, but is not limited to, S), α-alkyl derivatives, sulfon-NHS-ATMBA (sulfosuccinimidyl N-[3-(acetylthio)-3-methylbutyryl-β-alanine]), sulfodichlorophenol, 2-iminothiolane, 3-(2-pyridinyldithio)-propionylhydrazide, Ellman reagent, dichlorotriazine, and S-(2-thiopyridinyl)-L-cysteine.

[0071] In some embodiments, the complex formed by the anti-IL-4R antibody or its antigen-binding fragment and the toxin is in the form of a fusion protein.

[0072] In some embodiments, linker L1 is a protein linker and contains one or more amino acids or polypeptides, but is not limited thereto. Linker L1 typically contains about 2 to 50 amino acid residues, e.g., about 5 to 30 amino acid residues. Generally, protein linkers contain amino acid residues that are mostly polar, uncharged and / or charged, e.g., threonine, proline, glutamine, glycine, and alanine. Non-limiting examples of protein linkers include alanine-serine-glycine-glycine-proline-glutamic acid (ASGGPE) (SEQ ID NO: 50), valine-methionine (VM), alanine-methionine (AM), and AM(G2-4S)xAM, where G is glycine, S is serine, and x is an integer from 1 to 10.

[0073] In some embodiments, linker L1 is selected from peptides listed with standard single-letter codes: ASGCGPE (SEQ ID NO: 62), ASGCGPE (SEQ ID NO: 63), ASGCGSCPE (SEQ ID NO: 64), ASGTTGCPE (SEQ ID NO: 65), KASGKKYGCKKGPE (SEQ ID NO: 66), and KGGGCAGGPE (SEQ ID NO: 67).

[0074] Each component of the complex relating to this disclosure, for example, an anti-IL-4R antibody or antigen-binding fragment, linker L1, and toxin, can be appropriately linked or fused with one another as is well known in the art and / or as described herein.

[0075] In some embodiments, an anti-IL-4R antibody or antigen-binding fragment links or fuses with the toxin via linker L1.

[0076] In some embodiments, the C-terminus of the anti-IL-4R antibody or antigen-binding fragment is linked to or fused with linker L1. In some embodiments, the N-terminus of the anti-IL-4R antibody or antigen-binding fragment is linked to or fused with linker L1. In some embodiments, the C-terminus and N-terminus of the anti-IL-4R antibody or antigen-binding fragment are linked to or fused with homologous or heterologous linker L1, respectively.

[0077] In some embodiments, the toxin is a Pseudomonas aeruginosa exotoxin, a cleaved fragment of a Pseudomonas aeruginosa exotoxin, or a toxin variant.

[0078] In some embodiments, the toxin is PE38, a cleaved fragment of the Pseudomonas aeruginosa exotoxin.

[0079] In some embodiments, the toxin is PE38KDEL, represented by SEQ ID NO: 49, and variants such as PE38DKEL, PE38RDEL, and PE38KNEL, which have similar functions, may be used.

[0080] In some embodiments, L1 is a protein linker.

[0081] In some embodiments, L1 is ASGGPE (SEQ ID NO: 50), ASGCCGPE (SEQ ID NO: 63), ASGCGSCPE (SEQ ID NO: 64), ASCGTTGCPE (SEQ ID NO: 65), KASGKKYGCKKGPE (SEQ ID NO: 66), KGGGCAGGPE (SEQ ID NO: 67), AM(G 2-4 S) x Selected from AM, where x is an integer between 1 and 10.

[0082] In some embodiments, ScFv specifically binds to IL-4R, with VH and VL linked by the linker peptide L2, and VH and VL are modified to stabilize the antibody structure within the framework region. In some embodiments, the amino acids at positions VH-44 and VL-100 are mutated to cysteine.

[0083] In some embodiments, the ScFv configuration is VH-L2-VL or VL-L2-VH.

[0084] L2 consists of a repeating GGGGS amino acid sequence or a variant thereof, for example, (GGGGS)n, where n may be 0, 1, 2, 3, 4, 5 or more, for example n is 2, 3, 4, or for example n is 3.

[0085] In some embodiments, in the above ScFv, the above VH is (I) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or (II) comprising HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. and / or the above VL is, (I) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs. 6, 7 and 8, respectively, or (II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or (III) LCDR1, LCDR2 and LCDR3, respectively, as indicated by Sequence ID No. 38, Sequence ID No. 7 and Sequence ID No. 40, or (IV) Includes LCDR1, LCDR2, and LCDR3, respectively, as shown in Sequence ID No. 42, Sequence ID No. 39, and Sequence ID No. 8.

[0086] In some embodiments, the ScFv is (I) to (IV) below, namely, (I) VH containing HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, VL containing LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NOs. 6, 7, and 8, respectively. (II) VH containing HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and VL containing LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NOs. 14, 15, and 16, respectively. (III) VH containing HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, VL containing LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 38, SEQ ID NO: 7, and SEQ ID NO: 40, respectively. (IV) VH containing HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, VL containing LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 42, SEQ ID NO: 39, and SEQ ID NO: 8, respectively. It includes one of the following.

[0087] In some embodiments, the above ScFv, VH is (I) A sequence indicated by Sequence ID No. 1, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with Sequence ID No. 1, (II) A sequence indicated by sequence number 9, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 9, (III) A sequence that is represented by Sequence ID No. 43, or has at least 70%, 80%, 90%, 95%, 98%, or 99% identity with Sequence ID No. 43, or And / or, VL is (I) A sequence shown in Sequence ID No. 2, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with Sequence ID No. 2, (II) A sequence shown in SEQ ID NO: 10, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 10, (III) Sequences represented by Sequence ID No. 37, or sequences having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with Sequence ID No. 37, (IV) A sequence represented by sequence number 41, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 41.

[0088] Selectively, mutations are introduced into VH and VL to stabilize the antibody structure within the framework region, and in some embodiments, the amino acids at the VH-44 and VL-100 positions are mutated to cysteine.

[0089] In at least one embodiment, the VH sequence in the above ScFv is indicated by sequence number 1, the VL sequence is indicated by sequence number 2, or The VH sequence is indicated by sequence number 9, and the VL sequence is indicated by sequence number 10, or The VH sequence is shown in sequence number 43, and the VL sequence is shown in sequence number 37, or The VH sequence is shown as sequence number 43, and the VL sequence is shown as sequence number 41. Selectively, mutations that stabilize the antibody structure are introduced into the framework region of VH and VL.

[0090] In some embodiments, the amino acids at positions VH-44 and VL-100 are mutated to cysteine. The VH sequence is shown in SEQ ID NO: 47, and the VL sequence is shown in SEQ ID NO: 48.

[0091] In some embodiments, the above ScFv, VH is (I) A sequence represented by one of sequence numbers 25 to 27, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 25 to 27, (II) A sequence that is represented by one of sequence numbers 31 to 33, or has at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 31 to 33, And / or, VL is (I) A sequence represented by one of sequence numbers 28 to 30, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 28 to 30, (II) A sequence that is represented by one of sequence numbers 34 to 36, or has at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of sequence numbers 34 to 36, Selectively, mutations are introduced into VH and VL to stabilize the antibody structure within the framework region. In some embodiments, the amino acids at the VH-44 and VL-100 positions are mutated to cysteine.

