KR102652133B1 - IL-4Rα antibody and its uses - Google Patents

Abstract

The present invention belongs to the field of antibody technology. Provided is an antibody that specifically binds to the human interleukin-4 receptor (hIL-4R) or an antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or antigen-binding fragment, and a use thereof. Additionally, the present invention provides nucleic acid molecules encoding antibodies, vectors and host cells containing the nucleic acid molecules, and methods for producing the antibodies.

Description

IL-4Rα antibody and its uses

The present invention belongs to the field of antibodies and relates to humanized IL-4R antibodies and their use in the treatment of diseases associated with IL-4/IL-4Rα signaling.

Allergic diseases (allergic rhinitis, etc.) can be well treated with antihistamines and specific immune medications. However, for some more serious allergic diseases (such as atopic dermatitis and asthma) there is no specific treatment, and non-specific immunosuppression is still the main treatment.

There are over 235 million people worldwide, and approximately 10-20% of patients cannot be effectively controlled with existing medications (i.e., asthma worsens), and the treatment costs of these patients account for 80% of total asthma treatment expenditures. do. Atopic dermatitis affects 10-20% of children and 3-10% of adults, and approximately 20% of patients have moderate to severe atopic dermatitis, and there is currently no effective treatment.

Oral/intravenous administration of broad-spectrum immunosuppressants (steroids, cyclosporine A, methotrexate, azathioprine, and mycophenolate mofetil, etc.) can effectively alleviate the symptoms of the disease. The activity of these immunosuppressants is mainly derived from downstream regulatory factors (such as transcription factors) that target inflammation: for example, steroids bind to the glucocorticoid receptor and then inhibit the activation of key transcription factors (such as NF-κB) that trigger inflammation. inhibit expression; Cyclosporine A is a calcium-dependent phosphatase inhibitor that can prevent IL-2 expression required for T cell activation and proliferation through the transcription factor nuclear factor of activated T cells (NFAT).

Systemic administration of broad-spectrum immunosuppressants often leads to a series of side effects due to multiple effects such as fluid retention, glucose intolerance, hypertension, muscle weakness, osteoporosis, hypothalamic-pituitary-adrenal axis suppression, and increased risk of infection. Topical administration (inhalants, topical drops, skin sprays, ointments, etc.) can reduce the side effects of systemic administration, but it is difficult to effectively treat severe allergic diseases. For example, Eucrisa, a non-steroidal PDE4 inhibitor approved by Pfiser last December, is effective only in patients with mild to moderate atopic dermatitis.

In view of the high incidence of allergic diseases, especially atopic dermatitis and asthma, and the limitations (safety and effectiveness) of broad-spectrum immunosuppressive therapy, especially in some severely ill patients, unmet clinical needs call for safer and more effective specific treatments. do.

Type 2 inflammatory pathways describe inflammatory pathways in which Th2 cells play an important role. Th2 cells play an important role in the type 2 inflammatory pathway by secreting type 2 cytokines, namely IL-4, IL-5, and IL-13. IL-4 can promote Th cells to differentiate and proliferate into Th2 cells. Th2 cells additionally produce IL-4, IL-5, and IL-13. IL-5 can promote the differentiation of eosinophils in the bone marrow; IL-4, IL-5, and IL-13 can promote the migration of eosinophils in certain tissues; Additionally, IL-4 and IL-13 can induce serotype transformation of B cells and then produce IgE; Additionally, IL-13 also plays an important role in mucus secretion, goblet cell proliferation, smooth muscle contraction, and collagen production.

Typical signs of type 2 inflammatory pathway activation are elevated IgE, eosinophils, and TARC. When the type 2 immune response is excessively activated, severe allergic disease occurs and different disease symptoms appear in different tissues. Depending on the location of occurrence, it can be divided into atopic dermatitis, chronic sinusitis, nasal polyps, and asthma.

IL-4 and IL-13 are potent regulators of type 2 immune responses and exhibit corresponding functions depending on their different binding receptors. Although IL-4 and IL-13 have only 25% amino acid homology, their receptor complexes (type I receptor and type II receptor) have a common component, namely IL-4Rα, which is expressed and distributed in different It plays different roles depending on the cell. IL-4 can function through type I and type II receptors, whereas IL-13 can function only through type II receptors. While IL-4 binds IL-4Rα with high affinity and is independent of the γ-chain or IL-13Rα1, IL-4Rα can increase the affinity of IL-13 for 13Rα1 binding.

A direct sign of severe allergic disease is the presence of high concentrations of IgE and eosinophils. Therefore, although the antibody target for the type 2 inflammatory pathway in the early stages is IgE, subsequent studies have shown that targeting IgE is not universal in the treatment of severe allergic diseases. With a better understanding of the type 2 inflammatory pathway, the key triggers of the type 2 inflammatory pathway, namely IL-4 and IL-13, have become popular research targets for the next generation of antibody drugs to treat severe allergic diseases.

The biological activity of IL-4 is mediated by specific cell surface IL-4 receptors. Human IL-4 receptor α (hIL-4Rα) antibodies are described in US Pat. Nos. 5,717,072 and 7,186,809. Methods for using the hIL-4R antibody are described in US Pat. Nos. 5,714,146, 5,985,280, and 6,716,587. WO2005047331 relates to Altrakincept, an IL-4Rα recombinant protein developed by Immunex, which binds competitively to IL-4 by spray administration and then inhibits IL-4 binding to cell surface IL-4Rα in vivo. suppress; US6358509 discloses Pascolizumab, an IL-4 antibody developed by GSK that prevents IL-4 from binding to IL-4Rα. These two IL-4 inhibitors showed good efficacy in early studies (preclinical and phase 1 clinical trials), but did not show efficacy in large-scale late-stage clinical trials, so development was discontinued. In US8679487, Amgen also early developed the IL-4 receptor α antibody Amg-317, a fully human IL-4Rα antibody that inhibits the activity of IL-4 and IL-13, but is not yet in phase 2 clinical trials to treat asthma. It failed and development was halted.

Examples of anti-IL-13 antibody molecules include CAT-354 disclosed in US07/0128192 or WO2005007699; TNX-650 disclosed in WO2005062967 and WO2005062972, and Clinical Trails Gov. NTC00441818 disclosed in Identifier; Clinical Trails Gov. QAX-576 disclosed in Identifier (NTC532233); Includes anti-IL-13 antibodies disclosed in US6468528 by Amgen, US20060140948 filed under the name of Abgenix, or WO2006055638; WO2005091856 by Centocor, Inc. as applicant; and WO2007080174 filed under the name of Glaxo, and WO2007045477 filed under the name of Novartis.

Typically, for example, the IL-13 antibody tralokinumab developed by AstraZeneca is expected to receive phase 3 clinical trial results for the treatment of severe asthma in the second half of 2017 and is the world's first commercially available IL-13 antibody. 13 It is expected to be an antibody. At the same time, AstraZeneca and Abbott jointly developed a companion diagnostic method based on the biomarkers periostin and DPP4. Additionally, a phase 2b clinical trial of tralokinumab to treat atopic dermatitis has also been completed.

Lebrikizumab, an IL-13 antibody drug developed by Roche, had data from two phase 3 clinical trials released at the end of February 2016, resulting in 1 win and 1 loss. While lebrikizumab significantly reduced the asthma exacerbation rate in LavoltaI, similar results were not obtained in LavoltaII. According to information disclosed at the 2017 JP Morgan meeting, Roche has discontinued development of the drug for the asthma indication, and multiple phase 2 clinical trials are ongoing for COPD, atopic dermatitis, and idiopathic pulmonary fibrosis.

After many setbacks in the initial development of monofunctional IL-4 or IL-13 inhibitors (no products are currently commercially available), it is believed that blocking both IL-4 and IL-13 simultaneously may be a feasible approach. There are at least two methods to simultaneously block IL-4 and IL-13: one is the IL-4Rα antibody; The other is a bispecific antibody of IL-4 and IL-13.

Sanofi's SAR156597, a drug targeting both IL-4 and IL-13, is in development and disclosed in WO2009052081, a dual variable domain Ig (DVD-Ig) bispecific for the treatment of systemic scleroderma and idiopathic pulmonary fibrosis. It is an antibody and is currently undergoing phase 2 clinical trials.

WO2005023860 relates to Pitrakinra, an IL-4 mutant developed by Aerovance that can bind to IL-4Rα with high affinity, and is a dual antagonist of IL-4 and IL-13 that significantly reduces FEV1 (forced expiratory volume in 1 second) in asthma patients. It can be improved. PEG-modified Pitrakinra (Aeroderm) is the first dual blocker to undergo clinical trials for atopic dermatitis. Despite some improvement in symptoms, the improvement in disease endpoints was not statistically significant.

