阅读说明：本技术 针对il-33的抗体 (Antibodies to IL-33 ) 是由 钱峰 王军 段世鑫 于 2020-06-10 设计创作，主要内容包括：本发明提供单克隆抗体或其结合片段,所述非完全人源化单克隆抗体或其片段特异性识别IL-33,其包含至少1个选自以下的可变区或其突变序列：1)包含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的重链CDR区序列；和/或,2)包含SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示的轻链CDR区序列。本发明所提供的人源化单克隆抗体或其结合片段的亲和力高且稳定性强。(The present invention provides a monoclonal antibody or binding fragment thereof, which non-fully humanized monoclonal antibody or fragment thereof specifically recognizes IL-33, comprising at least 1 variable region or mutated sequence thereof selected from the group consisting of: 1) comprises the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, a heavy chain CDR region sequence; and/or, 2) comprises SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6, and a light chain CDR region sequence. The humanized monoclonal antibody or the binding fragment thereof provided by the invention has high affinity and strong stability.)

1. A monoclonal antibody or binding fragment thereof that specifically recognizes IL-33 comprising at least 1 variable region or mutated sequence thereof selected from the group consisting of:

1) comprises the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, a heavy chain variable region of a heavy chain CDR region sequence set forth in seq id no; and/or the presence of a gas in the gas,

2) comprises the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 in the sequence of the light chain CDR region.

2. The antibody or binding fragment thereof of claim 1, wherein the heavy chain CDR region sequence is in a framework sequence that differs from the framework sequence of SEQ ID NO: 47; preferably, the heavy chain CDR region sequence is in a framework sequence having 1-5 amino acid residues different from SEQ ID NO: 47; preferably, the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID NO: 47;

or 1-10 amino acid residues of the framework sequence in which the light chain CDR region is located are different from SEQ ID NO: 34; preferably, the light chain CDR regions are in a framework sequence differing from SEQ ID NO: 34; preferably, the light chain CDR regions are in a framework sequence differing from SEQ ID NO: 34.

3. the antibody or binding fragment thereof of claim 1, wherein the heavy chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 47: g044, S049, a075 and a 097.

4. The antibody or binding fragment thereof of claim 3, wherein the heavy chain CDR region sequences are in a framework sequence that differs from SEQ ID NO: 47: G044R, S049A, a075V and a 097T.

5. The antibody or binding fragment thereof of claim 1, wherein the heavy chain variable region sequence comprises the framework sequence set forth in seq id no:

(1) SEQ ID NO: 10 or SEQ ID NO: 11;

(2) SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15 or SEQ ID NO: 16;

(3) SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. or SEQ ID NO: 20; and

(4) SEQ ID NO: 21 or SEQ ID NO: 22.

6. the antibody or binding fragment thereof of claim 1, wherein the light chain CDR region sequences are in a framework sequence that differs from SEQ ID NO: 34: l004, G072 and T087.

7. The antibody or binding fragment thereof of claim 6, wherein the light chain CDR region sequences are in a framework sequence that differs from SEQ ID NO: 34: L004V, G072R and T087V.

8. The antibody or binding fragment thereof of claim 1, wherein the light chain variable region sequence comprises the framework sequence set forth in seq id no:

(1) SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25;

(2)SEQ ID NO：26；

(3) SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30, of a nitrogen-containing gas; and

(4) SEQ ID NO: 31 or SEQ ID NO: 32.

9. the antibody or binding fragment thereof of any one of claims 1-8, wherein the mutated sequence is N034 and/or S035;

preferably, the mutation sequence is N034Q or N034S;

further preferably, the mutation sequence is S035A.

10. The antibody or binding fragment thereof of claim 1, wherein the heavy chain thereof is selected from the group consisting of SEQ ID NOs: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51. SEQ ID NO: 52.

11. the antibody or binding fragment thereof of claim 1, wherein the light chain is selected from the group consisting of SEQ ID NOs: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 53.

12. the antibody or binding fragment thereof of claim 1, comprising the amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 33, or a variant thereof comprising SEQ ID NO: 47 and SEQ ID NO: 33, or a variant thereof comprising SEQ ID NO: 45 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 35, or which comprises SEQ id no: 48 and SEQ ID NO: 36, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 37, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 35, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 36, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 37, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 38, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 39, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 40, or which comprises SEQ ID NO: 46 and SEQ ID NO: 43, or a variant thereof comprising seq id NO: 46 and SEQ ID NO: 38, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 39, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 40, or which comprises SEQ ID NO: 47 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 42, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 49 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 50 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 52 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 43, or a variant thereof comprising seq id NO: 51 and SEQ ID NO: 42, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 53, or a variant thereof comprising SEQ ID NO: 52 and SEQ ID NO: 53, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 58, or a sequence shown in fig.