[0092] In some specific embodiments, the VH sequence is represented by one of SEQ ID NOs. 25 to 27, and the VL sequence is represented by one of SEQ ID NOs. 28 to 30, and the amino acids at positions VH-44 and VL-100 are mutated to cysteine, or The VH sequence is represented by one of sequence numbers 31 to 33, and the VL sequence is represented by one of sequence numbers 34 to 36, with the amino acids at positions VH-44 and VL-100 being mutated to cysteine.

[0093] Selectively, mutations are introduced into VH and VL to stabilize the antibody structure within the framework region. In some embodiments, the amino acids at the VH-44 and VL-100 positions are mutated to cysteine.

[0094] In some embodiments, the ScFv includes a heavy chain and a light chain, of which the heavy chain is (I) A sequence shown in sequence number 17, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 17, (II) A sequence shown in sequence number 19, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 19, (III) A sequence represented by sequence number 44, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 44, and / or, the light chain, (I) A sequence shown in sequence number 18, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 18, (II) A sequence shown in sequence number 20, or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with sequence number 20, (III) A sequence shown in SEQ ID NO: 45, or a sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 45, (IV) A sequence represented by sequence number 46, or having at least 90%, 95%, 98%, or 99% identity with sequence number 46, Selectively, mutations are introduced into VH and VL to stabilize the antibody structure within the framework region. In some embodiments, the amino acids at the VH-44 and VL-100 positions are mutated to cysteine.

[0095] In at least one embodiment, the VH sequence of the heavy chain variable region is shown in SEQ ID NO: 17, and the VL sequence of the light chain variable region is shown in SEQ ID NO: 18, or The VH sequence is shown in SEQ ID NO: 19, and the VL sequence is shown in SEQ ID NO: 20, where the amino acids at positions VH-44 and VL-100 are mutated to cysteine, or The VH sequence is shown in SEQ ID NO: 44, and the VL sequence is shown in SEQ ID NO: 45, where the amino acids at positions VH-44 and VL-100 are mutated to cysteine, or The VH sequence is shown in SEQ ID NO: 44, and the VL sequence is shown in SEQ ID NO: 46, in which the amino acids at positions VH-44 and VL-100 are mutated to cysteine.

[0096] In some embodiments, the anti-IL-4R antibody or the complex of its antigen-binding fragment has the sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53.

[0097] This disclosure further provides a complex of an anti-IL-4R antibody or an antigen-binding fragment thereof, comprising an anti-IL-4R antibody or an antigen-binding fragment thereof and one or more toxins, wherein the toxin is selected from pore-forming toxins, aerolysin, proaerolysin, bougain, lysine, Pseudomonas aeruginosa exotoxin, cholera toxin, or diphtheria toxin, preferably the toxin is selected from PE-LR, PE-LO10R456A, PE-T20, PE-T20-KDEL, PE4E, PE40, PE38, PE24, PE25, PE38QQR, PE35, PE38KDEL, PE38DKEL, PE38RDEL, PE38KNEL, and more preferably the toxin is PE38KDEL, represented by Sequence ID No. 49.

[0098] This disclosure provides polynucleotides encoding a complex of any one of the anti-IL-4R antibodies or its antigen-binding fragments described herein. In some embodiments thereof, the polynucleotide is DNA or RNA.

[0099] This disclosure provides a vector comprising the above-mentioned polynucleotide, which is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0100] This disclosure provides host cells containing the above vector, the host cells being selected from prokaryotic or eukaryotic cells. In at least one embodiment, the prokaryotic cells are selected from bacteria, for example, Escherichia coli. In at least one embodiment, the eukaryotic cells are selected from yeast or mammalian cells, for example, Pichia yeast or CHO cells or human embryonic kidney (HEK) 293 cells.

[0101] This disclosure relates to a method for preparing a complex of an anti-IL-4R antibody or its antigen-binding fragment, The steps include: expressing a complex of anti-IL-4R antibody or its antigen-binding fragment in the host cells; isolating the complex of anti-IL-4R antibody or its antigen-binding fragment from the host cells; and optionally purifying the complex of anti-IL-4R antibody or its antigen-binding fragment. Provides a method for including. Pharmaceutical composition

[0102] This disclosure further provides a pharmaceutical composition for use as a drug, comprising a complex of the above-mentioned anti-IL-4R antibody or its antigen-binding fragment as an active ingredient.

[0103] The pharmaceutical compositions of this disclosure may contain, in addition to the active ingredient, one or more pharmaceutically acceptable excipients, diluents, or vectors (vehicles). The pharmaceutical compositions may contain 0.1% to 99% by weight of the active ingredient.

[0104] This disclosure further provides uses in the preparation of any one or combination of anti-IL-4R antibodies or their antigen-binding fragments, complexes of anti-IL-4R antibodies or their antigen-binding fragments, or pharmaceutical compositions according to this disclosure, wherein the antibodies or their antigen-binding fragments or complexes are used to treat and / or prevent proliferative disorders or to delay the progression of proliferative disorders. The proliferative disorder may be cancer or a tumor, for example, the cancer or tumor being related to IL-4R expression. The cancers or tumors listed above are selected from prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma), lung cancer, kidney cancer, liver cancer (e.g., small cell lung cancer and non-small cell lung cancer), colorectal cancer (e.g., colon cancer), pancreatic cancer, stomach cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), brain cancer, and central nervous system tumors.

[0105] In some embodiments, the above-mentioned central nervous system tumors include glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, meningioma, neuroblastoma, retinoblastoma, and adult-type blastoma.

[0106] In some embodiments, the central nervous system tumor is a glioblastoma.

[0107] In some embodiments, the glioblastoma is a recurrent or refractory glioblastoma.

[0108] In some embodiments, the expression of O6-methylguanine-DNA methyltransferase (MGMT) in the glioblastoma is positive or negative. Treatment method

[0109] This disclosure provides a method for treating and / or preventing a proliferative disorder or delaying the progression of a proliferative disorder, the method comprising administering to a subject as needed an effective amount of an anti-IL-4R antibody or its antigen-binding fragment according to this disclosure, or a pharmaceutical composition according to this disclosure, or a complex of an anti-IL-4R antibody or its antigen-binding fragment according to this disclosure, wherein the proliferative disorder may be cancer or a tumor.

[0110] This disclosure provides a method for improving immune function in subjects who have, are at risk of having, or are susceptible to a cell-proliferative disorder. In some embodiments, the cell-proliferative disorder is cancer or a tumor.

[0111] For example, the cancers or tumors mentioned above are cancers or tumors associated with IL-4R expression. These cancers or tumors are selected from prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma), lung cancer, kidney cancer, liver cancer (e.g., small cell lung cancer and non-small cell lung cancer), colorectal cancer (e.g., colon cancer), pancreatic cancer, gastric cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), brain cancer, and central nervous system tumors.

[0112] In some embodiments, the above-mentioned central nervous system tumors include glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, meningioma, neuroblastoma, retinoblastoma, and adult-type blastoma.

[0113] In some embodiments, the central nervous system tumor is a glioblastoma.