Additionally, Roche is using its “Knobs into Holes” technology platform to create bispecific antibodies targeting IL-4 and IL-13, the antibodies have been modified based on lebrikizumab, but no clinical progress has been reported at present. . Regeneron's IL-4Rα and IL-13Rα fusion protein manufactured using Dual Trap technology achieved good results in early clinical studies (biomarker reduction), but the study was discontinued due to lack of funding.

WO2010053751 relates to the IL-4Rα antibody developed by Regeneron, which can regulate type 2 immunity by blocking both IL-4 and IL-13. The antibody in this patent application contains six complementarity-determining regions with the following sequences:

HCDR1 is Gly-Phe-Thr-Phe-X5-X6-Tyr-X8, where X5 is Asp or Arg (preferably Arg), X6 is Asp or Ser (preferably Asp), and X8 is Ala or Gly (preferably Ala).

HCDR2 is X1-Ser-X3-X4-X5-X6-X7-X8, where X1=Ile or Leu (preferably Ile), =Gly or Ser (preferably Gly), X6=Gly, Ser or Val, X7=Ser or Asn (preferably Asn), X8=Thr, Lys or Ile.

HCDR3 is Ala-Lys-X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ -X ¹² -X ¹³ -X ¹⁴ -X ¹⁵ -X ¹⁶ -X ¹⁷ -X ¹⁸ , where X3=Asp, Glu or Trp, X4=Gly or Arg, X5=Leu, Thr or Arg, X6=Gly, Arg or Ser, , X9=Ile, Asp or Phe, X10=Arg, Tyr or Asp, X11=Pro, Tyr or deletion, X12=Arg or deletion, X13=Tyr or deletion, =Leu or deletion, X17=Asp or deletion, X18=Val or deletion.

LCDR1 is Gln-X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ , where X2=Asp, Ser or Val, X3=Ile or Leu , X4=Ser, Leu or Asn, X5=Asn, Tyr or Ile, X6=Trp, Ser or Tyr, X7=Ile or deletion, X8=Gly or deletion, X9=Tyr or deletion, =Tyr or fruition.

LCDR2 is X ¹ -X ² -Ser, where X1=Leu, Ala or Val, X2=Ala or Gly.

LCDR3 is X ¹ -Gln-X ³ -X ⁴ -X ⁵ -X ⁶ -Pro-X ⁸ -Thr, where X1=Gln or Met, or Ser, X6=Thr, Phe or His, X8=Tyr, Ile or Trp.

As a protein drug, the amino acid sequence as the primary structure of the protein is the basis of the spatial structure of the protein, and the spatial structure directly determines its function. Small changes in the amino acid sequence can cause large changes in spatial structure, resulting in changes in function. The variable region of an antibody contains six complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3 in the heavy chain, and LCDR1, LCDR2, and LCDR3 in the light chain, with HCDR3 having the greatest high variability in structure and sequence; It is also a key region that reflects the diversity and specificity of antibody binding. The complementarity-determining region of an antibody is a key region for maintaining the activity of the antibody; In many cases, even replacing one of the amino acids with an amino acid with similar properties will have a significant impact on the binding of the entire antibody to the antigen and therefore its biological activity. It is difficult to predict what changes will occur in the activity of the antibody by modifying the CDR region.

The inventors of the present invention unexpectedly discovered that when R at position 108 in HCDR3 of the dupilumab antibody was changed to A or E, the binding ability of the antibody to the antigen could be increased (EC50 increased by more than 50%) ), when R at position 108 is changed to other representative amino acids such as Y, L or Q, the binding activity decreases by more than 50%. Similarly, when R is changed to A or E, especially when R at position 106 is changed to A or E, the binding activity (EC50) decreases 5-10 fold. Accordingly, the present invention provides a new IL-4Ra antibody whose binding activity is significantly superior to that of dupilumab; This antibody regulates type 2 immunity by binding to IL-4Rα while blocking IL-4 and IL-13.
According to WHO Drug Information, Vol.26, No.4, 2012, the amino acid sequence of dupilumab is as follows:
Heavy chain amino acid sequence:
EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
Light chain amino acid sequence:
DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

One of the purposes of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to human IL-4Ra. Compared to the existing dupilumab antibody, the affinity of the antibody or antigen-binding fragment thereof for IL-4R binding is further improved. Compared to dupilumab, this dupilumab variant contains the following amino acid substitutions: the amino acid Arg at position 24 of the LVR is mutated to Ala, or the amino acid Ser at position 28 is mutated to Arg, or the amino acid Ser at position 32 is mutated to Arg. The amino acid Ser is mutated to Arg or Lys; Arg at position 100 of the HVR is mutated to Ala, or Arg at position 106 is mutated to Ala or Glu, or Arg at position 108 is mutated to Ala, Glu, Gln, Tyr or Leu.

In particular, one aspect of the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor α (hIL-4Rα) comprising a heavy chain variable region (HVR) and a light chain variable region (LAR). , wherein the heavy chain variable region is CDR1 (HCDR1) with the amino acid sequence of Gly-Phe-Thr-Phe-Arg-Asp-Tyr-Ala; CDR2 (HCDR2) with the amino acid sequence Ile-Ser-Gly-Ser-Gly-Gly-Asn-Thr; and a CDR3 (HCDR3) having the amino acid sequence of Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val, wherein wherein Xaa1 is Arg or Ala, Xaa2 is Arg, Ala or Glu, and Xaa3 is Arg, Ala or Glu;

The light chain variable region is: CDR1 (LCDR1) with the amino acid sequence Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr, where Xaa4 is Ser or Arg and Xaa5 is Ser, Arg or Lys; CDR2 (LCDR2) with the amino acid sequence Leu-Gly-Ser; and CDR3 (LCDR3) with the amino acid sequence Met-Gln-Ala-Leu-Gln-Thr-Pro-Tyr-Thr.

In a preferred embodiment, the amino acid sequences of HCDR3 and LCDR1 are as follows: (a) HCDR3: Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr - Gly-Leu-Asp-Val, where Xaa1 is Arg, Xaa2 is Arg, and Xaa3 is Ala or Glu; and LCDR1: Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr, where Xaa4 is Ser and Xaa5 is Ser; (b) HCDR3: Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val, where Xaa1 is Arg and Xaa2 is Ala or Glu, and Xaa3 is Arg; and LCDR1: Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr, where Xaa4 is Ser and Xaa5 is Ser; (c) HCDR3: Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val, where Xaa1 is Ala and Xaa2 is is Arg, and Xaa3 is Arg; and LCDR1: Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr, where Xaa4 is Ser and Xaa5 is Ser; (d) HCDR3: Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val, where Xaa1 is Arg and Xaa2 is is Arg, and Xaa3 is Arg; and LCDR1: Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr, where Xaa4 is Arg and Xaa5 is Ser; (e) HCDR3: Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val, where Xaa1 is Arg and Xaa2 is is Arg, and Xaa3 is Arg; and LCDR1: Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr, where Xaa4 is Ser and Xaa5 is Arg or Lys.

In a preferred embodiment, the amino acid sequence of HVR is SEQ ID NO: 1. In a preferred embodiment, the amino acid sequence of LVR is SEQ ID NO:2. In a preferred embodiment, the amino acid sequence of the LVR is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9. In a preferred embodiment, the amino acid sequence of HVR is: SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19. SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25.

In a preferred embodiment, HVR and LVR have amino acid sequences: SEQ ID NO: 1 and SEQ ID NO: 3; SEQ ID NO: 1 and SEQ ID NO: 5; SEQ ID NO: 1 and SEQ ID NO: 7; SEQ ID NO: 1 and SEQ ID NO: 9; SEQ ID NO: 11 and SEQ ID NO: 2; SEQ ID NO: 13 and SEQ ID NO: 2; SEQ ID NO: 15 and SEQ ID NO: 2; SEQ ID NO: 17 and SEQ ID NO: 2; SEQ ID NO: 19 and SEQ ID NO: 2; SEQ ID NO: 21 and SEQ ID NO: 2; SEQ ID NO: 23 and SEQ ID NO: 2; or a combination of SEQ ID NO: 25 and SEQ ID NO: 2.

In the most preferred embodiment, HVR and LVR have amino acid sequences: SEQ ID NO: 17 and SEQ ID NO: 2; or a combination of SEQ ID NO: 11 and SEQ ID NO: 2.

In another aspect, the present invention also relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutically acceptable carrier. Meanwhile, the present invention also provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to the present invention; An expression vector containing a nucleic acid molecule according to the present invention; and a host cell containing the expression vector according to the present invention, preferably the host cell is a eukaryotic cell.

In another aspect, the present invention relates to a method of producing an antibody or antigen-binding fragment thereof that specifically binds to human IL-4R, the method comprising: helping the expression of the antibody and antigen-binding fragment thereof according to the present invention; It includes the step of expressing the nucleic acid molecule according to the present invention under these conditions, and the step of recovering the expressed antibody or antigen-binding fragment thereof.

