HK1212993B - Il-17a conjugate and uses thereof - Google Patents

Description

IL-17A conjugates and uses thereof

Technical Field

The present invention relates to an IL-17A conjugate and its use as a therapeutic agent, in particular as a therapeutic agent for various inflammatory or autoimmune diseases.

Background

The cytokines of the interleukin-17 family are named interleukin-17A through interleukin-17F, respectively. At the same time, one has also found their receptor family: interleukin-17 receptor a through interleukin-17 receptor E. These interleukin-17 cytokines can bind to corresponding receptor members and thereby mediate different inflammatory responses.

The most representative member of this family is interleukin-17A. Lymphocytes that migrate to infected or injured areas of the body secrete interleukin-17A. On one hand, the interleukin-17A can induce the expression of inflammatory factors and chemokines, so that more immune cells are recruited to reach an inflammatory part to intensify the inflammatory reaction; on the other hand, interleukin-17A also induces the expression of some factors related to tissue repair, thereby accelerating the recovery of the body. Although IL-17A serves to augment immune defense and protect the body during host infection and tissue repair, IL-17A is highly expressed in many autoimmune and neoplastic patients, and excessive IL-17A levels exacerbate pathological development because it induces the expression of many inflammatory factors. Many animal experiments also prove that the pathological degree of various autoimmune diseases can be effectively inhibited by the deletion of the interleukin-17A or the neutralization of the interleukin-17A by an antibody. There is evidence for efficacy in targeting IL-17 signaling in autoimmune diseases, including Rheumatoid Arthritis (RA), psoriasis, crohn's disease, Multiple Sclerosis (MS), psoriatic disease, asthma, and lupus erythematosus (see, e.g., Aggarwal et al, j.leukcc.biol., 71 (1): 1-8 (2002); Lubberts et al).

The human IL-17 gene codes for a polypeptide of up to 155 amino acids in length. The polypeptide comprises a 19 amino acid signal sequence and a 132 amino acid mature region. Human IL-17A is a glycoprotein with a relative molecular weight of 17,000Da (Spriggs et al, J.Clin.Immunol., 17: 366-369(1997)) in the form of a homodimer or a heterodimer. The homologue IL-17F may complex with IL-17A to form an IL-17A/F heterodimer. IL-17F (IL-24, ML-1) has up to 55% amino acid sequence similarity to IL-17A, and its receptor is also IL-17R. IL-17R is ubiquitously expressed in a variety of cells, including vascular endothelial cells, peripheral T cells, B cells, fibroblasts, myelomonocytes and bone marrow stromal muscle cells (Kolls et al, Immunity, 21: 467-476 (2004); Kawaguchi et al, J.Allergy Clin.Immunol., 114 (6): 1267-1273 (2004); Moseley et al, Cytokine Growth Factor Rev., 14 (2): 155-174 (2003)).

Since the discovery of interleukin-17A, various anti-IL-17A antibodies, such as CN101001645A, CN101326195A, CN101646690A, have been discovered so far, however, there is still a need to develop various improved antibodies effective in reducing or neutralizing IL-17 activity in inflammatory reactions and autoimmune diseases.

Disclosure of Invention

The invention provides an anti-IL-17A antibody with higher affinity and longer half-life.

The present invention provides an IL-17A conjugate comprising:

an antibody light chain variable region comprising 0-3 heavy chain variable regions selected from the group consisting of SEQ ID NOs: 13. SEQ ID NO: 14. SEQ ID NO: an LCDR region shown at 15; and

an antibody heavy chain variable region comprising 0-3 heavy chain variable regions selected from the group consisting of SEQ ID NOs: 10. SEQ ID NO: 11. SEQ ID NO:12, HCDR region;

wherein the number of CDR regions in the antibody light chain variable region and the heavy chain variable region cannot be 0 at the same time.

According to some embodiments of the invention, an IL-17A conjugate comprises SEQ ID NO: 13.

according to some embodiments of the invention, an IL-17A conjugate comprises SEQ ID NO: 14.

according to some embodiments of the invention, an IL-17A conjugate comprises SEQ ID NO: 15.

according to some embodiments of the invention, an IL-17A conjugate comprises SEQ ID NO: 10.

according to some embodiments of the invention, an IL-17A conjugate comprises SEQ ID NO: 11.

according to some embodiments of the invention, an IL-17A conjugate comprises SEQ ID NO: 12.

according to some embodiments of the invention, an IL-17A conjugate comprises 1 polypeptide selected from the group consisting of SEQ ID NO: 13. SEQ ID NO:14 or SEQ ID NO: LCDR area shown at 15.

According to some embodiments of the invention, an IL-17A conjugate comprises 1 polypeptide selected from the group consisting of SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, HCDR region shown.

According to some embodiments of the invention, an IL-17A conjugate comprising 2 polypeptide sequences selected from the group consisting of SEQ ID NO: 13. SEQ ID NO:14 or SEQ ID NO: LCDR area shown at 15.

According to some embodiments of the invention, an IL-17A conjugate comprising 2 polypeptide sequences selected from the group consisting of SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, HCDR region shown.

According to some embodiments of the invention, an IL-17A conjugate comprising 3 LCDR regions, wherein the amino acid sequence of LCDR1 is as set forth in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 15.

According to some embodiments of the invention, an IL-17A conjugate comprising 3 HCDR regions, wherein the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:10, the amino acid sequence of HCDR2 is shown in SEQ ID NO:11, the amino acid sequence of HCDR3 is shown in SEQ ID NO: shown at 12.

According to some embodiments of the invention, an IL-17A conjugate, the antibody light chain variable region of which further comprises a light chain FR region of a murine kappa, lambda chain or variant thereof. In some embodiments, the antibody light chain variable region sequence is: SEQ ID NO: 2. further, the IL-17A conjugates contain murine kappa, lambda chains or variants thereof light chain constant regions.

According to some embodiments of the invention, an IL-17A conjugate, the antibody heavy chain variable region of which further comprises a heavy chain FR region of heavy murine IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the antibody heavy chain variable region sequence is: SEQ ID NO: 1. further, the IL-17A conjugate comprises a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4, or a variant thereof.

According to some embodiments of the invention, an IL-17A conjugate, the antibody light chain variable region of which further comprises a light chain FR region of a human kappa, lambda chain or variant thereof; in some embodiments, the light chain FR region of the antibody light chain variable region is a light chain FR region of an amino acid sequence set forth in SEQ ID NO:4 or a variant thereof of human germline light chain a 10. In some embodiments, the FR region sequence variant in the variable region of the antibody light chain refers to an amino acid change from 0 to 10 in the light chain FR region of human germline light chain A10. In some embodiments, the amino acid change of the FR region sequence variant on the antibody light chain variable region is selected from one or more of: F71Y, K49Y, Y36F, L47W. In some embodiments, the antibody light chain is selected from the group consisting of: SEQ ID NO:9 and variants thereof. Further, the IL-17A conjugates contain a light chain constant region of a human kappa, lambda chain or variant thereof.