13. A polynucleotide encoding the antibody or binding fragment thereof of any one of claims 1-12;

further preferably, an expression vector capable of expressing the antibody or binding fragment thereof of any one of claims 1 to 12, or comprising said polynucleotide sequence;

further preferably, a host cell comprising said expression vector, or having said polynucleotide sequence integrated in its genome.

Technical Field

The invention relates to the field of biotechnology medicine. In particular, the invention relates to antibodies capable of specifically recognizing and binding to human IL-33 molecules.

Background

IL-33 is a key member of the IL-1 family, which plays an important role in tissue and metabolic homeostasis, infection, inflammation, cancer, and neurological diseases. IL-33 binds with high affinity to interleukin 1 receptor 4(IL-1R 4; also known as oncostatin 2[ ST2]), and forms a ternary complex with IL-1RAcP to form a signaling complex. This signaling complex results in a series of myddosomes (with MyD88 and IRAK family members) dependent downstream signaling. IL-33 acts as an alarm, produced by damaged barrier layer cells (endothelial and epithelial cells). When cells (e.g., mast cells or basophils) are stimulated by IL-33, type 2 cytokines, such as IL-4, IL-5, and IL-13, are produced.

Preclinical studies have shown that IL-33 has a key role in many asthmatics and allergic diseases. Blocking of the IL-33 signaling pathway can be achieved by neutralizing antibodies to IL-33 or IL-1R4, genetic deletions of IL-33 or IL-1R4, or soluble fusion proteins of IL-1R4 coupled to Fc (Coyleetal, 1999, J.Exper.Med190(7): 895-902). In models of inflammation induced by allergens (e.g., dust mites, cockroaches, or alternaria (alternaria) fungi), IL-33 plays an important role in driving inflammation and airway remodeling, among others. In pharmacological models that rely on adjuvants, such as aluminum hydroxide (alum) or sodium urate crystals, IL-33 plays an important role in the sensitization phase of the model and in inducing the production of type 2 cytokines, such as IL-5 and IL-13. IL-33 has also been found to play an important role in the inflammatory response associated with airway viral infections. Damage to airway epithelium caused by viral infection can trigger the release of IL-33 and alter the type of immune response.

Various cytokines are involved in pathogenesis in diseases such as chronic rhinosinusitis with nasal polyps (CRSwNP), Atopic Dermatitis (AD) and asthma. The IL-33 receptor ST2 is expressed in many cell types associated with type 2 inflammation, including mast cells, basophils, Th2 cells, type 2 innate lymphoid cells. These cells produce a number of inflammatory cytokines upon stimulation by IL-33, particularly cytokines associated with type 2 inflammation, including IL-5, IL-13, IL-4, IL-31, and IL-9. Other cytokines and chemokines are also produced, which have important roles in recruiting other inflammatory cells. Initial release of IL-33 is triggered by epithelial damage to the body or mucosal surface. Disease-related triggers include allergens with proteolytic activity, physical damage to the epithelium, viruses, and fungi and bacteria common to body surfaces. In diseased tissues rich in eosinophilic granulocytes and mast cells, damage to the epithelium initiates a cascade of reactions that release IL-33, acting on local target cells and driving the production of a variety of cytokines that are important to the type 2 inflammatory response.

Disclosure of Invention

Humanized antibodies are antibodies from non-human species (e.g., murine antibodies) whose protein sequences have been modified to increase their similarity to antibodies in humans, which substantially retain the affinity and specificity of the original antibody, while also reducing heterogeneity. The humanization of murine antibodies is achieved by genetic engineering to achieve a profile as similar as possible to that of the human antibody molecules, thereby avoiding the recognition by the human immune system. Humanization is carried out following two basic principles, namely to maintain or improve the affinity and specificity of the antibody and also to reduce or eliminate the immunogenicity of the antibody. Currently humanized antibodies can be divided into four classes: human-murine chimeric antibodies, modified antibodies, resurfaced antibodies, and fully humanized antibodies. In recent years, humanized antibodies have shown good application prospects in tumor treatment, autoimmune diseases, cardiovascular diseases and other aspects.