In another aspect, the present invention relates to the use of an antibody or antigen-binding fragment thereof in the manufacture of a drug for preventing or treating diseases or disorders associated with IL-4/IL-4Ra signaling in humans. In a preferred embodiment, the disease or disorder is: asthma, allergic dermatitis, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hypertrophy, COPD, atopic dermatitis, idiopathic pulmonary fibrosis, allergic reaction, Kawasaki disease, It is selected from the group consisting of sickle cell anemia, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, or kidney disease.

In another aspect, the present invention relates to a method for preventing or treating a disease or disorder related to IL-4/IL-4Rα signaling in the human body, using the antibody according to any one of claims 1 to 8 or the antibody according to any one of claims 1 to 8. and administering the antigen-binding fragment to the subject. In a preferred embodiment, the disease or disorder is: asthma, allergic dermatitis, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hypertrophy, COPD, atopic dermatitis, idiopathic pulmonary fibrosis, allergic reaction, Kawasaki disease, It is selected from the group consisting of sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, or kidney disease. In a preferred embodiment, the disease or disorder is allergic dermatitis or asthma. In a preferred embodiment, the antibody or antigen-binding fragment thereof is used in combination with other drugs to treat autoimmune diseases. In a preferred embodiment, the antibody or antigen-binding fragment thereof is used in combination with a hormonal drug, immunosuppressant, or antihistamine drug.

In another aspect, the invention relates to a formulation or kit comprising a container and a package insert, wherein the container contains an antibody or antigen-binding fragment thereof according to the invention, or a pharmaceutical composition according to the invention, The package insert contains instructions for use of the drug. In a preferred embodiment, the formulation or kit further comprises one or more containers containing one or more other drugs for preventing or treating diseases or disorders associated with IL-4/IL-4Ra signaling in humans. In a preferred embodiment, the other drug is a hormonal drug, immunosuppressant or antihistamine drug.

Figure 1 is an SDS-PAGE diagram of dupilumab variants D1-D9: lane 1 is the molecular weight marker, lane 2 is D1, lane 3 is D2, lane 4 is D3, lane 5 is D4, lane 6 is D5, and lane 7 is D6, lane 8 is D7, lane 9 is D8, and lane 10 is D9.
Figure 2 is a SEC detection graph of dupilumab variant D8.
Figure 3 is a graph showing the biological effect of dupilumab and dupilumab variants D5, D7, D8 and D9 in IL-4 neutralization in vitro, where dupilumab inhibits only 92% of cell growth, whereas D8 and D9 has an inhibition rate of 98%, which is clearly superior to dupilumab.
Figures 4A-4H show the comparative stability results of dupilumab and dupilumab variants under different storage conditions such as high temperature, strong light irradiation, repeated freeze-thaw, and strong acid conditions. Figure 4a is a graph of SEC results of three samples at 50°C, and Figure 4b is a graph of ELISA results of three samples at 50°C. Figure 4c is a graph of SEC results of three samples under repeated freeze-thaw conditions, and Figure 4d is a graph of ELISA results of three samples under repeated freeze-thaw conditions. Figure 4e is a graph of SEC results of three samples under 4500 lx conditions, and Figure 4f is a graph of ELISA results of three samples under 4500 lx conditions. Figure 4g is a graph of SEC results of three samples under strong acid conditions, and Figure 4h is a graph of ELISA results of three samples under strong acid conditions.

1. Definition:

“IL-4R” (i.e., CD124) is a type I transmembrane glycoprotein consisting of 825 amino acids (25 signal peptide domains, 207 extracellular domains, 24 transmembrane regions, and 569 intracellular domains). The extracellular domain has one CKR-SF domain and one type III fibrin domain, and is responsible for T cells, B cells, NK cells, monocytes/macrophages, neutrophils, hematopoietic progenitor cells, mast cells, endothelial cells, fibroblasts, and keratinocytes. There are six N-linked sugar chain attachment sites distributed throughout the cell. The α chains of IL-4R and IL-13R are equivalent, and binding of the α chain to IL-4 can cause tyrosine-mediated phosphorylation of several intracellular proteins.

“Disease associated with IL-4/IL-4Rα signaling” refers to a disease involving biological functions mediated by IL-4/IL-4Rα signaling. Examples of diseases associated with IL-4/IL-4Rα signaling include, for example, asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, multiple sclerosis, arthritis, allergic rhinitis, eosinophilic esophagitis, and psoriasis. Including inflammatory diseases or disorders such as:

In some embodiments, the disease associated with IL-4/IL-4Ra signaling is airway inflammation. In some embodiments, the disease associated with IL-4/IL-4Ra signaling is skin inflammation or atopic dermatitis.

“Allergic reaction,” “allergic disease,” or “allergic disorder” is considered an inflammatory disease caused by the interaction of genetic and environmental factors. Molecular and cellular immune mechanisms involved in the development of allergic diseases include changes in the molecular expression of the T cell immunoglobulin mucin domain due to attenuation of natural immune stimulation, changes in the balance of type 1 and type 2 T helper cells, and changes in the balance of regulatory T cells. A number of factors are involved. Among allergic diseases, atopic dermatitis, asthma, and allergic rhinitis are the most common inflammatory diseases in patients with allergic reactions; These patients often suffer from the onset of multiple clinical symptoms. The onset of allergic reactions is related to abnormal immune responses to external antigens (Mueller et al. (2002) Structure, binding, and antagonists in the IL-4/IL-13 receptor, system, Biochim Biophys Acta 1592: 237- 250). Overproduction of antigen-specific IgE is a necessary factor in causing allergic inflammation. Abnormal type 2 T helper cell (Th2) polarization contributes to increased IgE responses. Interleukin-4 (IL-4) and interleukin-13 (IL-13) produced by activated T cells are key Th2 cytokines that trigger and maintain immune and inflammatory responses in allergic reactions.

IL-4 and IL-13 signaling are mediated by two different receptor complexes with a shared subunit, IL-4 receptor α (IL-4Rα), which may contribute to the overlap in biological responses between these two cytokines. . IL-4Rα forms two different heterodimeric receptor complexes that mediate the biological functions of IL-4 and IL-13 in a tissue- and response-specific manner. Dupilumab is an antagonist monoclonal antibody against human IL-4Rα and inhibits the biological activities induced by IL-4 and IL-13. Dupilumab blocks IL-4 signaling by preventing IL-4 from binding to the receptor subunit, and its inhibitory effect on IL-13 signaling may be mediated by disrupting the interaction of the dimeric receptor.

Dupilumab, a fully human monoclonal antibody directed against the shared IL-4Rα subunit, was developed by Regeneron Pharmaceuticals, Inc. and is currently in clinical trials for the treatment of moderate to severe asthma and moderate to severe atopic dermatitis. .

An “antibody” as described herein is an immunoglobulin molecule composed of four peptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds; Each heavy chain includes a heavy chain variable region (HVR or VH) and a heavy chain constant region, where the heavy chain constant region includes three domains: CH1, CH2, and CH3; Each light chain includes a light chain variable region (LVR or VL) and a light chain constant region, where the light chain constant region includes a domain (CL1). The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the heavy chain constant region may be selected from IgG1, IgG2, IgG3 or IgG4, preferably IgG4, and the light chain constant region is selected from κ or λ.

The term “variable region” is used herein to describe a region of an antibody that differs between antibody sequences and is associated with the binding and specificity of a particular antibody for its particular antigen. However, variability is usually not evenly distributed throughout the variable region of an antibody. Typically the focus is on three sequences called the complementarity determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The relatively more conservative part of the variable region is called the framework region (FR). Each variable region of the natural heavy and light chains contains four FRs, most of which adopt a β-sheet configuration and are connected by three CDRs that form circular links, and in some cases form part of the β-sheet structure. . The CDRs of each chain are held in close proximity by FRs and, together with the CDRs of other chains, contribute to the formation of the antigen binding site of the antibody. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity.

The terms “complementarity determining region”, “hypervariable region” and “CDR” refer to one or more hypervariable regions or complementarity determining regions (CDRs) present in the variable region of an antibody light or heavy chain (see Kabat, E. A. et al ., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These terms refer to the hypervariable regions defined by Kabat et al. ("Sequences of Proteins of Immunological Interest," Kabat E., et al., USDept. of Health and Human Services, 1983) or the hypervariable regions in the three-dimensional structure of the antibody. Contains a variable loop (Chothia and Lesk, J. Mol. Biol. 196901-917 (1987)). The CDRs of each chain are held in close proximity by framework regions and, together with the CDRs from the other chain, contribute to the formation of an antigen binding site.

The terms “framework region” and “FR” refer to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody (see Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health , Bethesda, Md., (1987)). These terms include the amino acid sequence located between the amino acid terminus and the first CDR, the amino acid sequence located between the CDRs, and the amino acid sequence located between the third CDR and the start of the constant region in the light and heavy chains of the antibody.