[0114] In some embodiments, the glioblastoma is a recurrent or refractory glioblastoma.

[0115] In some embodiments, the expression of O6-methylguanine-DNA methyltransferase (MGMT) in the glioblastoma is positive or negative.

[0116] The dosage of a compound (e.g., a complex or composition) used in the therapeutic and / or preventive methods relating to this disclosure generally depends on the severity of the disease, the subject's body weight, and the relative efficacy of the compound. As a general guideline, a suitable unit dose may range from 0.1 mg to 1000 mg.

[0117] As is well known to those skilled in the art, the dosage of a drug depends on many factors, including, but not limited to, the activity of the specific compound used, the age of the subject, the subject's weight, the subject's health condition, the subject's behavior, the subject's diet, the time of administration, the method of administration, the rate of excretion, and the drug's composition, and can be determined according to known treatment plans. [Brief explanation of the drawing]

[0118] [Figure 1] These are schematic diagrams of exemplary complex structures, specifically the antibody-drug conjugates 25G7-scdsFv-PE38KDEL (Figure 1A) and 25G7-scFv-PE38KDEL (Figure 1B), which consist of the antibody 25G7 scdsFv and the toxin PE38KDEL. [Figure 2]The SDS-PAGE diagrams of the purified complexes 25G7-scdsFv-PE38KDEL and 25G7-scFv-PE38KDEL are shown, where M is the molecular weight marker. [Figure 3] This study involved detecting IL-4R expression levels in glioblastoma (GBM) cell lines using flow cytometry. Human IL-4R stable transformed LN229, U251, and U87 glioma cell lines were constructed using a lentiviral system, and IL-4R overexpression levels were detected by flow cytometry. Control cells were provided from a construct that had not been transfected with human IL-4R. [Figure 4] This study involved detecting the binding of the complex to the surface of glioblastoma cells using flow cytometry. Flow cytometry was used to measure the binding ability of 25G7-scdsFv-PE38KDEL and MDNA55 to the surface of IL-4R stable transformed cell lines LN229-IL4R, U251-IL4R, and U87-IL4R. [Figure 5] This study examined the endocytosis efficiency of complexes in glioblastoma cells. Flow cytometry was used to measure the endocytosis efficiency of 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55, respectively, in LN229-IL4R, U251-IL4R, and U87-IL4R cells from 0 to 24 hours. [Figure 6] This study describes the extracorporeal cytotoxicity of the complex against glioblastoma cells. The results were obtained by measuring the extracorporeal cytotoxicity of 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55 against LN229-IL4R, U251-IL4R, U87-IL4R, and control cells using the CTG method. [Figure 7] This report compares the extracorporeal cytotoxicity of 25G7-scFv-PE38KDEL, 25G7-scdsFv-PE38KDEL, and MDNA55 in LN229-IL4R, U251-IL4R, and control glioma cells using the CTG method. [Figure 8]This is a comparison of the in vivo efficacy of the U87-MG compound inoculated into nude mouse tumor models. The dosage of both 25G7-scdsFv-PE38KDEL (25G7-IT) and the MDNA55 drug was 0.5 mg / kg (mpk), administered intratumorally (it). [Figure 9] This is a comparison of the in vivo efficacy of the LN229-IL4R complex (IL-4R overexpressing LN229 cells) in a nude mouse tumor model. The dosage of 25G7-scdsFv-PE38KDEL (25G7-IT) and the MDNA55 drug was 0.25 mpk(it), or 0.25 mpk(iv) for 25G7-scdsFv-PE38KDEL. [Figure 10] These are the in vivo efficacy results in a nude mouse tumor model subcutaneously inoculated with the LN229-IL4R complex. The doses of the 25G7-scdsFv-PE38KDEL (25G7-IT) drug were 0.1 mpk (reduced to 0.05 mpk for the second and subsequent doses) and 0.3 mpk (reduced to 0.15 mpk for the second and subsequent doses), and the dose of the MDNA55 drug was 0.1 mpk (reduced to 0.05 mpk for the second and subsequent doses). These were administered intratumorally (it). [Modes for carrying out the invention]

[0119] term To make this application easier to understand, several technical and scientific terms are defined below. Unless otherwise specifically defined elsewhere in this specification, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0120] This disclosure incorporates all the contents of patent document WO2020 / 038454A1 into this application.

[0121] The three-letter and one-letter amino acid codes used in this application are as described in J. Biol. Chem, 243, p3558 (1968).

[0122] "Human IL-4R" (hIL-4R) refers to the human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4Rα. hIL-4R aims to cover various molecular forms of IL-4R at each stage in the body, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification processes of the IL-4R gene, such as precursor IL-4R, mature IL-4R, naturally occurring IL-4R splice variants, modified IL-4R, or fragments thereof.

[0123] An "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in their antigenicity because they have different amino acid compositions and sequence orders in the constant region of their heavy chains. As a result, immunoglobulins can be divided into five types, or immunoglobulin isotypes, called IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Identical Ig cells can be further divided into different subclasses based on differences in the amino acid composition of their hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. The light chains can be divided into κ or λ chains depending on the constant region. Each of the five types of Ig cells may have either a κ or λ chain.

[0124] The antibody light chain may further include a light chain constant region, the light chain constant region comprising human or mouse κ, λ chains or their variants.

[0125] The antibody heavy chain may further include a heavy chain constant region, the heavy chain constant region of which includes human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0126] In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is significantly altered, forming a variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable, forming a constant region (C region). The variable region includes three hypervariable regions (HVRs) and four skeletal regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged from the amino group terminus to the carboxyl group terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to light chain complementarity determination region 1 (LCDR1), light chain complementarity determination region 2 (LCDR2), and light chain complementarity determination region 3 (LCDR3), while the three CDR regions of the heavy chain refer to heavy chain complementarity determination region 1 (HCDR1), heavy chain complementarity determination region 2 (HCDR2), and heavy chain complementarity determination region 3 (HCDR3).

[0127] The antibodies include mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, which may be obtained by recombinant means, for example, recombinant fully human antibodies obtained by affinity maturation.

[0128] "Recombinant fully human antibodies" include fully human antibodies prepared, expressed, created, or isolated by recombinant methods, and the related techniques and methods are well known in this field, for example, (1) antibodies isolated from genetically modified human immunoglobulin genes, transmuted animals (e.g., mice), or hybridomas prepared thereby; (2) antibodies isolated from host cells transfected to express the antibody, e.g., transfectomas; (3) antibodies isolated from recombinant combined human antibody libraries; and (4) antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant fully human antibodies include variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur, for example, during antibody maturation.

[0129] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microorganism, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family such as Escherichia coli and Salmonella strains, Bacillaceae family such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0130] "Mouse antibody" is a monoclonal antibody against human IL-4R prepared in accordance with the knowledge and techniques of this art. During preparation, an IL-4R antigen (or a polypeptide containing an epitope) is injected into a test subject, and then hybridomas expressing an antibody having the desired sequence or functional properties are isolated. In some embodiments, the mouse antibody or antigen-binding fragment thereof that binds to human IL-4R further comprises the light chain constant region of the mouse κ, λ chain or its variants, or further comprises the heavy chain constant region of mouse IgG1, IgG2, IgG3, or IgG4 or its variants.