According to some embodiments of the invention, an IL-17A conjugate, wherein the heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof; in some embodiments, the heavy chain FR region in the antibody heavy chain variable region is a polypeptide having an amino acid sequence set forth in SEQ ID NO:3 or a variant thereof, of the FR region of human germline heavy chain VH 1-18; in some embodiments, the FR region sequence variant in the heavy chain variable region of the antibody is a 0-10 amino acid change in the FR region of the heavy chain of VH 1-18; in some embodiments, wherein the amino acid change of the FR sequence variant in the antibody heavy chain variable region is selected from one or more of: a93T, T71A, M48I, V67A, M69L, T73D, S76N; in some embodiments, wherein the antibody heavy chain sequence is selected from the group consisting of: SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. further, the IL-17A conjugate comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof.

Further, according to some embodiments of the present invention, there is provided a vector expressing the IL-17A conjugate described above. After transfection of the host cells with the vector, the host cells express and secrete the IL-17A conjugate.

According to some embodiments of the invention, the vector comprises nucleotides encoding the IL-17A conjugate of the invention.

Further, according to some embodiments of the present invention, there is also provided a pharmaceutical composition comprising an IL-17A conjugate as described above and a pharmaceutically acceptable excipient, diluent or carrier.

Further, according to some embodiments of the present invention, there is also provided a use of an IL-17A conjugate as described above, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment of an IL-17 mediated disease or disorder. The disease is inflammation or autoimmune disease; selected from psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory arthritis; the inflammation is preferably inflammatory arthritis. The inflammatory arthritis is selected from osteoarthritis, rheumatoid arthritis, rheumatic arthritis or osteoporosis, preferably rheumatic arthritis.

According to some embodiments of the invention, there is also provided the use of an IL-17A antibody or a humanized IL-17A antibody as described above and a pharmaceutical composition comprising the same in the manufacture of a medicament for the treatment of an IL-17 mediated disease or disorder. The diseases are inflammatory and autoimmune diseases. Wherein said inflammation is preferably inflammatory arthritis. The inflammatory arthritis is selected from: osteoarthritis, rheumatoid arthritis, or osteoporosis.

There is also provided, according to some embodiments of the invention, a method of treating an IL-17 mediated disease or disorder, comprising administering to a patient an effective amount of an IL-17A conjugate or a humanized IL-17A antibody or a pharmaceutical composition comprising the same as described above.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Term of

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j. diol. chem, 243, p3558 (1968).

As used herein, "conjugate" refers to a soluble receptor or fragment or analog thereof, or an antibody or fragment or analog thereof, capable of binding to a target. The term "IL-17A conjugate" as used herein refers to an antibody or fragment thereof or analog thereof capable of specifically recognizing IL-17A and binding to IL-17A.

The term "IL-17A" generally refers to native or recombinant human IL-17A, as well as non-human homologs of human IL-17A. Unless otherwise indicated, the molar concentration of IL-17A is calculated using the molecular weight of the homodimer of IL-17A (e.g., 30kDa for human IL-17A).

The antibody of the invention refers to immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as kappa or lambda chains by differences in the constant regions.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 FR Regions (FRs) which are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain, the light chain hypervariable region (LCDR), designated LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain, the light chain hypervariable region (LCDR), are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDE2-3) or in accordance with Kabat and chothia numbering convention (HCDR 1).

An "antigen-binding fragment" as used herein refers to an Fab fragment, an Fab 'fragment, an F (ab') 2 fragment, or a single Fv fragment having antigen-binding activity. Fv antibodies are the smallest antibody fragments that contain the variable regions of the heavy and light chains of an antibody, but no constant regions, and have all the antigen binding sites. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.

The term "antigenic determinant" of the invention refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.

"administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications. "treatment" when applied to a cell, tissue or organ in a human, veterinary or research subject includes contacting an IL-17A agonist or IL-17A antagonist with a human or animal subject, cell, tissue, physiological compartment or physiological fluid. "treatment of cells" also includes situations where an IL-17A agonist or IL-17A antagonist contacts the IL-17A receptor, e.g., in the fluid or colloidal phase, and includes situations where the agonist or antagonist does not contact the cell or receptor.

By "treating" is meant administering a therapeutic agent, such as a composition comprising any one of the conjugates of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient.

4 variants of the human IL-17A protein are indicated:

1) the terms "human IL-17A (huIL-17A)" and "native human IL-17A" as used herein refer to the mature forms of human IL-17A protein accession numbers NP-002181 and AAT22064 (i.e., residues 24-155), as well as native variants and polymorphisms thereof.

2) The term "rhIL-17A" as used herein refers to recombinant human IL-17A for convenience, and this nomenclature is used to indicate that various forms of IL-17A, may not match usage in the literature.

3) The term "His-huIL-17A" as used herein refers to recombinant human IL-17A with a His tag attached to the N-terminus. FLAG-huIL-17A refers to recombinant human IL-17A with a FLAG tag attached to the N-terminus. In some experiments, FLAG-huIL-17A was biotinylated.

4) The R & D Systems human IL-17A referred to herein refers to the recombinant human IL-17A purchased from R & D Systems.

The term "monoclonal antibody" as used herein refers to an antibody secreted by a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells are not limited to eukaryotic, prokaryotic, or phage clonal cell lines.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which portions of the heavy and/or light chain are identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains are identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they possess the desired biological activity.

The term "human source" as used hereinThe antibody "is a variable region engineered form of the murine antibody of the invention, having CDR regions derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and FR and constant regions substantially derived from human antibody sequences; that is, the CDR sequence of the mouse antibody is grafted to the framework sequences of different types of human germline antibodies. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA sequences of, for example, human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (at the Internet)www.mrccpe.com.ac.uk/vbaseAvailable), and is found in Kabat, EA. et al, 1991Sequences of proteins of Immunological Interest, 5 th edition. Because the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be present, and when present may be 1, 2 or 3.

The step of transforming a host cell with the recombinant DNA described in the present invention can be carried out by a conventional technique well known to those skilled in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell.

Antibodies specific for human IL-17

The invention provides anti-IL-17A antibodies and their use in treating inflammatory and autoimmune diseases. The diseases include Rheumatoid Arthritis (RA), osteoarthritis, rheumatoid arthritis osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), inflammatory bowel disease (e.g., crohn's disease, ulcerative colitis, and inflammatory bowel disease), asthma (including allergic asthma), allergy, COPD, multiple sclerosis, psoriasis, and cancer.

Any method suitable for producing monoclonal antibodies can be used to produce the anti-IL-17A antibodies of the invention. For example, recipient animals may be immunized with a linker or, for example, a naturally occurring IL-17A homodimer or fragment thereof. Suitable immunization methods may be used. Such methods may include an adjuvant. Other immunostimulants, repeated booster immunizations, and the use of one or more immunization routes.