CDR and FR residues are defined by standard sequence definitions (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md. (1987)) and structural definitions (Chothia and Lesk, J. Mot. Biol. 196: 901). -217 (1987)).

As referenced herein, reference is made to the numbering system of Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991). Kabat assigns a residue number to each amino acid in the listed sequence, and this method of assigning residue numbers has become standard in the field. When explicitly indicating that Kabat numbering is used, this specification follows the Kabat numbering system. Unless Kabat numbering is indicated, sequential amino acid sequence numbers are used (i.e., sequential amino acids are sequential integers from left to right from amino terminus to carboxy terminus (e.g., 1, 2, 3, etc.) are numbered).

The “affinity” of an antibody for an antigen or epitope is a well-known term in the art and refers to the degree or strength of the antibody's binding to the epitope. Affinity can be defined as the equilibrium dissociation constant (KD or Kd, the ratio of the dissociation rate of the antibody to the association rate, i.e. K _off /K _on ) and the apparent equilibrium dissociation constant (KD' or Kd') and IC50 (competitive can be measured and/or expressed by a variety of methods well known in the art, including, but not limited to, the amount required to achieve 50% inhibition in the assay); The relative affinity of a humanized antibody may be determined, for example, by comparison to a related murine or chimeric antibody. For the purposes of the present invention, affinity is the average affinity of a particular population of antibodies to bind to an antigen or epitope. The affinity of an antibody can be measured by enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS) analysis. The ELISA method is used in the examples herein to determine the affinity of antibodies of the invention for IL-4R (see Examples).

2. Method for producing antibodies according to the present invention

Antibodies according to the invention may be produced by any available method, such as recombinant expression techniques. Nucleic acids encoding light and heavy chain variable regions operably linked to constant regions can be inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. DNA segments encoding immunoglobulin chains can be operably linked to control sequences in an expression vector that ensure expression of the immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., native or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. In one embodiment, the expression control sequence is a prokaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is introduced into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the antibody.

Expression vectors can be replicated in any host organism, either as an episome or as an integrated part of the host chromosomal DNA. In one embodiment, the expression vector includes a selective marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow detection of cells transformed with the expected DNA sequence.

Expression vectors are used to express antibodies according to the invention from any host cell, including prokaryotic host cells (e.g., E. coli), yeast host cells, mammalian host cells, plant host cells, and insect host cells. can be used

In one embodiment, Escherichia coli is used to produce the antibodies of the invention. Other prokaryotic hosts suitable for this use include Bacteria, such as Bacillus, and other Enterobacteriaceae, such as Salmonella, Cellatia, and various Pseudomonas species. Expression vectors can also be prepared in these prokaryotic hosts, and expression vectors generally contain expression control sequences (e.g., origins of replication) that are compatible with the host cell. Additionally, there may be a variety of known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system of λ-bacteriophage. Optionally, a promoter typically has operator sequences to control expression, ribosomal binding site sequences to initiate and complete transcription and translation, etc.

Additionally, other microorganisms, such as yeast, can also be used to express the antibodies of the invention. For example, Saccharomyces can be used as a yeast host with a suitable promoter comprising expression control sequences (e.g., promoters), an origin of replication, a termination sequence, and other sequences of interest. Promoters used in yeast expression techniques include promoters for 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, but are not limited to, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

In another embodiment, mammalian tissue cell cultures can be used to express and produce antibodies of the invention. Any mammalian tissue cell can be used in these methods, including mammalian BHK or CHO cell lines, various Cos cell lines, HeLa cell lines, preferably myeloma cell lines, or transformed B cells, or hybridomas, including heterologous proteins ( A number of suitable host cell lines capable of secreting intact immunoglobulins (e.g., intact immunoglobulins) have been developed in this field. In one embodiment, the cell is a non-human cell. Expression vectors in mammalian cells may contain expression control sequences, such as origins of replication, promoters and enhancers, as well as necessary processing signal sites such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. . In one embodiment, the expression control sequence is a promoter derived from an immunoglobulin gene, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc.

Vectors containing polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred to host cells by well-known methods, depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipid transfection, bioballistics, or viral transfection can be used for other cell hosts. Other methods to transform mammalian cells include the application of polybrene, protoplast fusion, liposomes, electroporation and microinjection. To produce transgenic animals, the transgene can be microinjected into a fertilized egg, or introduced into the genome of an embryonic stem cell and transferred into an oocyte from which the nucleus of such cell has been removed.

If the nucleic acid molecules encoding the heavy and light chains are cloned into separate expression vectors, the vectors can be co-transfected such that intact immunoglobulins are expressed and assembled. Once expressed, intact antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified using ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, etc. It can be purified according to standard methods. Substantially pure immunoglobulins with at least about 90% to 95% homogeneity can be prepared for pharmaceutical use. In other embodiments, substantially pure humanized antibodies having a homogeneity of at least about 98% to 99% or greater may be prepared for pharmaceutical formulations and methods.

Accordingly, the present invention includes the steps of (a) transforming a host cell with a nucleic acid molecule encoding an antibody described herein, and (b) culturing the transformed host cell under conditions permitting expression of the antibody. , provides a method for expressing an antibody of the present invention. Known techniques can be used to include selectable markers in vectors to facilitate selection of labeled host cells expressing the antibody.

“Antigen-binding fragment” of an antibody or “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (hIL-4R, etc.). It has been shown that the antigen-binding function of an antibody can be achieved by specific fragments of the full-length antibody. Examples of “antigen-binding fragments” of antibodies include (i) Fab fragments, i.e. monovalent fragments consisting of the VL, VH, CL1 and CH1 domains; (ii) F(ab')2 fragment, i.e. a divalent fragment consisting of two F(ab') fragments connected by a disulfide bond in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the single arm VL and VH domains of the antibody; (v) dAb fragment consisting of VH domain; and (vi) CDRs. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be connected by a synthetic linker to form a single linked chain through recombination, in which the VL and VH regions are paired to form a monovalent molecule. (referred to as short-chain Fv, scFv). Such single chain antibodies are also included within the scope of “antigen-binding fragments” of antibodies.

The antibody of the present invention is a method comprising culturing a host cell capable of expressing the antibody under conditions capable of expressing the antibody and containing a DNA sequence encoding the antibody, and then recovering the produced antibody from the culture. can be manufactured.

The medium used for cell culture can be any conventional medium used for culturing host cells, such as basal medium or complex medium with appropriate additives. Suitable media are commercially available, or suitable media can be prepared according to the disclosed methods. Next, the polypeptides produced by the cells are separated from the host cells by centrifugation or filtration, the protein components of the supernatant or filtrate are precipitated with salts such as ammonium sulfate, and then, depending on the type of target peptide, ion exchange chromatography. It can be recovered from the medium by conventional methods, including purification by various chromatography methods such as chromatography, gel filtration chromatography, and affinity chromatography.

3. Treatment methods and agents

The present invention provides a pharmaceutical composition comprising an anti-IL-4Ra antibody or an antigen-binding fragment thereof according to the present invention. The pharmaceutical composition according to the present invention will be administered with appropriate carriers, excipients and other agents. These agents are included in the formulation to improve delivery and tolerability. A number of suitable formulations can be found in the pharmacopoeia known to every pharmacist (Remington's Pharmaceuticals, Mack Publishing, Easton, PA).

Adjust the dosage according to differences in the subject's age, body size, target disease, symptoms, route of administration, etc. When using the antibody of the present invention to treat various IL-4R-related disorders and diseases in adults, the antibody of the present invention is generally administered at about 0.01 to 20 mg/kg per body weight, more preferably about 0.1 to 15 mg/kg per body weight, It can be administered intravenously in a single dose of about 1 to 10, or about 3 to 10 mg/kg. Depending on the severity of the disorder, the frequency and duration of treatment may be adjusted.

Various types of known drug delivery systems, such as liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, may be used to administer the pharmaceutical composition according to the present invention. (e.g., Wu et al. (1987), J. Biol. Chem. 262: 4429-4432). Methods of drug administration include, but are not limited to: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, transthecal, and oral routes. The pharmaceutical composition may be administered by any convenient route, such as infusion or intravenous bolus injection, absorption into epithelial and mucosal layers (oral mucosa, rectal and small intestine mucosa, etc.), and may be administered in combination with other bioactive agents. The mode of administration may be systemic or topical.

Additionally, pharmaceutical compositions can be delivered through pockets, especially liposome pockets (see Langer (1990) Science 249: 1527-1533; Treat et al. (1989) in Liposomes in Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (Eds.), Liss, New York, pages 353-365; Lopez-Berestein, supra, pages 317-327).