[0131] "Fully human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies as used herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or by intracellular somatic mutation). However, the term "fully human antibodies" does not include antibodies (i.e., "humanized antibodies") obtained by transplanting CDR sequences derived from the germline of another mammalian species (e.g., mouse) into a human skeletal sequence.

[0132] A "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large amount of heterologous protein components. To avoid a decrease in activity due to reduced immunogenicity, the minimum possible reverse mutations can be performed on the human antibody variable region to maintain activity.

[0133] A "chimeric antibody" is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, and can reduce the immune response induced by the mouse antibody. To create a chimeric antibody, first, a hybridoma that secretes a mouse-specific monoclonal antibody is created, then the variable region gene is cloned from the mouse hybridoma cells, and if necessary, the constant region gene of a human antibody is cloned. The mouse variable region gene and the human constant region gene are then ligated to form a chimeric gene, which is then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell line. The constant region of the human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4 or their variants. For example, IgG1 containing the human IgG2 or IgG4 heavy chain constant region, or IgG1 that does not exhibit ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation, may be used.

[0134] "Antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment that binds to human IL-4R, and a polypeptide or protein containing the above fragments, all of which have antigen-binding activity. The above "antigen-binding fragment" includes one or more CDR regions of the antibody described herein. The Fv fragment includes an antibody heavy chain variable region and a light chain variable region, but lacks a constant region and has minimal antibody fragments of all antigen-binding sites. Generally, the Fv antibody further includes a polypeptide linker between the VH and VL domains and can form a structure necessary for antigen binding. Different linkers may be used to link two antibody variable regions into a single polypeptide chain called a single-chain antibody or single-chain Fv (scFv).

[0135] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to molecules comprising a heavy-chain variable domain (or region, VH) and a light-chain variable domain (or region, VL) linked by a linker (or linker peptide). Such scFv molecules may have the common structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Preferred prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, containing one to four repeating variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90:6444~6448). Other linkers available for use in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725~731, Choi et al. (2001), Eur. J. Immuno l. 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.

[0136] The term "antibody framework" refers to a portion of the variable domain (VL) or VH, used as a stent for the antigen-binding loop (CDR) of that variable domain. In effect, it is a variable domain that does not possess a CDR.

[0137] "Binding to IL-4R" means the ability to interact with human IL-4R (or its epitope or fragment). The term "antigen-binding site" as used herein refers to a three-dimensional spatial site recognized by the antibody or antigen-binding fragment as defined herein.

[0138] An "epitope" is a site in an antigen that specifically binds to an immunoglobulin or antibody. Epitopes may be formed by adjacent amino acids or by tertiary folding of non-adjacent amino acids. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes typically consist of at least 3 to 15 amino acids in a specific spatial conformation. Methods for determining which epitopes are bound by a given antibody are well known in this field and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of epitopes include techniques in this field and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0139] "Specific binding," "selective binding," "selective binding," and "specifically binding" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when recombinant human IL-4R is used as the analyte and an antibody as the ligand, and measured by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to the given antigen with an equilibrium dissociation constant (KD) of less than or equal to approximately 10⁻⁷ M, and its binding affinity to the given antigen is at least twice its binding affinity to non-specific antigens other than the given antigen or closely related antigens (e.g., BSA). The term "antigen-identifying antibody" may be used interchangeably with the term "specifically binding antibody" herein.

[0140] "Cross-reactivity" refers to the ability of the antibodies herein to bind to IL-4R from different species. For example, an antibody herein that binds to human IL-4R may also bind to IL-4R from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or by detecting binding or functional interaction with cells that physiologically express IL-4R. Methods for determining cross-reactivity include standard binding assays described herein, such as surface plasmon resonance (SPR) analysis or flow cytometry.

[0141] A “neutralizing” or “inhibitory” antibody refers to an antibody that, upon binding to hIL-4R, suppresses the biological activity of hIL-4 and / or hIL-13. Such suppression of the biological activity of hIL-4 and / or IL-13 can be evaluated by one or more indicators of hIL-4 / or hIL-13 biological activity known in this field, such as hIL-4 / or hIL-13-induced cell activation and the binding of hIL-4 to hIL-4R. See, for example, the description in CN103739711A. “Inhibition of growth” (e.g., for cells) is intended to include any measurable reduction in cell growth.

[0142] "Induction of an immune response" and "enhancement of an immune response" may be used interchangeably and refer to stimulation of a specific antigen by an immune response (i.e., passive or adaptive). Regarding the term induction of CDC or ADCC, "induction" refers to stimulating a specific direct cell-killing mechanism.

[0143] ADCC (antibody-dependent cell-mediated cytotoxicity) refers to the direct killing of antibody-coated target cells by cells expressing the Fc receptor recognizing the Fc portion of the antibody. Modification of the Fc portion in IgG can reduce or eliminate the ADCC effector function of the antibody. The above modifications refer to mutations in the constant region of the antibody's heavy chain, and are selected from, for example, the N297A, L234A, L235A, IgG2 / 4 chimera, F235E, or L234A / E235A mutations in IgG1.

[0144] A fusion protein is a protein product obtained by DNA recombination that co-expresses two genes. For example, the anti-IL-4R antibody-PE38KDEL fusion protein is a fusion protein obtained by DNA recombination that co-expresses the anti-IL-4R antibody and the toxin molecule PE38KDEL. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art and can be found, for example, in Chapters 5-8 and 15 of the Cold Spring Harbor Manual of Antibody Experimental Techniques. For example, mice can be immunized with human IL-4R or its fragments, and the resulting antibodies can be restored, purified, and subjected to amino acid sequencing by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described herein are obtained by genetic engineering by adding one or more human FRs to a non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website or from the Journal of Immunoglobulins, 2001 ISBN 012441351.

[0145] Engineered antibodies or antigen-binding fragments can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombined immunoglobulin expression vector can stably transfect CHO cells. By molecular cloning techniques, the sequences of the humanized antibodies described herein can be inserted into the corresponding expression vectors and produced by expression using the HEK293 cell expression system to obtain the corresponding humanized antibodies. For example, mammalian expression systems will induce glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones can be obtained by expressing antibodies that specifically bind to human antigens. Positive clones produce antibodies by expanding the culture in serum-free medium in a bioreactor. The culture medium from which the antibodies have been secreted can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving or ion exchange. The obtained product must be immediately frozen, for example, at -70°C, or lyophilized.

[0146] The antibody may be a monoclonal antibody (mAb) and refers to an antibody obtained from a single clonal cell line, the cell line being not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques, for example, hybridoma technology, recombinant technology, phage display technology, synthesis technology (e.g., CDR-grafting), or other conventional techniques.

[0147] Antibodies may be monospecific, bispecific, or multispecific antibodies. Multispecific antibodies may exhibit specificity to different epitopes of the target peptide, or may contain antigen-binding domains that exhibit specificity to one or more target peptides. Human anti-IL-4R antibodies can be ligated to or co-expressed with other functional molecules (e.g., other peptides or proteins). For example, an antibody or fragment thereof can be functionally ligated (e.g., by chemical bonding, gene fusion, non-covalent bonding, or other means) to one or more other molecules (e.g., another antibody or antigen-binding fragment) to produce a bispecific or multispecific antibody having at least one binding specificity.