Any suitable form of IL-17A may be used as an immunogen (antigen) for the production of non-human antibodies specific for IL-17A, which antibodies may be screened for biological activity. The challenge immunogen may be full-length mature human IL-17A, including a linked native homodimer, or a peptide thereof containing a single epitope or multiple epitopes. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculovirus vectors, plasmids and non-viral vectors.

An exemplary method for producing an anti-human IL-17A antibody of the invention is described in example 1.

III, humanization of IL-17A specific antibodies

The humanized antibody may be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE. In one embodiment, the antibody is an IgG antibody. Any isotype of IgG may be used, including IgG1, IgG2, IgG3, and IgG 4. Variants of the IgG isotype are also envisaged. Humanized antibodies may comprise sequences from more than one species or isotype. Optimization of the sequence of the essential constant domains to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.

Likewise, any type of light chain can be used in the compounds and methods herein. In particular, κ, λ or variants thereof are useful in the compounds and methods of the invention.

An exemplary method for humanizing an anti-human IL-17A antibody of the present invention is described in example 2.

Detailed Description

The present invention is further described below with reference to examples, but these examples do not limit the scope of the present invention.

The experimental method of the present invention, in which no specific conditions are specified in the examples or test examples, is generally performed under conventional conditions or conditions recommended by manufacturers of raw materials or commercial products. See Sambrook et al, molecular cloning, A laboratory Manual, Cold spring harbor laboratory; contemporary molecular biology methods, Ausubel et al, Greene publishing Association, Wiley Interscience, NY. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

EXAMPLE 1 mouse monoclonal antibody against human IL-17A

A monoclonal antibody to human IL-17A (hIL-17A) was obtained as follows. 6-8 week-old female BALB/c mice (Shanghai Sepal-Bikay laboratory animals Co., Ltd., animal production license number: SCXK (Shanghai) 2008-0016) and 6-8 week-old female SJL mice (Beijing Witonglihua laboratory animals technology Co., Ltd., animal production license number: SCXK (Jing) 2012-0001) were divided into two groups of high and low doses, each group consisting of 10 BALB/c mice and 10 SJL mice.

The high and low dose groups were separately immunized with a series of N-terminally His-tagged variants of native hIL-17A (His-hIL-17A, wherein the amino acid sequence of hIL-17A is as described in Genbank human IL-17A protein accession NP-002181) which had been produced by the HEK293E (293-EBNA, Invitrogen, Lot Num: 493985) expression system, and the expressed proteins were purified using Ni affinity column (Superdex)75SEC sequential chromatography. The inoculation time is 0, 14, 35 and 56 days.

The high dose group on day 0 was given a subcutaneous (sc) injection of 500. mu.g/mouse His-huIL-17A, together with an Intraperitoneal (IP) injection of Freund's complete adjuvant (CFA). On days 14 and 35, sc injections of 25. mu.g/mouse His-hIL-17A were performed, together with IP injection of Freund's incomplete adjuvant (IFA). Day 56 the immunization was boosted prior to splenocyte fusion and IP injected with 25. mu.g/mouse His-hIL-17A in saline. The time and method of immunization injection in the low dose group were the same as those in the high dose group except that the dose of His-hIL-17A administered on day 0 was 10. mu.g/mouse and the doses of His-hIL-17A administered on days 14, 35 and 56 were 5. mu.g/mouse.

Blood tests were carried out on days 22 and 43, and the antibody titer in the mouse serum was determined by testing the mouse serum by the ELISA method of test example 1. Mice with high antibody titers in sera were selected for splenocyte fusion at day 56, and splenic lymphocytes were fused with myeloma cells Sp2/0 cells using an optimized PEG-mediated fusion procedure (seeCRL-8287^TM) And carrying out fusion to obtain hybridoma cells.

The immunization protocol steps were as follows:

protocol 1, high dose, 10 BALB/c and 10 SJL mice, the procedure was as follows:

day 0	Pre-hemospastic 15-30 μ L serum/mouse; primary immunization, IP, CFA 50. mu.g/mouse
		14	Boost 1 (Boost immunization)1): IP, IFA 25. mu.g/mouse
21	Blood drawing (15-30 μ L serum/mouse)
		22	ELISA test
35	Boost 2 (Boost 2): IP, IFA 25. mu.g/mouse
		42	Blood drawing (15-30 μ L serum/mouse)
43	ELISA test
		44	Data analysis and phase conclusions
56	Pre-fusion boosting, IP, saline 25. mu.g/mouse

Scheme 2, low dose, procedure was as follows:

day 0	Pre-hemospastic 15-30 μ L serum/mouse; primary immunization, IP, CFA 10. mu.g/mouse
		14	Boost 1 (Boost 1): IP, IFA 5. mu.g/mouse
21	Blood drawing (15-30 μ L serum/mouse)
		22	ELISA test
35	Boost 2 (Boost 2): IP, IFA 5. mu.g/mouse
		42	Blood drawing (15-30 μ L serum/mouse)
43	ELISA test
		44	Data analysis and phase conclusions
56	Pre-fusion boosting, IP, saline 5. mu.g/mouse

Test example 1 antigen antibody indirect ELSIA assay was used for primary screening of hybridoma cells. And (4) carrying out limiting dilution on the cell strain with positive detection to obtain a monoclonal cell strain.

The obtained monoclonal cell strain is further screened, and comprises the following steps:

1. a receptor blocking test is carried out, see test example 2, the result is shown in table 5, and a monoclonal cell strain IL17-mAb049 with the activity superior to that of a positive control is obtained by screening;

2. and (3) affinity detection: see test example 3, the results are shown in Table 6, which shows that the activity of the monoclonal cell strain IL17-mAb049 obtained by screening of the invention is equivalent to or superior to that of the positive control;

3. cell level bioassay (GRO α analysis) see test example 4 and results in Table 8, which shows that the monoclonal cell strain IL17-mAb049 obtained by the screening of the present invention has comparable or superior activity to the positive control.

The invention further studies 12 monoclonals after the first and second screening, and selects 1 lead monoclonals (lead mAb) IL17-mAb049 through epitope grouping and biological activity detection. The following are the specific sequences of the (IL-17mAb) heavy chain (VH) and light chain (LH) of the IL-17A murine antibody mAb 049:

IL-17mAb049VH SEQ ID NO：1

HVQLQQSGADLVRPGASVTLSCKASGYIFTDYEVHWVKQTPVHGLEWIGVIDPGTGGVAYNQKFEGKATLTADDSSNTAYMELRSLTSEDSAVYYCTRYSLFYGSSPYAMDYWGQGTSVTVSS

IL-17mAb049VL SEQ ID NO：2

QIVLTQSPAIMSASPGEKVTITCSASSSVNYMHWFQQKPGTSPKLWIYRTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPWTFGGGTNLEIK

example 2 humanization of murine anti-human IL-17A antibodies

Humanization of the murine anti-human IL-17A monoclonal antibody mAb049 was essentially carried out as described in many publications in the art. Briefly, human constant domains are used in place of parent (murine) constant domains, and human antibody sequences are selected for humanization based on the homology of murine and human antibodies.