In certain cases, pharmaceutical compositions may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201). In other embodiments, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (Eds.), CRC Pres., Boca Raton, Florida (1974)). For a discussion of other controlled release schemes, see Langer (1990) Science 249:1527-1533.

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, drip infusion, etc. These injectable formulations can be prepared by the disclosed methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous or oily medium commonly used for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like. These include alcohols (e.g. ethanol), polyols (e.g. propylene glycol, polyethylene glycol), nonionic surfactants [e.g. polysorbate 80, HCO-50 (polyoxyethylene of hydrogenated castor oil (50 moles) ) It can be used in combination with a suitable solubilizer such as an adduct). The oily medium used is sesame oil or soybean oil. They may be used in combination with solubilizing agents such as benzylbenzoate and benzyl alcohol. The injection prepared in this way is preferably packaged in an appropriate ampoule.

Monotherapy and combination therapy of antibodies and fragments thereof according to the present invention are useful for treating diseases and disorders that can be improved, inhibited or improved by reducing the activity of IL-4. These diseases include diseases characterized by abnormal expression or overexpression of IL-4, or an abnormal host response to IL-4 production. Diseases related to IL-4 treated with the antibody or fragment thereof according to the present invention include, for example, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, and asthma (mild , moderate and severe asthma), inflammatory diseases such as inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell anemia, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune Includes immune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis (disease), atopic dermatitis, ulcerative colitis, fibrosis and kidney disease (see US Patent 7,186,809).

The present invention includes combination therapy in which an anti-IL-4Rα antibody or fragment thereof is administered together with one or more therapeutic agents (or referred to as second therapeutic agents). Co-administration and combination therapy are not limited to simultaneous administration, but include treatment regimens in which the anti-IL-4Rα antibody or fragment thereof is administered to the patient at least once during a course of treatment that includes the administration of at least one other therapeutic agent. The second therapeutic agent may be other IL-4 antagonists, such as other antibodies/fragments thereof, or soluble cytokine receptors, IgE antagonists, anti-asthma drugs (e.g., corticosteroids, non-steroidal drugs), which may be administered by inhalation or other suitable manner. , β agonists, leukotrienes, antagonists, xanthines, fluticasone, salmeterol, albuterol). In certain embodiments, the anti-IL-4R antibody or fragment thereof according to the present invention may be administered together with an IL-1 antagonist such as rilonacept or an IL-13 antagonist. The second agent may include one or more leukotriene receptor antagonists to treat conditions such as allergic inflammation, such as asthma and allergies. Examples of leukotriene receptor antagonists include, but are not limited to, montelukast, pranlukast, and zafirukast. The second agent may include a cytokine inhibitor, such as one or more TNF (etanercept, ENBRELTM), IL-9, IL-5, or IL-17 antagonists.

The present invention also includes the use of any of the anti-IL-4Rα antibodies or antigen-binding fragments thereof described herein in the manufacture of a drug for treating a disease or condition, wherein the disease or condition is human interleukin-4 (hIL-4). 4) It is enhanced, improved or inhibited by eliminating, suppressing or reducing the activity of. Examples of such diseases or conditions include, for example, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, pulmonary disease, asthma, inflammatory diseases, allergic reactions, Kawasaki disease, sickle cell disease, chro- Includes Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis (disease), kidney disease, atopic dermatitis, and asthma.

In one aspect of the present invention, a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human IL-4R according to the present invention and a pharmaceutically acceptable carrier is provided.

In this application, drug, pharmaceutical composition, and pharmaceutical agent (agent) may be used interchangeably with each other, unless contradictory or otherwise specified. In this context, pharmaceutically acceptable excipients refer to non-toxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients. Excipients such as diluents, microcrystalline cellulose, mannitol, etc.; Fillers such as starch, sucrose, etc.; binders such as starch, cellulose derivatives, alginates, gelatin, and/or polyethylenepyrrolidone; disintegrants such as calcium carbonate and/or sodium bicarbonate; Uptake enhancers such as quaternary ammonium compounds; surfactants such as hexadecanol; solvents such as carriers, water, physiological saline, kaolin, bentonite, etc.; These are lubricants such as talc, calcium/magnesium stearate, and polyethylene glycol. Additionally, the pharmaceutical composition according to the present invention is preferably administered by injection.

In some embodiments of the present invention, the antibody or antigen-binding fragment thereof in the pharmaceutical composition according to the present invention is administered at a concentration of 1 to 1000 mg/ml, preferably at a concentration of 10 to 1000 mg/ml, more preferably 50 mg/ml. It is present at a concentration of ~500 mg/ml, and even more preferably at a concentration of 100-300 mg/ml.

The pharmaceutical composition according to the present invention preferably has a pH of 3.0 to 9.0, and may further include a buffering system, preservative, surface tension agent, chelating agent, stabilizer, and surfactant. In one embodiment of the invention, the pharmaceutical composition according to the invention is an aqueous formulation. These preparations are generally solutions or suspensions. In certain embodiments of the invention, the pharmaceutical composition is a stable aqueous solution. In another specific embodiment of the invention, the pharmaceutical composition is a lyophilized formulation, and the solvent and/or diluent are added by the physician or patient to dissolve the lyophilized formulation prior to use.

Example 1: Construction of expression vector for dupilumab variant

HindIII cleavage site ( AAGCCT ), KoZAK sequence ( GCCGCCACC ), ATG, signal peptide gene ATGGAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT and dupilumab heavy chain coding gene (SEQ ID NO: 38, heavy chain variable region coding gene SEQ ID NO: 27 and constant region IgG4 encoding gene) ), the terminator code TAATAATAA and the EcoRI encoding gene GAATCC were fused sequentially in the sequence, and the gene fragments were obtained by chemical synthesis. Through the EcoRI and HindIII sites, the above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.1(+) and sequence verification was performed to obtain the expression plasmid PCDNA3.1(+)-DH for dupilumab heavy chain.

HindIII cleavage site ( AAGCCT ), KoZAK sequence ( GCCGCCACC ), ATG, signal peptide gene ATGGAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT and dupilumab light chain coding gene (SEQ ID NO: 39, light chain variable region coding gene SEQ ID NO: 28, and fractionation region κ encoding gene) ), the terminator code TAATAATAA and the EcoRI encoding gene GAATCC were fused sequentially in sequence, and the gene fragments were obtained by chemical synthesis. Through the EcoRI and HindIII sites, the above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.1(+) and sequence verification was performed to obtain the expression plasmid PCDNA3.1(+)-DL for the dupilumab light chain.

According to the same method as above, a series of expression plasmids of dupilumab variants were prepared, respectively pCDNA 3.1(+)-D1L, pCDNA 3.1(+)-D2L, pCDNA 3.1(+)-D3L, pCDNA 3.1(+)- D4L, pCDNA3.1(+)-D5H, pCDNA3.1(+)-D6H, pCDNA3.1(+)-D7H, pCDNA3.1(+)-D8H, pCDNA3.1(+)-D9H, pCDNA3.1 They were called (+)-D10H, pCDNA 3.1(+)-D11H, pCDNA 3.1(+)-D12H, pCDNA 3.1(+)-DH, and pCDNA 3.1(+)-DL.

For variant D1, the amino acid Arg at position 24 of the LVR of dupilumab is mutated to Ala, and the gene sequence of the LVR encoding D1 is SEQ ID NO: 8; For variant D2, the amino acid Ser at position 28 of the LVR of dupilumab is mutated to Arg, and the gene sequence of the LVR encoding D2 is SEQ ID NO: 10; For variant D3 or D4, the amino acid Ser at position 32 of the LVR of dupilumab is mutated to Arg or Lys, respectively, and the gene sequences of the LVR encoding D3 and D4 are SEQ ID NO: 4 and 6, respectively; For variant D5, Arg at position 100 of the HVR of dupilumab is mutated to Ala, and the gene sequence of the HVR encoding D5 is SEQ ID NO: 14; For variant D6 or D7, the amino acid Arg at position 106 of dupilumab HVR is mutated to Ala or Glu, respectively, and the gene sequences of HVR encoding D6 and D7 are SEQ ID NO: 16 and 20, respectively; For variant D8-D12, the amino acid Arg at position 108 of dupilumab HVR is mutated to Ala, Glu, Gln, Tyr or Leu, respectively, and the gene sequence of HVR encoding D8-D12 is 18, 12, 22, respectively. 24 and 26. The heavy chain constant region of each variant is IgG4 and the light chain constant region is κ.

Example 2: Expression and purification of dupilumab and its variants

Eukaryotic expression cells FreeStyle™ 293-F cells (Invitrogen Corporation, R790-07) were used to express the antibody of interest with the DNA construct described in Example 1. According to the operation manual of the FreeStyle™ 293 Expression System, the cell density was adjusted to 1×10 ⁶ cells/ml the day before plasmid transfection. On the day of plasmid transfection, plasmids were combined according to the table below, mixed with transfection reagent and added to the cell culture medium; After continuous culture at 37°C and 8% CO ₂ for 5 to 7 days, the cell culture supernatant was collected for antibody purification.