[0148] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biofluids, “administration,” “giving,” and “processing” mean the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biofluids. “Administration,” “giving,” and “processing” may also mean, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Processing of cells includes contact of reagents with cells and contact of reagents with fluids, where the fluids come into contact with the cells. “Administration,” “giving,” and “processing” also mean the treatment of, for example, cells in vitro and in vitro with reagents, diagnostics, or compositions, or through other types of cells. When applied to humans, veterinary medicine, or research subjects, “processing” means therapeutic treatment, preventive or precautionary measures, research, and diagnostic applications.

[0149] "Treatment" means administering an oral or topical therapeutic agent, such as any one of the compound or composition described herein, to a subject who has (or is likely to have or is susceptible to) one or more disease symptoms, and the therapeutic agent has a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the subject or population being treated in a dose that effectively relieves one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms from progressing to any clinically measurable degree. The dose of the therapeutic agent that effectively relieves any specific disease symptom (also called the "therapeutic dose") can vary depending on several factors, including the subject's disease state, age and weight, and the agent's ability to produce the therapeutic effect required by the subject. The reduction in disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity and progression of the symptoms. While embodiments of this specification (e.g., treatment methods or products) may be ineffective in achieving remission of a single target disease symptom, any statistical test known in the art, such as the Student t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test, can confirm that they can reduce the target disease symptom in a statistically significant number of subjects.

[0150] The term "naturally occurring" applied to an object means that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in living organisms (including viruses) that can be isolated and obtained from naturally occurring sources, and that has not been artificially or intentionally modified, is considered naturally occurring.

[0151] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medical disorder. The effective dose also refers to an amount sufficient to allow or promote a diagnosis. The effective dose used for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method and route of administration and dosage, and the severity of side effects. The effective dose may also be the maximum dose or administration plan that avoids significant side effects or toxic effects.

[0152] "Exogenous" can refer to substances produced outside of living organisms, cells, or the human body, depending on the context.

[0153] "Endogenous" can refer to substances produced within an organism, cell, or even within the human body.

[0154] "Homologousity" or "identity" refers to the similarity between two polynucleotide sequences or two polypeptides. If the positions in the two sequences being compared are both occupied by the same base or amino acid monomer subunit—for example, if the positions in both DNA molecules are occupied by adenine—then the molecules are homologous at those positions. The percentage of homology between two sequences is a function of dividing the number of shared matching or homologous positions in the two sequences by the number of positions being compared and multiplying by 100%. For example, if the sequences are optimally aligned, and 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. Generally, the two sequences are compared when the highest possible homology percentage is obtained by aligning them. As used herein, “at least 85% sequence identity” means that, when comparing a mutant with a parent sequence, the two sequences have at least 85% homology, and in some forms, they have at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology, in some specific forms they have 90%, 95%, or 99% or more, and in other specific forms they have at least 95% sequence homology. The amino acid sequences having at least 85% sequence identity include those obtained by making one or more amino acid deletions, insertions, or substitution mutations in the parent sequence.

[0155] The terms “cell,” “cell line,” “cell culture,” and “cell culture” as used herein may be used interchangeably, and all such terms include their offspring. Accordingly, the terms “transformed organism” and “transformed cell” include primary test cells and cultures derived therefrom, without regard to passage number. It should also be understood that, due to intentional or unintended mutations, offspring and parental cells may not be exactly the same in terms of DNA content. The terms include mutant offspring having similar function or biological activity to the parental cells, of which the parental cells were screened from the initial transformed cells.

[0156] "Optionally" or "at will" means that the event or situation described below may occur, but is not necessarily required, and this description includes cases where the event or situation occurs and cases where it does not. For example, "optionally containing 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions may be present, but are not necessarily required.

[0157] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA ring to which other DNA segments can be ligated. In another embodiment, the vector is a viral vector, to which other DNA segments can be ligated to a viral genome. The vectors disclosed herein can self-replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors), or, after being introduced into a host cell, can be adapted to the host cell's genome and replicate together with the host genome (e.g., non-episomal mammalian vectors).

[0158] "Pharmaceutical composition" refers to a mixture comprising one or more of the compound substances described herein or their physiologically / pharmaceutically acceptable salts or prodrugs, and other components such as physiologically / pharmaceutically acceptable vectors (vehicles) or excipients. The pharmaceutical composition is intended to facilitate administration to a living organism, contribute to the absorption of the active ingredient, and further exert biological activity.

[0159] The term "excipient" refers to an adduct other than the active ingredient in a pharmaceutical preparation, and may also be called an additive. For example, binders, fillers, disintegrants, and lubricants in tablets; the matrix portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations can all be called excipients.

[0160] The term "diluent," also known as a "filler," primarily serves to increase the weight and volume of tablets. The addition of a diluent not only ensures a consistent volume but also reduces dose variations in the main component and improves the compressibility of the drug. When a drug in a tablet contains oily components, an absorbent is added to absorb the oily substance and maintain a "dry" state, aiding in tablet preparation. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0161] "Immunohistochemical scoring" is a histological scoring method for processing immunohistochemical results, which converts the number of positive cells and their staining intensity in each section into corresponding numerical values ​​for semi-quantification of tissue staining. A score of 0 indicates no staining or membrane staining in less than 10% of tumor cells; a score of +1 indicates slight / barely noticeable membrane staining in 10% or more tumor cells; a score of +2 indicates moderate complete membrane staining in 10% or more tumor cells; and a score of +3 indicates strong complete membrane staining in 10% or more tumor cells. In this disclosure, samples in which IL-4R is scored 0 or +1 may be considered not to be overexpressing IL-4R, and samples in which IL-4R is scored 2 or +3 may be considered to be overexpressing IL-4R.

[0162] The present application will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the claims.

[0163] Experimental methods in examples or test cases where specific conditions are not specified generally follow normal conditions or conditions recommended by the raw material or product manufacturer. See Sambrook et al., *Molecular Cloning*, Cold Spring Harbor Laboratory, and *Modern Molecular Biology Methods*, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents whose specific source is not specified are standard commercially available reagents. Example 1. Preparation of an exemplary immunotoxin

[0164] The single-chain variable segment (scFv) of the humanized IL-4R monoclonal antibody hu25G7 (the heavy chain variable region sequence is shown in SEQ ID NO: 43 and is hu25G7-VH, and the light chain variable region sequence is shown in SEQ ID NO: 37 and is hu25G7-A LCVR) is fused and expressed with the mutated and cleaved Pseudomonas aeruginosa exotoxin (PE38KDEL) (SEQ ID NO: 49) using the linker ASGGPE (SEQ ID NO: 50). Here, the amino acids at positions VH-44 and VL-100 of the hu25G7 antibody are mutated to cysteine ​​(e.g., SEQ ID NOs: 47-48), and VH and VL are linked by the linker peptide (GGGGS)3 (SEQ ID NO: 51). This yields the IL-4R-targeting immunotoxin 25G7-(scdsFv)-PE38KDEL (hereinafter referred to as 25G7-IT), the complete sequence of which is shown in SEQ ID NO: 52.