1. CDR regions of murine anti-IL-17A antibodies

The amino acid residues of the VH/VL CDRs were determined and annotated by the Kabat numbering system. The CDR sequences of murine mAb049 of the invention are shown in the following table:

table 1: CDR sequence of murine anti-IL-17A antibody

2. Selection of human germline FR region sequences

Based on the typical structure of the obtained VH/VL CDR of the murine antibody, the heavy and light chain variable region sequences were compared with the antibody database to obtain the human germline heavy chain with high homology of VH1-18(SEQ ID NO: 3) and the light chain of A10(SEQ ID NO: 4) as the humanized FR region sequence. The specific sequence is as follows:

VH1-18 SEQIDNO：3

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR

A10 SEQ ID NO：4

EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLP

3. design of humanized antibodies:

amino acid residues that form loop conformation and VH interface are identified, and Q1E mutations to eliminate N-terminal pyroglutamic acid formation are contemplated, as are mutations that maintain the identity within the selected VH family to maintain the typical structure of the CDRs and the VH/VL interface, to avoid the occurrence of the N-glycosylation pattern (N- { P } -S/T) in the humanized structure, and the like.

The humanized mutation design of the variable region of murine anti-mAb 049 is summarized below:

table 2: murine antibody mAb049 humanization site design

Note: e.g., A93T, indicating a mutation of position 93A back to T according to the Kabat numbering system.

Represents the murine antibody CDR-grafted human germline FR region sequences.

Table 3: murine antibody mAb049 humanized sequences

Note: the table shows the sequences obtained by combining the various mutations. As shown by Hu049-8, there are two Hu049VK.1A and Hu049VH.1B mutations on the humanized murine antibody mAb 049. And so on.

4. Expression and purification of humanized antibodies

The antibodies are cloned, expressed and purified by a gene recombination method, and the humanized antibody with good performance is finally selected by ELISA, receptor binding inhibition experiment, Biacore, cell activity detection and the like. Specific antibodies are as follows:

table 4: humanized antibody IL-17A composition

The mAb049 humanized sequence is specifically listed below:

Hu049-17.VH SEQ ID NO：5

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEVHWVRQAPGQGLEWMGVIDPGTGGVAYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRYSLFYGSSPYAMDYWGQGTLVTVSS

Hu049-18.VH SEQ ID NO：6

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEVHWVRQAPGQGLEWMGVIDPGTGGVAYNQKFEGRVTMTADTSTSTAYMELRSLRSDDTAVYYCTRYSLFYGSSPYAMDYWGQGTLVTVSS

Hu049-19.VH SEQ ID NO：7

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEVHWVRQAPGQGLEWIGVIDPGTGGVAYNQKFEGRVTMTADTSTSTAYMELRSLRSDDTAVYYCTRYSLFYGSSPYAMDYWGQGTLVTVSS

Hu049-20.VH SEQ ID NO：8

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEVHWVRQAPGQGLEWIGVIDPGTGGVAYNQKFEGRATLTADDSTNTAYMELRSLRSDDTAVYYCTRYSLFYGSSPYAMDYWGQGTLVTVSS

Hu049VL SEQ ID NO：9

EIVLTQSPDFQSVTPKEKVTITCSASSSVNYMHWFQQKPDQSPKLWIYRTSNLASGVPSRFSGSGSGTDYTLTINSLEAEDAATYYCQQRSSYPWTFGQGTKLEIKR

example 3 in vivo pharmacodynamic drug test of humanized anti-IL-17 antibody

Human IL-17 binds to and stimulates the mouse IL-17 receptor, resulting in an increase in the male mouse KC (CXCL1) chemokine and subsequent secretion. Time and dose variation experiments were performed to identify the optimal dose of human IL-17 and the optimal timing of KC induction in mice (see test example 5). These experiments indicate that a 150mg/kg dose of human IL-17 and a time of 2 hours after IL-17 administration resulted in the highest levels of KC in mouse serum. The full length antibodies of the invention were administered intravenously to mice at 3, 30, 300, 3000 μ g/kg, 20 hours prior to subcutaneous injection of human IL-17. 2 hours after administration of human IL-17, mice were sacrificed and KC levels were determined by ELISA using a commercially available Kit according to the manufacturer's instructions (Mouse CXCL1/KC Quantikine ELISA Kit, R & D SYSTEM, # SMKC 00B). Isotype matched antibodies were used as negative controls. The antibody blocks the ability of human IL-17 to stimulate the mouse IL-17 receptor, inhibiting the increase of mouse KC in a dose-dependent manner. The antibody Hu049-18 of the invention reduced the mean KC level to about 1/6 at a dose of 3000. mu.g/mouse under the conditions described, compared to a non-effector control antibody.

The serum pharmacokinetics of the antibody Hu049-18 of the present invention was determined after intravenous or subcutaneous administration in rats and macaques (see test example 6). The half-life of the rats was 9.91 days after 5mg/kg intravenous administration and 11.5 days after 5mg/kg subcutaneous administration. After intravenous administration at 1mg/kg, the half-life of the macaque is 24.4 days.

Test example

Test example 1 Indirect ELSIA

Purpose of the experiment:

an indirect ELISA method was selected to ensure the selection of antibodies recognizing conformational epitopes and mouse hybridoma cells were screened in example 1 of the present invention.

Experimental materials:

human IL-17A (hIL-17A): genbank human IL-17A protein accession NP-002181 was cloned and expressed by transient transfection of HEK293E cells according to methods known in the art.

Human IL-17A/F (heterodimer, hIL-17A/F): genbank human IL-17A protein accession NP-002181 and human IL-17F protein accession NP-443104 were cloned and expressed by transient transfection of HEK293E cells according to methods known in the art.

Murine anti-IL-17 antibodies from Lilly and Novartis (Lilly mAb, Novartis mAb) as positive controls were cloned according to the murine sequences provided in US7,838,638B2(LY 2439821) and US7,807,155B 2(AIN 457), respectively, and expressed by transient transfection of HEK293E cells.

Murine antibodies mAbs of the mouse hybridoma cells of example 1 of the present invention.