In view of this, the present application provides humanized antibodies that recognize IL-33. The humanized antibodies selected according to the present application contain a number of specific "back mutations" in their variable region sequences, i.e., mutations from the humanized sequence back to the mouse sequence. These back mutations are important in maintaining the conformation and binding affinity of the original antibody, while they are performed on residues with the lowest incidence of potential T cell epitopes, thereby minimizing the risk of adverse immune responses to the antibody.

To generate a humanized antibody recognizing IL-33 with the desired and human framework having specific CDR region sequences, the inventors of the present application identified key residues in the human framework (outside the CDR region sequences) that affect CDR region display and designed a series of mutations of these key residues to restore the correct display of the CDR regions while minimizing the immunogenicity of the antibody and partially mutating the CDR region sequences of the humanized antibody to improve its affinity and specificity.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

in one aspect, the present application provides a monoclonal antibody or binding fragment thereof capable of specifically recognizing IL-33 comprising at least 1 variable region or mutated sequence thereof selected from the group consisting of:

1) comprises the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, a heavy chain CDR region sequence; and/or the presence of a gas in the gas,

2) comprises the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6, and a light chain CDR region sequence.

In one embodiment of the present application, wherein the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID NO: 47; or 1-10 amino acid residues of the framework sequence in which the light chain CDR region is located are different from those in SEQ ID NO: 34.

in a specific embodiment of the present application, wherein the framework sequence in which the heavy chain CDR region sequences are located differs from SEQ ID NO: 47.

in one embodiment of the present application, the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID NO: 47.

in one embodiment of the present application, the heavy chain CDR region sequence is in a framework sequence differing from SEQ ID NO: 47.

in one embodiment of the present application, the heavy chain CDR region sequence is located in a framework sequence of 3 amino acid residues different from SEQ ID NO: 47.

in a specific embodiment of the present application, the light chain CDR regions are in a framework sequence that differs from SEQ ID NO: 34.

in one embodiment of the present application, the light chain CDR regions are in a framework sequence differing from SEQ ID NO: 34.

in one embodiment of the present application, the light chain CDR regions are in a framework sequence differing from SEQ ID NO: 34.

in one embodiment of the present application, the light chain CDR regions differ from the framework sequences of SEQ ID NOs: 34.

in a specific embodiment of the present application, wherein the framework sequence in which the heavy chain CDR region sequences are located differs from SEQ ID NO: 47: g044, S049, a075 and a 097.

In a specific embodiment of the present application, wherein the framework sequence in which the heavy chain CDR region sequences are located differs from SEQ ID NO: 47: G044R, S049A, a075V and a 097T.

In one embodiment of the present application, the heavy chain variable region sequence comprises the framework sequence set forth in seq id no: (1) SEQ ID NO: 10 or SEQ ID NO: 11; (2) SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15 or SEQ ID NO: 16; (3) SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. or SEQ ID NO: 20; and (4) SEQ ID NO: 21 or SEQ ID NO: 22.

in one embodiment of the present application, the framework sequences in which the heavy chain CDR region sequences are located are selected from the framework sequences represented by the following sequences: SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51 or SEQ ID NO: 52.

in a specific embodiment of the present application, the light chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 34: l004, G072 and T087.

In a specific embodiment of the present application, the light chain CDR region sequence is in a framework sequence that differs from SEQ ID NO: 34: L004V, G072R and T087V.

In one embodiment of the present application, the light chain variable region comprises the framework sequence set forth in seq id no: (1) SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25; (2) SEQ ID NO: 26; (3) SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30, of a nitrogen-containing gas; and (4) SEQ ID NO: 31 or SEQ ID NO: 32.

in one embodiment of the present application, the light chain CDR region sequences are selected from the framework sequences set forth in seq id nos: SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44 or SEQ ID NO: 53.

the present invention also provides a mutated sequence of the monoclonal antibody or binding fragment thereof.

In one embodiment of the invention, the mutation of the mutated sequence occurs in a light chain CDR region.

In a particular embodiment of the invention, the mutation is N034 and/or S035.

In a specific embodiment of the invention, the mutation is N034Q or N034S.

In one embodiment of the invention, the mutation is S035A.

In one embodiment of the invention, the heavy chain is selected from the group consisting of SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51. SEQ ID NO: 52.