[0165] Figures 1A and 1B are schematic diagrams of the structures of the two immunotoxins. 25G7-IT was expressed in E. coli BL21, and the protein was purified and reconstructed from the inclusion bodies. Then, the endotoxin was removed to obtain the purified 25G7-IT protein, which has a molecular weight of approximately 63 kDa (SDS-PAGE results are shown in Figures 2A and 2B).

[0166] The humanized anti-human IL-4R antibody 25G7 has its heavy chain sequence shown in SEQ ID NO: 17 and its light chain sequence shown in SEQ ID NO: 18. The antibody can be expressed and purified using conventional antibody expression and purification methods in this art, for example, by the method described in patent application WO2020038454A1. The aforementioned patent application is incorporated herein by reference in its entirety.

[0167] [ka] [ka] [ka] [ka] [ka] [ka]

[0168] Furthermore, an immunotoxin fusion protein, 25G7-(scFv)-PE38KDEL, which does not have the additional interchain disulfide bond structure, has been developed, and its amino acid sequence is shown in SEQ ID NO: 53.

[0169] [ka] Example 2. Measurement of interacting affinity dynamics

[0170] Using the Biacore biomolecular interaction analyzer (biacore 8K, GE), the affinity dynamics of 25G7-IT and the reference sample MDNA55 (expressed and purified in an E. coli system) interacting with hrIL-4R-Fc (ILR-H5253, Acro) were measured using surface plasmon resonance (SPR) technology.

[0171] In this experiment, an anti-human IgG antibody (29234600, GE) was covalently bound to a CM5 chip (29149603, GE) using a capture method. After capturing rhIL-4R-Fc as a ligand on the chip, 25G7-IT and the reference sample MDNA55 were injected as analytes, and affinity analysis and calculations were performed.

[0172] Experimental results showed that 25G7-IT and the reference sample MDNA55 exhibited strong affinity for rhIL-4R-Fc, with KD values ​​of 1.01E-10 and 4.39E-10 M, respectively (Table 2). The affinity of 25G7-IT for rhIL-4R-Fc was significantly stronger than the affinity of the reference sample MDNA55 for rhIL-4R-Fc.

[0173] [Table 2] Example 3. Measurement of binding ability to GBM target cells

[0174] Since endogenous IL-4R expression is relatively low in GBM cell lines U87, U251, and LN229, human IL-4R was overexpressed in these three GBM cell lines using a lentiviral system. After screening with G418, the effect of IL-4R overexpression was identified by flow cytometry (Figure 3), and the results showed that IL-4R was stably overexpressed in these three cell lines.

[0175] Complete culture medium was prepared by adding 10% FBS and 1% penicillin / streptomycin to DMEM medium, and U87-, U251-, and LN229-IL4R stable transformed cell lines were cultured in an incubator containing 5% CO2 at 37°C. Cells in a T75 culture flask were digested with 1.5 mL of TrpLE (Gibco, #12605-010) at 37°C for 3 to 5 minutes, then neutralized with FBS-containing DMEM medium, and after centrifugation, the concentration was 2 × 10⁶. 6Cell suspensions were prepared at a concentration of cells / mL and added to a 96-well round-bottom plate at a volume of 50 μL per well. 25G7-IT, MDNA55, and hIgG1 isotype controls were each diluted in DMEM medium to eight concentration points (3x gradient: 20, 6.67, 2.22, 0.74, 0.25, 0.08, 0.03, 0 μg / mL), added to the 96-well plate at a volume of 50 μL per well, and homogeneously mixed with the cells. The cells and controls were then incubated together on ice for 40 minutes. After incubation, the cells were washed three times with 200 μL of flow detection buffer. Anti-PE38 antibody (Sigma-aldrich, Cat:P2318-1ML) was diluted 1:300 with flow detection buffer and added to the 96-well plate at a volume of 50 μL per well to resuspend the cells. The antibody and cells were incubated together on ice for 40 minutes. Next, the cells were washed three times with 200 μL of flow detection buffer, and the anti-rabbit IgG Fab2 Alexa Fluor 647 (Cell Signaling Technology, #02 / 2020) was diluted 1:500 with flow detection buffer. The cells were then resuspended in a 96-well plate at a volume of 50 μL per well, and the antibody and cells were incubated together on ice for 30 minutes. After centrifugation and washing three times with 200 μL of flow detection buffer, the cells were resuspended, and the signal for single-cell Alexa Fluor647 in each well was detected using a flow cytometer BD FACSCelesta™.

[0176] Analysis of average fluorescence intensity (MFI) revealed that, as shown in Table 3 and Figures 4A-4C, both 25G7-IT and the reference sample MDNA55 were capable of binding to IL-4R-positive GBM cell lines. Furthermore, 25G7-IT exhibited stronger binding ability to U87-, U251-, and LN229-IL4R stable transformed cell lines than MDNA55.

[0177] [Table 3-1] [Table 3-2] Example 4. Measurement of endocytosis efficiency in GBM target cells

[0178] To measure the endocytosis efficiency of 25G7-IT and the reference sample MDNA55 in GBM target cells, the endocytosis efficiency of 25G7-IT and MDNA55 was compared in the three IL-4R stable transformed glioma cells described above.

[0179] Based on the experimental results of Example 3, a final concentration of 2 μg / mL was selected as the concentration for detecting endocytosis efficiency, as this concentration nearly saturated the endocytosis efficiency in the three cell samples. 25G7-IT, MDNA55, and hIgG1 isotype controls were diluted to 2 μg / mL in DMEM medium. These were added to a 96-well plate in volumes of 50 μL per well and homogeneously mixed with the cells, then incubated together on ice for 40 minutes. After washing three times with DMEM complete medium, the cells were resuspended in 100 μL of DMEM complete medium. 100 μL of cells labeled 0 h in the 96-well plate were transferred to another 96-well round-bottom plate, and 100 μL of 4% paraformaldehyde fixative was added, followed by fixation at 4°C. The remaining cells were placed in a 37°C incubator and cultured for 1 hour, 2 hours, 4 hours, and 24 hours, respectively. Then, the corresponding cells were removed, transferred to another 96-well plate, and fixed at 4°C using the same method.

[0180] Cells endocytosed for 0 to 4 hours were stained with flow antibody on the same day, and cells endocytosed for 24 hours were incubated until the end of the experiment, after which flow antibody staining and flow analysis were performed. After fixing cells endocytosed for 4 hours for 30 minutes, all fixed cells were centrifuged and washed twice with flow detection buffer. Anti-PE38 antibody (Sigma-aldrich, Cat:P2318-1ML) was diluted 1:300 with flow detection buffer and added to a 96-well plate at a volume of 50 μL per well to resuspend the cells. Then, the antibody and cells were incubated together on ice for 40 minutes. The subsequent experimental procedure was the same as in Example 3, and the analytical results are shown in Table 4 and Figures 5A to 5C.

[0181] Experimental results showed that both 25G7-IT and the reference sample MDNA55 could effectively endocytose IL-4R-overexpressing GBM cells. At 4 hours, the endocytosis efficiency in all three cell types reached 60% to 80%, and 25G7-IT showed higher endocytosis efficiency than MDNA55 in LN229- and U251-IL4R stable transformed cell lines.