The experimental method comprises the following steps:

1. directly coating 1 mug/ml streptomycin avidin and standing overnight at 4 ℃;

2. blocking microtiter plates with 300. mu.l PBST containing 2% BSA (v/v), blocking at 37 ℃ for 1h, while blocking uncoated blank wells as a control;

3. PBST washes 3 times, all washes were run on a Biotek (Elx 405) automatic plate washer;

4. adding 100 μ l of PBS containing hIL-17A or hIL-17A/F (1 μ g/ml) into each well, and incubating at 37 deg.C for 1 h;

5. PBST wash 3 times;

6. titration of positive control Lilly mAb and Novartis mAb or murine antibodies mAbs of the invention: diluted according to the 1: 5 times ratio, the initial concentration is 1 mug/ml, 100 mug/l of diluted positive control or the murine antibody of the invention is added into each hole, and the incubation is carried out for 1h at the constant temperature of 37 ℃. Each titration concentration is provided with a repeat hole;

7. PBST wash 3 times;

8. mu.l of HRP anti-mouse secondary antibody (Santa Cruz Cat. No. sc-2005) (1: 5000) was added to each well, and incubated at 37 ℃ for 1 h;

9. PBST was washed 3 times. Mu.l TMB substrate per well, incubation at 37 ℃ for 5min, followed by 100. mu.l 2M H per well₂SO₄Stopping the reaction;

10. an ELISA plate reader (Molecular Devices, Spectra Max) reads the OD at a wavelength of 450 nm.

11. Comparing the OD value measured by the murine antibody mAbs with the positive control, and screening to obtain monoclonal cell strains with the ratio of more than 1, wherein the monoclonal cell strains comprise IL17-mAb 049.

Test example 2IL-17 Receptor Blockade Assay (RBA)

Purpose of the experiment:

the purpose of the receptor blocking assay is to select antibodies that block IL-17 binding to the IL-17 receptor (e.g., hIL-17 RA). The assay is based on functional testing and can be used for high throughput screening of hybridoma cells.

Experimental materials and instruments:

anti-human Fc antibody (goat anti-human IgG-Fc fragment specific antibody (purchased from Jackson Immunoresearch, 109-005-008))

The human IL-17RA-Fc used in the present invention was cloned according to the method known in the art using the amino acid sequence of human IL-17 receptor provided by Genbank ID No. ADY18334.1, and expressed by transient transfection of HEK293E cells, and the Fc fragment thereof was taken from human IgG 1.

Murine anti-IL-17 antibodies from Lilly and Novartis (Lilly mAb, Novartis mAb) as positive controls were cloned according to the murine sequences provided in US7,838,638B2(LY 2439821) and US7,807,155B 2(AIN 457), respectively, and expressed by transient transfection of HEK293E cells.

mIgG: murine IgG (Millipore Cat. No. PP54) as a blank control

An ELISA microplate reader: molecular Devices, Spectra Max

The murine monoclonal cell line obtained in example 1 of the present invention.

The experimental method comprises the following steps:

1. directly coating 10 mug/ml of anti-human Fc antibody for incubation and staying overnight at 4 ℃;

2. blocking the microtiter plate with 300. mu.l PBST containing 2% BSA at 37 ℃ for 1 hour, and blocking the uncoated blank well as taking the control;

3. PBST washes 3 times, all washes were run on a Biotek (Elx 405) automatic plate washer;

4. mu.l of PBS containing IL-17RA-Fc (60ng/ml) was added to each well and incubated at a constant temperature of 37 ℃ for 2 h;

5. PBST wash 3 times;

6. the positive control Lilly mAb and the Novartis mAb or the antibody of the invention are diluted according to a 1: 5-fold ratio, the initial concentration is 40 mu g/ml, mIgG is diluted by the same method, 50 mu l of the diluted positive control or the antibody of the invention or mIgG is added into each hole, at the same time, 50 mu l of 0.2nM biotin-labeled IL-17A is added into the diluted positive control or the antibody of the invention, the mixture is mixed evenly and incubated for 1h at a constant temperature of 37 ℃.

7. PBST wash 3 times;

8. adding 100 μ l of HRP-labeled streptomycin avidin complex (1: 5000) into each well, and incubating at 37 deg.C for 1 h;

9. PBST was washed 3 times. Mu.l TMB substrate per well, incubation at 37 ℃ for 5min, followed by 100. mu.l 2M H per well₂SO₄Stopping the reaction;

10. the OD value at the wavelength of 450nm was read by an ELISA reader.

11. Calculating the IC of the test antibody for blocking IL-17 binding to the IL-17 receptor₅₀The value is obtained.

From the curve of the OD value versus the antibody concentration gradient, the IC50 value (antibody concentration (nM) at which the OD value decreased by 50%, namely RBA) was obtained.

The experimental results are as follows:

the hybridoma obtained in example 1 was screened as described above to obtain a murine monoclonal antibody, designated IL17-mAb049, with the following results:

table 5:

antibody numbering	huIL-17RBA(nM)
		Lilly mAb	0.17
Novartis mAb	1.56
		IL17-mAb049	0.07

And (4) conclusion: the activity of the murine antibody IL17-mAb049 obtained by hybridoma screening is better than that of the positive antibodies Lilly mAb and Novartis mAb.

Test example 3 affinity assay

Purpose of the experiment:

the BIACORE method was used in this experiment to determine antigen-antibody binding kinetics and affinity.

Experimental materials and instruments:

1.1 protein:

human IL-17A (hIL-17A): genbank human IL-17A protein accession NP-002181 was cloned and expressed by transient transfection of HEK293E cells according to methods known in the art.

Human IL-17A/F (heterodimer, hIL-17A/F): cloning was performed using Genbank human IL-17A protein accession NP _002181 and human IL-17F protein accession NP _443104 according to methods known in the art and expression was performed by transient transfection of HEK293E cells.

Mouse IL-17A (Mu IL-17A), Rat IL-17A (Rat IL-17A): cloning was performed using Genbank mouse IL-17A protein accession NP _034682 and rat IL-17A protein accession NP _001100367, respectively, according to methods known in the art, and expression was performed by transient transfection of HEK293E cells.

Murine anti-IL-17 antibodies from Lilly and Novartis (Lilly mAb, Novartis mAb) as positive controls were cloned according to the murine sequences provided in US7,838,638B2(LY 2439821) and US7,807,155B 2(AIN 457), respectively, and expressed by transient transfection of HEK293E cells.

A Lilly humanized anti-IL-17 antibody (Lilly mAb (hu)) as a positive control was cloned according to the humanized sequence provided in US7,838,638B2(LY 2439821) and expressed by transient transfection of HEK293E cells.

The murine monoclonal cell line obtained in example 1 of the present invention.

The humanized IL-17 antibody obtained in example 2 of the present invention.