In one embodiment of the invention, the light chain is selected from the group consisting of SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 53.

in one embodiment of the invention, the antibody or binding fragment thereof comprises SEQ ID NO: 45 and SEQ ID NO: 33, or a variant thereof comprising SEQ ID NO: 47 and SEQ ID NO: 33, or a variant thereof comprising SEQ ID NO: 45 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 35, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 36, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 37, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 41, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 35, or which comprises seq id NO: 46 and SEQ ID NO: 36, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 37, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 38, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 39, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 40, or which comprises SEQ ID NO: 46 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 38, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 39, or a variant thereof comprising SEQ ID NO: 46 and SEQ ID NO: 40, or which comprises SEQ ID NO: 47 and SEQ ID NO: 41, or which comprises seq id NO: 48 and SEQ ID NO: 42, or a variant thereof comprising SEQ ID NO: 48 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 49 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 50 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 52 and SEQ ID NO: 44, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 43, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 42, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 53, or a variant thereof comprising SEQ ID NO: 52 and SEQ ID NO: 53, or a variant thereof comprising SEQ ID NO: 51 and SEQ ID NO: 58, or a sequence shown in fig.

In another aspect, the invention provides a polynucleotide encoding an antibody or binding fragment thereof as described above.

The invention also provides an expression vector capable of expressing the antibody or binding fragment thereof as described above, or the expression vector can comprise the polynucleotide as described above.

In another aspect, the present invention provides a host cell comprising the above-described expression vector, or a host cell having the above-described polynucleotide integrated into its genome.

Illustratively, the present invention has at least one of the following advantages:

the antibody or the binding fragment thereof provided by the invention has high affinity and high stability, has great potential medicinal value for preparing antibody medicines, and can treat, prevent or alleviate diseases related to IL-33, such as asthma, rheumatoid arthritis, septicemia, heart diseases, atherosclerosis, malignant tumors and the like.

Drawings

FIG. 1 is a graph showing the results of SDS-PAGE after purification of ProteinA provided in the examples of the present invention.

FIG. 2 is a chart showing the results of SDS-PAGE experiments after SEC secondary purification provided in the examples of the present invention.

FIGS. 3-1 to 3-6 are graphs showing the results of experiments in which non-specific binding of antigens to the chip is detected by SPR. Wherein, fig. 3-1: the antibody is W3759-cAb1-xIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIG. 3-2: the antibody is W3759-cAb1-z1-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3-3: the antibody is W3759-cAb1-sz8-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3-4: the antibody is W3759-cAb1-sz9-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3 to 5: the antibody is W3759-cAb1-sz10-p1-uIgG4K.SP, the antigen is W3759-hPro1-WT, and the ratio is 1: 1; FIGS. 3 to 6: the antibody is W3759-cAb1-xIgG4K.SP, the antigen is W3759-hPro1-Mut, and the ratio is 1: 1.

FIG. 4-1 is a graph showing the results of an experiment in which the binding affinity of an antibody to an antigen is determined by the antigen coating method.

FIG. 4-2 is a graph showing the results of an experiment for detecting the affinity of an antibody for wild-type and mutant antigens by ELISA.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention discloses monoclonal antibodies recognizing IL-33 that are almost fully humanized, thereby avoiding adverse immune responses to the antibody and are therefore safe for use in vivo. The antibodies of the present application are characterized by unique CDR region sequences and novel non-fully humanized framework sequences and designs, and partial mutations of the CDR region sequences that result in high affinity antibodies with the non-fully humanized framework sequences.

Description of the drawings: the variable regions of the antibodies provided herein comprise CDR regions and framework sequences, at least one residue in at least one of which is different from the corresponding fully human framework sequence.

The antibodies of the invention are molecules comprising at least the antigen-binding portion of an antibody, including in particular intact antibodies, such as polyclonal or monoclonal antibodies, as well as proteolytic fragments thereof, such as Fab or F (ab')2 fragments. Single chain antibodies are also within the scope of the invention.

The term "amino acid residue mutation" as used herein refers to substitution, insertion or deletion of a single amino acid residue. The term "back-mutation" as used herein refers to the substitution of a single amino acid residue found in the human antibody backbone to the corresponding amino acid residue found in the murine antibody backbone.

In the invention, W3759-hPro1-WT I is human IL-33, and W3759-hPro1-Mut is human IL-33 with epitope mutation.

The reference fully humanized heavy chain sequence to which back mutations were introduced was SEQ ID NO: 47; the fully humanized light chain sequence is SEQ ID NO: 34.