The expression supernatant was filtered through a 0.22 μM filter membrane, and the expressed antibody was equilibrated using a Mabpurix affinity chromatography column (purchased from Sepax, 65008), i.e., the chromatography column was equilibrated with equilibration buffer (120mM Tris+100mM NaCl, pH 7.5). After equilibration, the filtrate was loaded and passed through an affinity chromatography column, and eluted with an elution buffer (0.15M glacial acetic acid, pH 2.8). The purified antibody was detected by SDS-PAGE and SEC, and the purity was over 95%.

Example 3: Measurement of antigen binding affinity

The purified antibody described in Example 2 was used for in vitro affinity measurements. The microplate was coated with IL-4R (R&D, 7700-4R-050) at 2 μg/ml, 100 μl/well, attached to the plate membrane, placed overnight at 4°C, and the next day, the plate was washed three times with washing solution. Next, prepare 10% bovine serum albumin solution as a washing solution, add 200㎕/well to the Elisa plate, place at 37°C for 2 hours, and wash the plate three times with the washing solution; Add 100 μl/well of gradient diluted antibody solution, place at 37°C for 2 hours, and wash the plate three times with washing solution; HRP-labeled secondary antibody (catalog: ab6858, manufactured by Abcam) was diluted at a ratio of 1/5000 with a diluent (2% bovine serum albumin solution was prepared as a washing solution) and incubated in a microplate at 37°C for 1 hour. Add at 100㎕/well; Wash the plate 5 times with washing solution; TMB coloring solution was added at 100㎕/well, placed in a dark place at room temperature to react for 5-10 minutes, and 50㎕/well of stop solution was added to stop the reaction. Absorbance was measured at a wavelength of 450 nm.

The results show that D8 and D9 have improved EC50 compared to dupilumab, 3.200nM and 1.997nM, respectively; In particular, for D8, the EC50 was about 60% of dupilumab.

Example 4: Biological effects on IL-4 neutralization in vitro

The purified antibody described in Example 2 was used to determine its biological effect on IL-4 neutralization in vitro. Using RPMI1640 basal medium (C22400500BT from GIBCO), TF-1 cells were cultured under starvation at 37°C and 5% carbon dioxide for 24 hours. The next morning, TF-1 cells were collected, washed three times with RPMI1640 medium, and resuspended in complete medium (RPMI1640 basal medium containing 10% fetal bovine serum) at a cell concentration of 4.0 × 10 ⁵ cells/ml. The cell suspension, antibody sample, and gradient dilution of IL-4 were each added to a 96-well cell culture plate, mixed well, and cultured at 37°C in 5% carbon dioxide for 2 days. According to the operation manual of CCK-8 (CK04, manufactured by Dojindo), CCK-8 diluent was added and cultured at 37°C in 5% carbon dioxide for 2.5 hours; When detected with a microplate reader, cell viability was detected by measuring absorbance at a reference wavelength of 630 nm and a determination wavelength of 450 nm.

The results in Figure 3 show that dupilumab inhibits cell growth by 92%, while D8 and D9 show a better inhibition effect with an inhibition rate of 98%, which is much better than dupilumab.

Example 5: Stability of dupilumab variants

To further investigate the properties of the mutated antibody, we designed different storage conditions to determine changes in stability and activity of the mutated antibody compared to the primary antibody dupilumab, and analyzed the mutated antibody by SEC-HPLC method (abbreviated as SEC) after storage. The main peak purity of the antibody was detected, and the relative activity of the antibody after storage was detected by ELISA method.

The purified antibody of the present invention and dupilumab were each added to a buffer solution of 12.5mM sodium acetate (pH 6.15) at a final concentration of about 2.7 mg/ml. According to the experimental results of the preliminary experiment, 35.1㎕ of 0.2M Tris-Base was added to 1.5㎖ of sample to bring the pH to 9.0, filtered and divided at 200㎕/piece, each filtrate was divided into 7 pieces; 288 μl of 0.2M citric acid was added to 2.5 ml of sample for acidification to pH about 3.0, filtered and divided at 200 μl/piece, each filtrate divided into 12 pieces; The last remaining sample was filtered and sterilized at 200㎕/piece, and each filtrate was divided into 30 pieces. All samples were stored in clean, sterilized vials sealed with sterilized aluminum caps and covered with rubber stoppers.

SEC-HPLC analysis: Materials with different molecular weights were separated by gel filtration chromatography to quantify the purity of the samples. Column was TSK G3000SWXL, mobile phase: 0.2 mol/L potassium phosphate buffer, 0.25 mol/L potassium chloride (pH 6.2±0.1); The flow rate was 0.5 mL/min and the washing time was 30 minutes.

ELISA method (detection with microplate reader): 2 μg/ml IL-4R coated microplate, 500 ng/ml test sample was diluted 5-fold to initial concentration to obtain 8 concentration gradients, then transferred to microplate and IL-4R coated microplate. It binds to 4R, adds a goat anti-human Fab segment secondary antibody (product: Jackson Immuno), develops color, reads the plate, and calculates the relative activity of the antibodies according to the present invention (relative activities are: refers to the relative activity of the antibody of the invention when the activity of dupilumab on the same plate sample at the time of testing is taken as 1).

Storage conditions include high temperature, strong light irradiation, repeated freezing and thawing, and strong acid. Specific operations, testing points, and testing items are shown in the table below.

High temperature test: Samples were placed in a constant temperature incubator at 50°C for 20 days and tested on days 0, 5, 10, 15 and 20. The results are shown in Figures 4A and 4B, and the SEC-HPLC experiment results showed that the stability change trend of the test samples was consistent with dupilumab. The purity of the dupilumab samples was higher than that of the antibody samples of the invention at time point zero. As the high temperature storage time increased, both showed a decrease in purity of less than 5% and there was no significant difference in thermal stability. Ignoring the abnormal results of the ELISA results (second point of HC-108A in Figure 4b), the activity of the antibody according to the present invention is always higher than that of dupilumab, and the difference increases as the high-temperature storage time is prolonged, and the activity of the antibody according to the present invention increases. The thermal stability of the antibody's binding activity was shown to be better than that of dupilumab.

Repeated freeze-thaw cycle: To detect changes in the antibody and dupilumab according to the present invention, repeated freeze-thaw cycles were performed 3 to 5 times at -80°C and room temperature, and the results are shown in Figures 4c and 4d. From the results of SEC-HPLC, it was found that the purity of dupilumab did not change and was close to 100% even as the freeze-thaw cycle increased. The purity of the antibody according to the present invention at time point 0 was lower than that of dupilumab, and the purity of the main peak decreased by only 5% after repeated freeze-thaw cycles. However, in terms of the effect on activity after repeated freezing and thawing, ELISA showed that the antibody of the invention was superior to dupilumab in preventing decline in activity due to repeated freezing and thawing. The antibody of the present invention had higher relative activity after repeated freezing and thawing under the same conditions.

Effect of strong acids: Samples were adjusted to pH 3.0 with citric acid, placed at 25°C for 3 days, observed and tested at time point 0, day 1 and day 3 respectively. From the SEC experiment results in Figure 4e, it was seen that the purity of the three test samples decreased by about 10% from time point 0. After excluding the abnormal detection spot for HC-R108E on day 1 in Figure 4g, the purity of all samples did not decrease further within 3 days. From the SEC peak diagram, it is assumed that strong acid treatment will destroy the sample from time point 0 through partial polymerization and partial decomposition. In Figure 4h, it was found that the binding activity of the antibody according to the present invention after treatment with strong acid was significantly higher than that of dupilumab under the same conditions. It is assumed that after structural modification of the test sample, the isoelectric point is lowered and the acid resistance is increased.

Effect of illumination: Samples were placed in a drug stability test chamber at 25°C and 4500lx light intensity for 10 days and tested at time point 0, day 5 and day 10, respectively. The results are shown in Figures 4E and 4F. From the SEC results, it was found that there was little change in the purity of the target peak for dupilumab and the antibody according to the present invention. The ELISA results show that the activity of the antibody according to the present invention does not decrease even when the illumination time is extended, indicating that the light stability of the antibody according to the present invention is slightly better than that of dupilumab.

Based on the results of high temperature, strong acid, illumination, and repeated freeze-thaw experiments, it can be seen that the influence of various factors on the stability of antibodies is not significantly different; In terms of activity, the antibody according to the invention has better tolerance compared to dupilumab, which is more advantageous in the later storage and release process.