1.2BIACORE model: BIACORE X100, GE;

1.3BIACORE chip and reagents (trade names, no recognized intermediate), see below:

materials and reagents	Company(s)	Catalog of products
			1.Sensor Chip CM5Research Grade	GE Healthcare	BR-1000-14
2.Amine Coupling Kit	GE Healthcare	BR-1000-50
			3.HBS buffer BIA Certified	GE Healthcare	BR-1001-88
4. Acetate (100ml)	GE Healthcare	BR-1003-51
			5.Mouse Antibody Capture Kit	GE Healthcare	BR-1008-38
6.Regeneration buffer Glycine 1.5	GE Healthcare	BR-1003-54
			7.BIAmaintenance Kit	GE Healthcare	BR-1006-66

The experimental method comprises the following steps:

1. immobilization of the antibody of the present invention on a CM5 chip: ready-prepared 1: 150mM NHS: 200mM EDC was injected into FC2(Flow cell 2) channel at a rate of 10. mu.l/min for 7min, activating the CM5 sensor chip. The antibody of the present invention was dissolved in 10mM sodium acetate buffer at a concentration of 30. mu.g/ml, pH 5.0, and injected into the activated chip at 5. mu.l/min (HBS-EP mobile phase buffer: 10mM HEPES, 150mM NaCl, 3.4mM EDTA, 0.005% surfactant P20, pH 7.4). The remaining active coupling sites were blocked by injection with 1 methanomine at a rate of 10. mu.l/min for 7 min. Approximately 8000 RU.

2. Binding kinetics assay: FC1(Flow cell 1) was used as a reference channel, FC2(Flow cell 2) was used as a sample channel, and the antibody of the invention or murine or humanized control antibody was captured in FC2 channel at 300RU, respectively, followed by injection of different concentrations of IL-17 (including hIL-17A, MuIL-17, Rat IL-17). The circulation conditions are as follows: surface regeneration was performed by injecting 30. mu.l/min 3min analyte in all channels of FCs with a dissociation time of 20min, 10mM Glycine, pH 1.5 for 60s (rate of 10. mu.l/min). The difference between the signal of the captured antibody and the signal without the captured antibody was calculated by Biacore X100evaluation software ver 2.0(Biacore) in running buffer (running buffer) 10mM Hepes, 650mM NaCl, 3mM EDTA, 0.05% Tween-20.

The experimental results are as follows:

1. the hybridoma obtained in example 1 was selected as described above, and the results were as follows:

table 6:

antibodies	Human IL-17A KD (M)
		Lilly mAb	2.18E-11
Novartis mAb	4.24E-10
		IL17-mAb049	2.62E-11

And (4) conclusion: the affinity of the murine antibody IL17-mAb049 obtained by hybridoma screening is equivalent to that of the human IL-17A and the positive antibody Lilly mAb, and is better than that of Novartis mAb.

2. The humanized IL-17 antibody obtained in example 2 was tested as described above, and the results are as follows:

table 7:

and (4) conclusion: the affinity of the humanized antibody was increased 10-fold over that of positive Lilly's antibody (1.48E-11M).

test example 4 bioassay at cell level (GRO. alpha. assay)

Purpose of the experiment:

the following experiment used anti-IL-17A antibodies to inhibit IL-17 stimulation of GRO α secretion by Hs27 cells to test the cellular biological activity of anti-IL-17A antibodies.

Experimental materials and instruments:

hs27 cells: ATCC Cat.No. CRL-1634 (note: cell culture over 6 weeks is not recommended for bioassay experiments);

hs27 cell culture medium: DMEM + 10% FBS

DMEM：ATCC Cat.No.30-2002；

FBS：GIBCO Cat.No.10099，lot 8122818；

Recombinant human IL-17A (rhIL-17A): r & D Systems Cat. No.317-ILB, lot SOA 161109B;

recombinant human IL-17A/F (rhIL-17A/F): r & D System Cat No.5194-IL/CF, lotRXT 101109A;

human CXCL1/GRO alpha Quantikine PharmPak kit: r & D system Cat.No. PDGR00

Equipment: biotek ELx808 microplate reader.

The murine monoclonal cell line obtained in example 1 of the present invention.

The humanized IL-17 antibody obtained in example 2 of the present invention.

The experimental method comprises the following steps:

1. hs27 cell culture:

hs27 cells were cultured in T175 flasks with 50ml DMEM + 10% FBS medium; cells (density about 90%) are diluted and cultured according to the proportion of 1: 3 every 3 days; the cultured cells were subjected to bioassay experiments within one month, or re-frozen and thawed from liquid nitrogen; freshly frozen and thawed cells were cultured for approximately one week before they were available for bioassay experiments.

2. Bioassay (IL-17A) Experimental procedure

2.1950rpm for 4min (Trypsin-EDTA was completely removed) and Hs27 cells were collected. Trypan blue staining is used for analyzing the cell viability, and the cell viability is more than 80 percent and can be used for experiments;

2.2 adding the culture medium into a 96-well plate according to 50 mul/well;

2.3Hs27 was diluted with DMEM + 10% FBS and added to a 96 well plate at 10000 cells/50. mu.l/well;

2.4 adding 25 mul IL-17 human antibody into the repeat hole, diluting the antibody according to 1: 3 times ratio, the initial concentration is 10 nM;

2.5 mu.l of recombinant human IL-17A was added per well. The final concentration of IL-17A was 0.3 nM. Centrifuging the 96-well plate at 500rpm for 1 min;

2.6 cells were incubated at 37 ℃ for 17 h;

2.7 collecting cell culture medium supernatant, detecting the concentration of GRO α in the supernatant by using human CACL1/GRO α Quantikine kit (according to the operation of manufacturer's instructions);

3. bioassay (IL-17A/F) Experimental procedure:

the operation procedure for IL-17A/F bioassay is similar to that for IL-17 Abioassay. Except that IL-17A was replaced with IL-17A/F.

The experimental results are as follows:

1. the hybridoma obtained in example 1 was selected as described above, and the results were as follows:

table 8:

and (4) conclusion: the biological activity of the antibody IL17-mAb049 obtained by hybridoma screening is equivalent to that of a positive antibody Lilly mAb, and is better than that of a Novartis mAb.

2. The humanized antibody obtained in example 2 was tested as described above, and the results were as follows:

table 9:

and (4) conclusion: the above results indicate that the humanized antibody has cell biological activity. The IC50(0.04nM to 0.066nM) of Hu049-17, 18, 19, 20 is the same as the positive antibody (0.04 nM). In addition, these antibodies cross-reacted with cynomolgusIL-17A (IC50 between 0.03nM and 0.039 nM). Human IL-17A/F activity is approximately 10-fold weaker than IL-17A.

Test example 5 in vivo neutralization assay of human IL-17

Purpose of the experiment:

the purpose of the in vivo neutralization assay is to verify that the antibodies of the invention block IL-17 binding to the IL-17 receptor (e.g., hIL-17RA) in vivo, thereby inhibiting IL-17-induced expression of CXCR 1.

Experimental materials and instruments:

protein: human IL-17A (hIL-17A): genbank human IL-17A protein accession NP-002181 was cloned and expressed by transient transfection of HEK293E cells according to methods known in the art.