[0182] [Table 4] Example 5. Measurement of in vitro killing efficiency in GBM target cells

[0183] To measure the in vitro killing efficiency of 25G7-IT and reference sample MDNA55 in GBM target cells, the in vitro efficacy of both drugs was compared in the three stable transformed human IL-4R brain tumor cells using the CTG (CELL TITER-GLO) method.

[0184] Complete culture medium was prepared by adding 10% FBS and 1% penicillin / streptomycin to DMEM medium. Stable transformed cell lines U87-IL4R, U251-IL4R, and LN229-IL4R, as well as U87, U251, and LN229 control cells, were cultured in an incubator containing 5% CO2 at 37°C.

[0185] Cells were seeded in 96-well plates at a density of 4000 cells per well and 130 μL of culture medium. After one night, once the cells had completely adhered to the cell walls, 20 μL of gradient-concentration reagents containing 25G7-IT or MDNA55 (final concentrations: 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.00128 nM, 0.000256 nM, 0 nM) were added to each well, and the cells were incubated for 3 days. After 3 days, the 96-well plates were removed from the incubator and allowed to equilibrate to room temperature. 50 μL of CTG reagent (CellTiter-Glo Luminescent, Promega, #G7573) was added to the cell suspension, mixed homogenously with a shaker for 2 minutes, and then allowed to stand at room temperature for 10 minutes. Cell viability was detected using the Luminescence mode built into the EnVision2105 Multimode Plate Reader (PerkinElmer). After converting the absorbance results to percentages, the results were analyzed using Prism 8.

[0186] According to the experimental results (Figures 6A to 6F), both 25G7-IT and the reference sample MDNA55 were able to efficiently kill target cells in IL-4R-overexpressing GBM cells, and showed relatively high efficacy against LN229-IL4R in particular, achieving an in vitro killing effect of 80% at a concentration of 0.1 nM. Overall, 25G7-IT showed higher in vitro killing efficiency than MDNA55 in three IL-4R-stable transformed glioma cell lines: LN229, U251, and U87.

[0187] Furthermore, the in vitro killing efficiencies of the drugs 25G7-scFv-PE38KDEL, 25G7-scdsFv-PE38KDEL (25G7-IT), and MDNA55, without interchain disulfide bonds, were further compared in LN229 and U251 IL-4R stable transformed glioma cell lines. Figures 7A to 7D show that the in vitro killing efficiencies of 25G7-scFv-PE38KDEL, 25G7-IT, and MDNA55 are equivalent in glioma cells. Example 6. In vivo efficacy of IL-4R-targeting drugs in a GBM tumor model.

[0188] To measure the in vivo efficacy of 25G7-IT and the reference sample MDNA55 in a GBM tumor model, we first constructed a model of subcutaneous injection of U87-MG with IL-4R expression of 1+ (immunohistochemical score).

[0189] 2 × 10⁶ 6 Individual U87-MG cells were subcutaneously inoculated, and the cells were resuspended in PBS and Matrigel in a 1:1 ratio (0.1 mL / cell). The tumor growth was observed periodically. The average size of the subcutaneously inoculated tumors was approximately 100 mm. 3 ~150 mm 3 At this point, the mice were divided into groups of 8 per group. Multi-site intratumoral administration was performed, with the drugs 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55 administered at a dose of 0.5 mg / kg (mpk) once a week for a total of 4 doses. After administration began, the body weight and tumor size of the mice were measured twice a week, and the tumor volume was calculated using the formula: tumor volume (mm²). 3 ) = 1 / 2 × (a × b 2 (where a represents the major axis and b represents the minor axis.)

[0190] As shown in Figures 8A-8C and Table 5, in a U87-MG tumor model with low IL-4R expression (immunohistochemical score of 1+), after four weekly administrations at a dose of 0.5 mpk, the TGI (tumor growth inhibition rate) of 25G7-IT was 80%, while the TGI of the reference sample MDNA55 was 51%. These results demonstrate that in a glioma model with low IL-4R expression, the tumor growth inhibition effect of 25G7-IT is significantly superior to that of MDNA55.

[0191] [Table 5] Note: s.c.: subcutaneous, mpk: mg / kg (milligrams per kilogram of body weight), i.t.: intratumoral injection, i.v.: intravenous injection, QW: once a week. ** indicates p < 0.01 and *** indicates p < 0.001. Example 7. In Vivo Pharmacodynamic Effects of an Agent Targeting IL-4R in a GBM Tumor Model (IL-4R 3+)

[0192] To further measure the in vivo pharmacodynamic effects of 25G7-IT and the reference sample MDNA55 in a GBM tumor model, a subcutaneous inoculation model was constructed using the LN229 cell line with overexpressed IL-4R (designated LN229-IL4R, with an immunohistochemical score of 3+).

[0193] 1 × 10 7 LN229-IL4R cells were subcutaneously inoculated into the right dorsal area of experimental mice. The cells were resuspended in 1:1 PBS (0.1 mL / mouse), and the tumor growth status was observed regularly. When the average size of the subcutaneously inoculated tumor reached approximately 100 mm 3 ~150 mm 3 the mice were grouped at 6 - 7 mice per group. The dosage of MDNA55 was 0.25 mpk (i.t.), and the dosage of 25G7-scdsFv-PE38KDEL (25G7-IT) was 0.25 mpk (i.t. or i.v.). The drugs were administered once a week for a total of 4 times. Monitoring and calculation of tumor growth were the same as in Example 6.

[0194] As shown in FIGS. 9A - 9C and Table 6, in the LN229-IL4R tumor model with high expression of IL-4R (immunohistochemical score of 3+), the TGI (tumor growth inhibition rate) of 25G7-IT and MDNA55 at 0.25 mpk (i.t.) both reached 99% (complete remission). However, the TGI of 25G7-IT at 0.25 mpk (i.v.) with the same dosage was 68%, indicating that the intratumoral administration effect is superior to intravenous administration.

[0195] In addition, in the LN229-IL4R tumor model with high IL-4R expression (immunohistochemical score is 3+), the dose of the immunotoxin was further reduced. The dosage of 25G7-scdsFv-PE38KDEL (25G7-IT) was 0.1 (reduced to 0.05 for the second and subsequent administrations), 0.3 (reduced to 0.15 for the second and subsequent administrations) mpk, and the dosage of MDNA55 was 0.1 (0.05 for the second and subsequent administrations) mpk, and they were administered intratumorally.

[0196] As shown in FIGS. 10A-10C and Table 6, in the LN229-IL4R tumor model (immunohistochemical score is 3+), it was administered once a week at 0.1 (first administration) or 0.05 (second to fourth administrations) mpk. After four administrations, the TGI (tumor growth inhibition rate) of 25G7-IT was 77%, and the TGI of the reference sample MDNA55 was 59%. Moreover, with the dosage of 25G7-IT at 0.3 (first administration) or 0.15 (second to fourth administrations) mpk, the tumors of the tumor-bearing mice achieved complete remission (TGI = 99%). From these results, in the glioma model with high IL-4R expression, the tumor growth inhibitory effect of low-concentration 25G7-IT is superior to that of MDNA55 at the same dosage, and the high-dose 25G7-IT drug can completely relieve the tumor-bearing mice, indicating an obvious dose-effect relationship.