SEQUENCE LISTING <110> BEIJING KAWIN TECHNOLOGY SHARE-HOLDING., LTD. <120> IL-4R┒ ANTIBODY AND USE THEREOF <130> FA00011PCT <140> PCT/CN2018/074890 <141> 2018-02-01 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 124 < 212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 1 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Val Thr Val Ser 115 120 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 2 Ala Ile Val Met Thr Gly Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gly Pro Ala Ser Ile Ser Cys Ala Ser Ser Gly Ser Leu Leu Thr Ser 20 25 30 Ile Gly Thr Ala Thr Leu Ala Thr Thr Leu Gly Leu Ser Gly Gly Ser 35 40 45 Pro Gly Leu Leu Ile Thr Leu Gly Ser Ala Ala Ser Gly Val Pro 50 55 60 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Ile 65 70 75 80 Ser Ala Val Gly Ala Gly Ala Val Gly Pro Thr Thr Cys Met Gly Ala 85 90 95 Leu Gly Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 100 105 110 <210> 3 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 3 Ala Ile Val Met Thr Gly Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gly Pro Ala Ser Ile Ser Cys Ala Ser Ser Gly Ser Leu Leu Thr Ser 20 25 30 Ile Gly Thr Ala Thr Leu Ala Thr Leu Gly Leu Ser Gly Gly Ser 35 40 45 Pro Gly Leu Leu Ile Thr Leu Gly Ser Ala Ala Ala Ser Gly Val Pro 50 55 60 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Ile 65 70 75 80 Ser Ala Val Gly Ala Gly Ala Val Gly Pro Thr Thr Cys Met Gly Ala 85 90 95 Leu Gly Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 100 105 110 <210> 4 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 4 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60 atcagctgca ggagcagcca gagcctgctg tacaggat cg gctacaacta cctggactgg 120 tacctgcaga agagcggcca gagcccccag ctgctgatct acctgggcag caacagggcc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagaccccc 300 tacaccttcg gccagggcac caagctggag atcaag 336 <210> 5 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 5 Ala Ile Val Met Thr Gly Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gly Pro Ala Ser Ile Ser Cys Ala Ser Ser Gly Ser Leu Leu Thr Ser 20 25 30 Ile Gly Thr Ala Thr Leu Ala Thr Thr Leu Gly Leu Ser Gly Gly Ser 35 40 45 Pro Gly Leu Leu Ile Thr Leu Gly Ser Ala Ala Ser Gly Val Pro 50 55 60 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Ile 65 70 75 80 Ser Ala Val Gly Ala Gly Ala Val Gly Pro Thr Thr Cys Met Gly Ala 85 90 95 Leu Gly Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 100 105 110 <210> 6 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 6 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60 atcagctgca ggagcagcca gagcctgctg tacaagatcg gctacaacta cctggactgg 120 tacctgcaga agagc ggcca gagcccccag ctgctgatct acctgggcag caacagggcc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagaccccc 300 tacaccttcg gccagggcac caagctggag atcaag 336 <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 7 Ala Ile Val Met Thr Gly Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gly Pro Ala Ser Ile Ser Cys Ala Ser Ser Gly Ser Leu Leu Thr Ser 20 25 30 Ile Gly Thr Ala Thr Leu Ala Thr Thr Leu Gly Leu Ser Gly Gly Ser 35 40 45 Pro Gly Leu Leu Ile Thr Leu Gly Ser Ala Ala Ala Ser Gly Val Pro 50 55 60 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Ile 65 70 75 80 Ser Ala Val Gly Ala Gly Ala Val Gly Pro Thr Thr Cys Met Gly Ala 85 90 95 Leu Gly Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 100 105 110 <210> 8 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 8 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60 atcagctgcg ccagcagcca gagcctgctg tacagcatcg gctacaacta cctggactgg 120 tacctgcaga agagcggcca gagcccccag ctgctgatct acctgggcag caacagggcc 1 80 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagaccccc 300 tacaccttcg gccagggcac caagctggag atcaag 336 <210> 9 <2 11> 112 <212 > PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 9 Ala Ile Val Met Thr Gly Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gly Pro Ala Ser Ile Ser Cys Ala Ser Ser Gly Ser Leu Leu Thr Ser 20 25 30 Ile Gly Thr Ala Thr Leu Ala Thr Leu Gly Leu Ser Gly Gly Ser 35 40 45 Pro Gly Leu Leu Ile Thr Leu Gly Ser Ala Ala Ala Ser Gly Val Pro 50 55 60 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Ile 65 70 75 80 Ser Ala Val Gly Ala Gly Ala Val Gly Pro Thr Thr Cys Met Gly Ala 85 90 95 Leu Gly Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 100 105 110 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 10 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60 atcagctg ca ggagcagcca gaggctgctg tacagcatcg gctacaacta cctggactgg 120 tacctgcaga agagcggcca gagcccccag ctgctgatct acctgggcag caacagggcc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagaccccc 300 tacaccttcg gccagggcac caagctggag atcaag 336 <210> 11 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 11 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 12 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 12 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgcgccg gcagc ggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caaggacagg 300 ctgagcatca ccatca ggcc cgagtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 13 <211> 124 <212> PRT <213> Artificial Sequence <220> < 223> Artificially synthesized sequence <400> 13 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 14 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 14 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgc gccg gcagcggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actact gcgc caaggacgcc 300 ctgagcatca ccatcaggcc caggtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 15 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 15 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 16 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 16 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgcgccg gcagcggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgca gatga acagcctgag ggccgaggac accgccgtgt actactgcgc caaggacagg 300 ctgagcatca ccatcgcccc caggtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 17 <211> 124 <212> PRT < 213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 17 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 18 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 18 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgcgccg gcagcggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggcag gttcaccatc ag cagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caaggacagg 300 ctgagcatca ccatcaggcc cgcctactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 19 < 211> 124 < 212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 19 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Val Thr Val Ser 115 120 <210> 20 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 20 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgcgccg gcagcggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctact ac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caaggacagg 300 ctgagcatca ccatcgagcc caggtactac ggcctggacg tgtggggcca gggcacca cc 360 gtgaccgtga gc 372 <210> 21 < 211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 21 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 22 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 22 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgcgcctg 60 agctgcgccg gcagcggctt caccttccgc gactacgcca tgacctgggt gcgccaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagc ggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caaggaccgc 300 ctgagcatca ccatccgccc ccagtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 < 210> 23 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 23 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 24 <211> 372 <212> DNA <213> Artificial Sequence <220> <223 > Artificially synthesized sequence <400> 24 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgcgcctg 60 agctgcgccg gcagcggctt caccttccgc gactacgcca tgacctgggt gcgccaggcc 120 cccggcaagg gcctggagtg ggtga gcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caaggaccgc 300 ctgagcatca ccatccgcc c ctactactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 25 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 25 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 26 <211> 372 <212> DNA <213> Artificial Sequence < 220> <223> Artificially synthesized sequence <400> 26 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgcgcctg 60 agctgcgccg gcagcggctt caccttccgc gactacgcca tgacctgggt gcgccaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa cacc ctgtac 240 ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caaggaccgc 300 ctgagcatca ccatccgccc cctgtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 27 <211> 372 < 212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 27 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgcgccg gcagcggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 ccc ggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caaggacagg 300 ctgagcatca ccatcaggcc caggtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gc 372 <210> 28 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 28 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60 atcagctgca ggagcagcca gagcctgctg tacagcatcg gctacaacta cctggactgg 120 tacctgcaga agagcggcca gagcccccag ctgctgatct acctgggcag caacagggcc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagacccccc 300 tacaccttcg gccagggcac caagctggag atcaag 336 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 29 Gly Pro Thr Pro Ala Ala Thr Ala 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence < 400> 30 Ile Ser Gly Ser Gly Gly Ala Thr 1 5 <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 31 Leu Gly Ser Gly 1 <210 > 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 32 Met Gly Ala Leu Gly Thr Pro Thr Thr 1 5 <210> 33 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 33 Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Gly Gly Pro Gly Gly 1 5 10 15 Ser Leu Ala Leu Ser Cys Ala Gly Ser Gly Pro Thr Pro Ala Ala Thr 20 25 30 Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val 50 55 60 Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr Leu Thr 65 70 75 80 Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys 85 90 95 Ala Leu Ala Ala Leu Ser Ile Thr Ile Ala Pro Ala Thr Thr Gly Leu 100 105 110 Ala Val Thr Gly Gly Gly Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Leu Gly Pro Ser Val Pro Pro Leu Ala Pro Cys Ser Ala Ser Thr Ser 130 135 140 Gly Ser Thr Ala Ala Leu Gly Cys Leu Val Leu Ala Thr Pro Pro Pro Gly 145 150 155 160 Pro Val Thr Val Ser Thr Ala Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Pro Pro Ala Val Leu Gly Ser Ser Gly Leu Thr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Leu Thr Thr Cys 195 200 205 Ala Val Ala His Leu Pro Ser Ala Thr Leu Val Ala Leu Ala Val Gly 210 215 220 Ser Leu Thr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Gly Pro Leu 225 230 235 240 Gly Gly Pro Ser Val Pro Leu Pro Pro Pro Leu Pro Leu Ala Thr Leu 245 250 255 Met Ile Ser Ala Thr Pro Gly Val Thr Cys Val Val Val Ala Val Ser 260 265 270 Gly Gly Ala Pro Gly Val Gly Pro Ala Thr Thr Val Ala Gly Val Gly 275 280 285 Val His Ala Ala Leu Thr Leu Pro Ala Gly Gly Gly Pro Ala Ser Thr 290 295 300 Thr Ala Val Val Ser Val Leu Thr Val Leu His Gly Ala Thr Leu Ala 305 310 315 320 Gly Leu Gly Thr Leu Cys Leu Val Ser Ala Leu Gly Leu Pro Ser Ser 325 330 335 Ile Gly Leu Thr Ile Ser Leu Ala Leu Gly Gly Pro Ala Gly Pro Gly 340 345 350 Val Thr Thr Leu Pro Pro Ser Gly Gly Gly Met Thr Leu Ala Gly Val 355 360 365 Ser Leu Thr Cys Leu Val Leu Gly Pro Thr Pro Ser Ala Ile Ala Val 370 375 380 Gly Thr Gly Ser Ala Gly Gly Pro Gly Ala Ala Thr Leu Thr Thr Pro 385 390 395 400 Pro Val Leu Ala Ser Ala Gly Ser Pro Pro Leu Thr Ser Ala Leu Thr 405 410 415 Val Ala Leu Ser Ala Thr Gly Gly Gly Ala Val Pro Ser Cys Ser Val 420 425 430 Met His Gly Ala Leu His Ala His Thr Thr Gly Leu Ser Leu Ser Leu 435 440 445 Ser Leu Gly 450 <210> 34 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 34 Ala Ile Val Met Thr Gly Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gly Pro Ala Ser Ile Ser Cys Ala Ser Ser Gly Ser Leu Leu Thr Ser 20 25 30 Ile Gly Thr Ala Thr Leu Ala Thr Thr Leu Gly Leu Ser Gly Gly Ser 35 40 45 Pro Gly Leu Leu Ile Thr Leu Gly Ser Ala Ala Ala Ser Gly Val Pro 50 55 60 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Ile 65 70 75 80 Ser Ala Val Gly Ala Gly Ala Val Gly Pro Thr Thr Cys Met Gly Ala 85 90 95 Leu Gly Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 100 105 110 Ala Thr Val Ala Ala Pro Ser Val Pro Ile Pro Pro Pro Pro Ser Ala Gly 115 120 125 Gly Leu Leu Ser Gly Thr Ala Ser Val Val Cys Leu Leu Ala Ala Pro 130 135 140 Thr Pro Ala Gly Ala Leu Val Gly Thr Leu Val Ala Ala Ala Leu Gly 145 150 155 160 Ser Gly Ala Ser Gly Gly Ser Val Thr Gly Gly Ala Ser Leu Ala Ser 165 170 175 Thr Thr Ser Leu Ser Ser Thr Leu Thr Leu Ser Leu Ala Ala Thr Gly 180 185 190 Leu His Leu Val Thr Ala Cys Gly Val Thr His Gly Gly Leu Ser Ser 195 200 205 Pro Val Thr Leu Ser Pro Ala Ala Gly Gly Cys 210 215 <210> 35 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 35 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgaggctg 60 agctgcgccg g cagcggctt caccttcagg gactacgcca tgacctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caaggacagg 300 ctgagcatca ccatca ggcc caggtactac ggcctggacg tgtggggcca gggcaccacc 360 gtgaccgtga gcagcgccag caccaagggc cccagcgtgt tccccctggc cccctgcagc 420 aggagcacca gcgagagcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag 480 cccgtgaccg t gagctggaa cagcggcgcc ctgaccagcg gcgtgcacac cttccccgcc 540 gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc 600 ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccagcaacac caaggtggac 660 aagagggtgg agagcaagta cggccccccc tgccccccct gccccgcccc cgagttcctg 720 ggcggccccca gcgtgttcc t gttccccccc aagcccaagg acaccctgat gatcagcagg 780 accccgagg tgacctgcgt ggtggtggac gtgagccagg aggaccccga ggtgcagttc 840 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag ggaggagcag 900 ttcaacagca cctacagggt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 960 ggcaaggagt acaagtgcaa ggtgagcaac aagggcctgc ccagcagcat cgagaagacc 1020 atcagcaagg ccaagggcca gcccagggag ccccaggtgt acaccctgcc ccccagccag 1080 gaggagatga ccaagaacca ggtgagcctg acctgcctgg tgaagggctt ctaccccagc 1140 gacatcgccg tggagtggga gagcaacggc cagcccgaga a caactacaa gaccaccccc 1200 cccgtgctgg acagcgacgg cagcttcttc ctgtacagca ggctgaccgt ggacaagagc 1260 aggtggcagg agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1320 tacacccaga agagcctgag cctgagcctg ggc 1353 <210> 36 <211> 657 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 36 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60 atcagctgca ggagcagcca gagcctgctg tacagcatcg gctacaacta cctgg actgg 120 tacctgcaga agagcggcca gagcccccag ctgctgatct acctgggcag caacagggcc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagacccccc 300 tacaccttcg gccagggcac caagctggag atcaagagga ccgtggccgc ccccagcgtg 360 ttcatcttcc cccccagcga cgagcagctg aagagcggca ccgccagcgt gg tgtgcctg 420 ctgaacaact tctaccccag ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480 agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg 540 agcagcaccc tgaccctgag caaggccgac tacgagaagc acaagg tgta cgcctgcgag 600gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagggg cgagtgc 657