Lilly humanized anti-IL-17 antibody (Lilly mAb (hu)) as a positive control was cloned according to the humanized sequence provided by US7,838,638B2(LY 2439821), respectively, and expressed by transient transfection of HEK293E cells.

Human IgG (HuIgG): (Millipore Cat. No. AG711).

Animals: 7-week-old male mice C57/B6 (Shanghai Sepal-BiKai laboratory animals Co., Ltd., animal production license number: SCXK (Shanghai) 2008-0016) were provided, and 6 mice were administered per group.

Reagent: ab dilution solution: citrate buffer (pH 5.0): 10mM citric acid sodium salt, 50mM NaCl

hIL-17A dilution: PBS (sodium phosphate buffer, pH 7.2).

Mouse CXCL1/KC Quantikine ELISA Kit, 6-well plate, R & D SYSTEM, # SMKC 00B.

The experimental method comprises the following steps:

1) mice were randomized into 15 groups of 6 mice each.

2) Each mouse was dosed intraperitoneally (I.P.) with 100uL of Hu049-18 or control antibody (HuIgG or Lilly mAb (Hu)) or diluted solutions at 3000. mu.g/kg, 300. mu.g/kg, 30ug/kg and 3. mu.g/kg, respectively.

3) After 20 hours, 150 μ g/kg hIL-17A was injected subcutaneously (S.C.) and 100uL per mouse.

4) After 2 hours, blood samples were collected, left at room temperature for 2 hours to agglutinate, or at 2-8 ℃ overnight to agglutinate, and then centrifuged at 2000Xg for 20 minutes. The supernatant was discarded and the experiment was immediately performed or the samples were stored in aliquots at-20 ℃. Repeated freeze thawing is avoided.

5) The samples obtained in step 4 were assayed using the mouse CXCL1/KC Quantikine ELISA Kit.

The experimental results are as follows:

the humanized antibody Hu049-18 obtained in example 2 was tested as described above, and the results were as follows:

table 10:

antibody (injection concentration 3000 mug/mouse)	KC mean value (pg/ml)
		HuIgG	937
Lilly mAb(hu)	158
		Hu049-18	145

And (4) conclusion: the antibody Hu-049-18 of the invention reduced the mean KC level to about 1/6 at a dose of 3000. mu.g/mouse under the conditions described, compared to a non-effector control antibody. The antibody Hu049-18 of the invention has comparable inhibitory capacity against mouse KC levels at a dose of 3000. mu.g/mouse under the conditions described, compared to the control antibody.

Test example 6 half-life (T1/2) detection of in vivo antibodies

Purpose of the experiment:

in order to detect the pharmacokinetic parameters of the antibody Hu049-18 in rats or macaques.

Experimental materials and reagents:

protein: human IL-17A (hIL-17A): genbank human IL-17A protein accession NP-002181 was cloned and expressed by transient transfection of HEK293E cells according to methods known in the art.

Lilly humanized anti-IL-17 antibody (Lilly mAb (hu)) as a positive control was cloned according to the humanized sequence provided by US7,838,638B2(LY 2439821), respectively, and expressed by transient transfection of HEK293E cells.

Human IgG (HuIgG): human IgG, Polyclonal, Millipore Cat.No. AG711

Animals: 230-250g SD male rats (Shanghaisi Leike laboratory animals, Inc., animal production license number: SCXK (Shanghai) 2007-0005) were divided into two groups of dorsal foot Intravenous (IV) group and subcutaneous injection (SC) group, each group containing 5 rats

The macaque: 2-3kg of cynomolgus monkey (Hainan Jingang biotech GmbH, animal production license number: SCXK (HN)2010-0001, 0000152.)

Reagent: antibody dilution solution: citrate buffer (pH 5.0): 10mM citric acid sodium salt, 50mM NaCl

hIL-17A dilution: PBS (sodium phosphate buffer, pH 7.2)

Goat anti-human IgG (Fab-specific) peroxidase conjugated antibody, Sigma Cat. No.121M4811

The experimental method comprises the following steps:

1. rat in vivo detection step:

(1) in vivo administration

The SD rats were randomly divided into 2 groups (dorsal foot Intravenous (IV) and Subcutaneous (SC) groups) of 5 rats each;

under aseptic conditions, Hu049-18 was dissolved in citrate buffer (pH 5.0) to a final concentration of 2.5 mg/mL;

IV or SC administration is carried out on each rat, and the administration dose is 5 mg/kg;

group IV blood was taken at time points 0, 5, 15, 30min, 1, 2, 4, 8, 24hr, 2, 4, 7, 10, 14, 21, 28d tail vein, 200uL each time (equivalent to 80uL serum); SCs groups were bled at time points 0, 30min, 1, 2, 4, 8, 12, 24hr, 2, 4, 7, 10, 14, 21, 28d tail vein, 200uL each time (equivalent to 80uL serum);

the collected blood samples were left at room temperature for half an hour to agglutinate and then centrifuged at 10000 Xg for 5 minutes at 4 ℃. The supernatant was collected and either immediately subjected to the experiment or the samples were stored in aliquots at-80 ℃. Repeated freeze thawing is avoided.

(2) ELISA detection using serum sample obtained in step (1)

1) Standard curve

a) Directly coating 1 mug/ml streptomycin avidin and standing overnight at 4 ℃;

b) blocking the microtiter plate with 300. mu.l PBST containing 2% BSA at 37 deg.C for 1h, and blocking the uncoated blank well as controlling;

c) PBST washes 3 times, all washes were run on a Biotek (Elx 405) automatic plate washer;

d) adding 100ul PBS containing hIL-17A (0.2 mu g/mL) into each well, and incubating at the constant temperature of 37 ℃ for 1 h;

e) PBST wash 3 times;

f) hu049-18 titration: diluted 1: 2 fold with antibody diluent at an initial concentration of 0.8. mu.g/ml, 100. mu.l of diluted Hu049-18 per well was added for standard curve plotting. Incubation was performed at 37 ℃ for 1 h.

g) PBST wash 3 times;

h) mu.l of goat anti-human IgG (Fab-specific) peroxidase conjugated antibody (SigmaCat. No.121M4811) (1: 5000) was added to each well, and incubated at 37 ℃ for 1 hour;

i) PBST was washed 3 times. Adding 100 μ l TMB substrate to each well, incubating at 37 deg.C for 5min, and adding 100 μ l 1M HCl to each well to stop reaction;

j) an ELISA plate reader (Molecular Devices, Spectra Max) reads OD values at 450nm/630nm wavelength.