[0197]

Table 6

[0198] The use and welfare of experimental animals in this disclosure followed the regulations of the "Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)". The health status and death situation of the animals were monitored daily. Routine examinations included observing the effects of the test substances and drugs on the daily behaviors of the animals, such as behavioral activities, body weight changes, and appearance symptoms.

Claims

1. A complex of an anti-IL-4R antibody or its antigen-binding fragment, - Anti-IL-4R antibody or its antigen-binding fragment, - Containing one or more toxin molecules, The anti-IL-4R antibody or its antigen-binding fragment covalently or noncovalently binds to the toxin molecule. The anti-IL-4R antibody or its antigen-binding fragment is Heavy chain variable region including HCDR1, HCDR2, and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and The light chain includes a variable region containing LCDR1, LCDR2, and LCDR3, whose amino acid sequences are shown in SEQ ID NO: 38, SEQ ID NO: 7, and SEQ ID NO: 40, respectively. A complex of an anti-IL-4R antibody or its antigen-binding fragment.

2. The aforementioned anti-IL-4R antibody is one selected from mouse antibodies, chimeric antibodies, or humanized antibodies. A complex of the anti-IL-4R antibody or its antigen-binding fragment according to claim 1.

3. The anti-IL-4R antibody or its antigen-binding fragment comprises a FR derived from the human germline light chain IGKV3-11*01 or a FR having at least 95% sequence identity therewith, and / or The anti-IL-4R antibody or its antigen-binding fragment comprises a FR derived from the human germline heavy chain IGHV3-48*01 or a FR having at least 95% sequence identity therewith. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 2.

4. The anti-IL-4R antibody or its antigen-binding fragment comprises a FR derived from the human germline light chain IGKV2D-29*01 or a FR having at least 95% sequence identity therewith, and / or The anti-IL-4R antibody or its antigen-binding fragment comprises a FR derived from the human germline heavy chain IGHV1-2*02 or a FR having at least 95% sequence identity therewith. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 2.

5. The anti-IL-4R antibody or its antigen-binding fragment comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and / or The antigen-binding fragment is Fab, Fv, scFv, F(ab')2, dsfv, or ScdsFv. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 2.

6. The scFv contains, in order from the N-terminus to the C-terminus, a heavy chain variable region, a linker peptide, and a light chain variable region, or The scFv sequentially includes a light chain variable region, a linker peptide, and a heavy chain variable region from the N-terminus to the C-terminus. The aforementioned linker peptide is (GxS) ｎ (SxG) ｎ (GGGGS) ｎ (G) ｎ One of the following or a combination thereof is selected, where x is one integer from 1 to 6 and n is one integer from 1 to 30, or The linker peptide is selected from any one or a combination thereof from GKSGSGSESKS, EGKSGSGSESKEF, GSTSGSGSESEGKG, GSTSGSGSGSESEGGSTKG, GSTSGSGKPGGSGEGSTKG, SRSSG, SGSSC, and (GGGGGS) 3. A complex of the anti-IL-4R antibody or its antigen-binding fragment according to claim 5.

7. The following (I) and (V), namely, (I) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 43, and A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 37, (V) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 47, and A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 48 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 48, Includes one of the following: A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 6.

8. The heavy chain variable region includes an amino acid sequence represented by one of SEQ ID NOs. 25 to 27, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with one of SEQ ID NOs. 25 to 27. The light chain variable region includes the amino acid sequence shown in SEQ ID NO:

37. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 6.

9. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein the heavy chain variable region and / or light chain variable region include mutations for structural stabilization.

10. The 44th amino acid residue in the heavy chain variable region is mutated to a cysteine ​​residue, and / or The 100th amino acid residue in the light chain variable region is mutated to a cysteine ​​residue. A complex of the anti-IL-4R antibody or its antigen-binding fragment according to claim 9.

11. The anti-IL-4R antibody or its antigen-binding fragment, A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 44, and A light chain comprising the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 45, A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 10.

12. The anti-IL-4R antibody or its antigen-binding fragment is linked to the toxin molecule via a linker. The linker is either a proteinaceous or non-proteinaceous linker. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 11.

13. The complex is a fusion protein, wherein the complex is a complex of an anti-IL-4R antibody or an antigen-binding fragment thereof, as described in Claim 12.

14. The linker is ASGGPE, VM, AM, AM(G ２－４ S) ｐ Selected from any one or a combination of AM, ASGCGPE, ASGCCCGPE, ASGCGSCPE, ASCGTTTGCPE, KASGKKYGCKKGPE, KGGGCAGGPE, Among them, p is one integer between 1 and 10. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to claim 12.

15. The toxin molecule is selected from one or a combination thereof from among pore-forming toxins, aerolysin, proaerolysin, bougain, lysine, Pseudomonas aeruginosa exotoxin, cholera toxin, or diphtheria toxin. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 14.

16. The aforementioned toxin is selected from any one or a combination thereof from PE-LR, PE-LO10R456A, PE-T20, PE-T20-KDEL, PE4E, PE40, PE38, PE24, PE25, PE38QQR, PE35, PE38KDEL, PE38DKEL, PE38RDEL, and PE38KNEL. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to claim 15.

17. The anti-IL-4R antibody or antigen-binding fragment thereof complex according to claim 16, wherein the toxin is PE38KDEL, represented by Sequence ID No.

49.

18. The sequence includes the sequence shown in sequence number 52 or sequence number 53, A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 17.

19. Encoding a complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 18, Polynucleotide.

20. A vector comprising a polynucleotide as described in claim 19, wherein the vector is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector. vector.

21. A host cell comprising the vector according to claim 20, wherein the host cell is a bacterium, yeast, or mammalian cell.

22. A complex of an anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 15, Optionally, including with pharmaceutically acceptable excipients, diluents, or vectors, Pharmaceutical composition.

23. For preparing medicines for the treatment and / or prevention of cancer or tumors, The use of an anti-IL-4R antibody or a complex of its antigen-binding fragment according to any one of claims 1 to 18, a polynucleotide according to claim 19, or a pharmaceutical composition according to claim 22, The cancer or tumor used is selected from any one or a combination of the following: prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, kidney cancer, liver cancer, colorectal cancer (e.g., colon cancer), pancreatic cancer, stomach cancer, leukemia, and central nervous system tumors.

24. The central nervous system tumor is selected from any one or a combination thereof from glioma, glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, meningioma, and retinoblastoma. The use described in claim 23.

25. The central nervous system tumor is a recurrent or refractory glioblastoma, or a glioblastoma in which O6-methylguanine-DNA methyltransferase expression is positive or negative. The use described in claim 23.

26. A method for preparing a complex of an anti-IL-4R antibody or its antigen-binding fragment, The steps of expressing an anti-IL-4R antibody or a complex of its antigen-binding fragment in the host cells described in claim 21, The step of separating the anti-IL-4R antibody or the complex of its antigen-binding fragment from the host cells, method.

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