Claims (24)

An antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor α (hIL-4Rα),
Contains a heavy chain variable region (HVR) and a light chain variable region (LVR),
Here, the heavy chain variable region is:
CDR1 (HCDR1) with the amino acid sequence Gly-Phe-Thr-Phe-Arg-Asp-Tyr-Ala;
CDR2 (HCDR2) with the amino acid sequence Ile-Ser-Gly-Ser-Gly-Gly-Asn-Thr; and
Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val (where Xaa1 is Arg, Xaa2 is Arg, and Xaa3 is Ala or Glu) and includes a CDR3 (HCDR3) having the amino acid sequence of
The light chain variable region is:
CDR1 (LCDR1) having the amino acid sequence Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr (where Xaa4 is Ser and Xaa5 is Ser);
CDR2 (LCDR2) with the amino acid sequence Leu-Gly-Ser; and
An antibody or antigen-binding fragment thereof comprising a CDR3 (LCDR3) having the amino acid sequence Met-Gln-Ala-Leu-Gln-Thr-Pro-Tyr-Thr. According to claim 1,
It is a dupilumab variant, and Arg at position 108 of the heavy chain variable region (HVR) of dupilumab is mutated to Ala or Glu, or an antigen-binding fragment thereof. A pharmaceutical composition for preventing or treating diseases or disorders related to IL-4/IL-4Rα signaling in the human body, comprising the antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier,
The above diseases or disorders include asthma, allergic dermatitis, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, COPD, atopic dermatitis, idiopathic pulmonary fibrosis, allergic reactions, Kawasaki disease, sickle cell anemia, chlo- A pharmaceutical composition selected from the group consisting of Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, or kidney disease. delete According to claim 3,
A pharmaceutical composition wherein the disease or disorder is allergic dermatitis or asthma. According to claim 3 or 5,
A pharmaceutical composition in which an antibody or an antigen-binding fragment thereof is used in combination with other drugs to treat autoimmune diseases. According to claim 3 or 5,
A pharmaceutical composition in which the antibody or antigen-binding fragment thereof is used in combination with a hormonal drug, an immunosuppressant, or an antihistamine drug. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to claim 1. An expression vector comprising the nucleic acid molecule according to claim 8. A host cell into which the expression vector according to claim 9 has been introduced in vitro. According to claim 10,
Host cell, which is a eukaryotic cell. A method of producing an antibody or antigen-binding fragment thereof that specifically binds to human IL-4R, comprising:
Expressing the nucleic acid molecule according to claim 8 under conditions conducive to expression of the antibody or antigen-binding fragment thereof according to claim 1 or 2, and
A method comprising recovering the expressed antibody or antigen-binding fragment thereof. A product comprising a container and package insert,
The container is a product containing the antibody or antigen-binding fragment thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 or 5, and a package insert containing instructions for use of the drug. According to claim 13,
A product further comprising one or more containers containing one or more other drugs for preventing or treating diseases or disorders associated with IL-4/IL-4Rα signaling in the human body. According to claim 14,
Products where other drugs are hormonal drugs, immunosuppressants, or antihistamine drugs. delete delete delete delete delete delete delete delete delete