2) Sample assay

a) Directly coating 1 mug/ml streptomycin avidin and standing overnight at 4 ℃;

b) blocking the microtiter plates with 300. mu.l of PBST containing 2% BSA at 37 ℃ for 1h (1 h) while blocking the uncoated blank wells as a control;

c) PBST washes 3 times, all washes were run on a Biotek (Elx 405) automatic plate washer;

d) add 100. mu.l of PBS containing hIL-17A (0.2. mu.g/mL) into each well, incubate 1h at 37 ℃;

e) PBST wash 3 times;

f) titration of serum samples: a rat serum sample is taken before an experiment and diluted according to different proportions to obtain an optimal dilution proportion of the antibody concentration in the serum at the middle position of a standard curve, the serum sample is diluted according to the optimal dilution proportion, and meanwhile, Hu049-18 is diluted to 25 ng/mL. Mu.l of diluted sample serum and Hu049-18 were added to each well and incubated at 37 ℃ for 1 h. Each titration concentration is provided with a repeat hole;

g) PBST wash 3 times;

h) mu.l of goat anti-human IgG (Fab-specific) peroxidase conjugated antibody (SigmaCat. No.121M4811) (1: 5000) was added to each well, and incubated at 37 ℃ for 1 hour;

i) PBST was washed 3 times. Adding 100 μ l TMB substrate to each well, incubating at 37 deg.C for 5min, and adding 100 μ l 1M HCl to each well to stop reaction;

j) an ELISA plate reader (Molecular Devices, Spectra Max) reads OD values at 450nm/630nm wavelength.

2. In vivo detection of macaques:

the in vivo detection procedure of macaca mulatta (cynomolgus monkey) is similar to that of rat except that the cynomolgus monkey is administrated by intravenous Injection (IV) only, the dosage is 1mg/kg, the intravenous blood taking time is 0, 5, 15, 30min, 1, 2, 4, 8, 24, 32hr, 3, 4, 5, 6, 9, 12, 14, 17, 21, 28 and 35d, 500 mu L of blood is taken each time, the serum sample obtained by centrifugation is divided into 3 parts (ensuring that the content of 2 parts of serum sample is 60 mu L), and the serum sample is frozen at 80 ℃ for detection.

The experimental results are as follows:

the humanized antibody Hu049-18 obtained in example 2 was tested as described above, and the results were as follows:

table 11:

and (4) conclusion: the above results show that the antibody Hu049-18 of the invention has a significantly increased in vivo half-life under the conditions described, compared to the control antibody of Lilly (positive antibody reported as T1/2 in cynomolgus monkeys at 6.5 days (iv) and 10.3 days (sc).

Claims (25)

1. An IL-17A conjugate comprising:

an antibody light chain variable region comprising an LCDR region as shown in SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15; and

an antibody heavy chain variable region comprising an HCDR region as shown in SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12.

2. The IL-17A conjugate of claim 1, wherein the antibody light chain variable region further comprises a light chain FR region of a murine kappa chain or murine kappa chain variant, or a light chain FR region of a murine lambda chain or murine lambda chain variant.

3. The IL-17A conjugate of claim 2, wherein the antibody light chain variable region amino acid sequence is set forth in seq id No. 2.

4. The IL-17A conjugate of any one of claims 2-3, further comprising a light chain constant region of a murine kappa chain or variant thereof, or a light chain constant region of a lambda chain or variant thereof.

5. The IL-17A conjugate of claim 1, wherein the antibody heavy chain variable region further comprises a heavy chain FR region of murine IgG1 or a variant thereof, a heavy chain FR region of IgG2 or a variant thereof, a heavy chain FR region of IgG3 or a variant thereof, a heavy chain FR region of IgG4 or a variant thereof.

6. The IL-17A conjugate of claim 5, wherein the antibody heavy chain variable region amino acid sequence is set forth in SEQ ID NO. 1.

7. The IL-17A conjugate of any one of claims 5-6, further comprising a heavy chain constant region of murine IgG1 or a variant thereof, a heavy chain constant region of IgG2 or a variant thereof, a heavy chain constant region of IgG3 or a variant thereof, a heavy chain constant region of IgG4 or a variant thereof.

8. The IL-17A conjugate of claim 1, wherein the antibody light chain variable region further comprises a light chain FR region of a human kappa chain or a variant thereof, or a light chain FR region of a lambda chain or a variant thereof.

9. The IL-17A conjugate of claim 8, wherein the light chain FR region is the FR region of human germline light chain a10 or a variant thereof having the amino acid sequence set forth in SEQ ID No. 4.

10. The IL-17A conjugate of claim 9, wherein the FR region variant of human germline light chain a10 refers to an amino acid change of 0-10 in the FR region of human germline light chain a 10.

11. The IL-17A conjugate of claim 10, wherein the amino acid change is selected from one or more of: F71Y, K49Y, Y36F, L47W.

12. The IL-17A conjugate of claim 8, wherein the antibody light chain variable region is selected from the group consisting of the light chain variable region set forth in SEQ ID No. 9 and variants thereof.

13. The IL-17A conjugate of any one of claims 8-12, further comprising a light chain constant region of a human kappa chain or variant thereof, or a light chain constant region of a lambda chain or variant thereof.

14. The IL-17A conjugate of claim 1, wherein the heavy chain variable region further comprises a heavy chain FR region of human IgG1 or a variant thereof, a heavy chain FR region of IgG2 or a variant thereof, a heavy chain FR region of IgG3 or a variant thereof, a heavy chain FR region of IgG4 or a variant thereof.

15. The IL-17A conjugate of claim 14, wherein the heavy chain FR region is the FR region of human germline heavy chain VH1-18 or a variant thereof having the amino acid sequence set forth in SEQ id No. 3.

16. The IL-17A conjugate of claim 15, wherein the variant refers to an amino acid change of 0-10 in the FR region of the heavy chain of VH 1-18.

17. The IL-17A conjugate of claim 16, wherein the amino acid change is selected from one or more of: a93T, T71A, M48I, V67A, M69L, T73D and S76N.

18. The IL-17A conjugate of claim 15, wherein the antibody heavy chain variable region is selected from the group consisting of: 5, 6, 7,8 or a variant thereof.

19. The IL-17A conjugate of claim 14, further comprising a heavy chain constant region of human IgG1 or a variant thereof, a heavy chain constant region of IgG2 or a variant thereof, a heavy chain constant region of IgG3 or a variant thereof, a heavy chain constant region of IgG4 or a variant thereof.

20. A vector expressing an IL-17A conjugate according to any one of claims 1 to 19.

21. The vector of claim 20, comprising nucleotides encoding an IL-17A conjugate of any one of claims 1-19.

22. A pharmaceutical composition comprising: the IL-17A conjugate of any one of claims 1 to 19; and a pharmaceutically acceptable excipient, diluent or carrier.

23. Use of an IL-17A conjugate according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 22, in the manufacture of a medicament for the treatment of an IL-17 mediated disease or disorder.

24. The use of claim 23, wherein:

the disease is selected from: inflammation and autoimmune diseases.

25. The use of claim 23, wherein:

the disease is selected from: psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory arthritis.