WO2023035272A1 - Il17 antibody, preparation method therefor and application thereof - Google Patents

Abstract

An IL17 antibody, a preparation method therefor and an application thereof. The IL17 antibody comprises a heavy chain variable region, which comprises CDR1, the sequence of which is shown in any one of SEQ ID NO: 1-24; CDR2, the sequence of which is shown in any one of SEQ ID NO: 25-47; and CDR3, the sequence of which is shown in any one of SEQ ID NO:48-71. The IL17 antibody obtained in the present invention can bind, with specificity and high affinity, to one or more of human cytokines IL17A, IL17F and IL17A/F, and can also bind, with high affinity and specificity, to cynomolgus monkey IL17A and IL17F.

Description

A kind of IL17 antibody and its preparation method and application technical field

The invention belongs to the field of biomedicine, and in particular relates to an IL17 antibody and its preparation method and application.

Background technique

The cytokine IL17 can come from a variety of different immune cells, among which the secretion of Th17 helper T cells is the main source, and CD8 ⁺ T cells, NK T cells, LLC3 (Type 3 innate lymphoid cells) and γδ T cells can also secrete IL17 (Nat Rev Immunol. 2010, 10, 479). The cytokine IL17 protein family mainly includes IL17A, IL17B, IL17C, IL17D, IL17E (IL25) and IL17F, among which IL17A and IL17F have a large sequence similarity and can be expressed simultaneously in Th17 cells. IL17A and IL17F can exist as a homodimer, or as an IL17A/F heterodimer composed of IL17A and IL17F monomers (Cold Spring Harb Perspect Biol. 2018, 10, a028522). There are multiple subunits of IL17 receptor protein, including five receptor subunits including IL17RA, IL17RB, IL17RC, IL17RD and IL17RE, among which IL17RA is often used as the common core subunit of IL17 receptor and participates in the binding of different IL17 molecules, such as IL17A , IL17F and IL17A/F both bind to the receptor complex composed of IL17RA/IL17RC, IL17C binds to the receptor complex composed of IL17RA/IL17RE, IL17E binds to the receptor complex composed of IL17RA/IL17RB (Cytokine 2013,64,477; Trends Immunol. 2017, 38, 310).

IL17RA is expressed in both hematopoietic stem cells and non-hematopoietic cells, but IL17RC is commonly expressed in non-hematopoietic stem cells, so dimers such as IL17A, IL17F and IL17A/F activate non-hematopoietic stem cells, such as epithelial cells, by binding to the IL17RA/IL17RC receptor complex cells and stromal cells, etc. After the cells are activated, they produce a series of pro-inflammatory factors, such as cytokines such as IL-1, IL-6, G-CSF and TNF, and also induce the expression of CXCL1, CXCL2, CXCL5, CCL2, CCL7, CCL20 and IL-8. It also promotes the expression of matrix metalloproteinases MMP1, MMP3, MMP9 and MMP13, and produces some antimicrobial peptides, such as β-defensins, S-100 protein, etc. (Immunity 2011, 34, 149). The cytokine IL17 plays an important role in resisting external bacteria and fungal infections, maintaining the integrity of the mucosa and epidermis, and promoting wound healing (Immunol Rev. 2008, 226, 57). Conversely, once the activity of IL17 is not normally regulated, it will cause chronic inflammation and autoimmune diseases, and may even induce the formation of tumors.

The increased expression of IL17A and related cytokines can cause a series of autoimmune diseases, including psoriasis, rheumatoid arthritis, scleroderma and lupus. In the process of psoriasis formation, DC cells that can secrete TNF-α/iNOS (TIP-DC) play an important role. TIP-DC cells are activated by binding TNF-α, and the activated TIP-DC cells Further secrete TNF-α, IL23, IL12 and other cytokines, IL23 activates Th17 cells, prompts Th17 cells to secrete excessive cytokines such as IL17A, IL17F and IL22, and these cytokines further drive abnormal differentiation and proliferation of keratinocytes, thus forming psoriasis disease (J Am Acad Dermatol.2014,71,141; J Dermatol.2018,45,264; Int J Mol Sci.2020,21,1690). Inhibition of the IL23-IL17 immune axis (Nat. Rev. Immunol. 2014, 14, 585) has achieved good results in the treatment of psoriasis and other related diseases. In addition, elevated serum concentrations of IL17A and IL17F were positively correlated with the severity of atopic dermatitis in infants. Recent studies have also shown that IL17A is also a potential therapeutic target for sepsis (Front Immunol. 2020, 11, 1558). In the field of neurological diseases, in addition to multiple sclerosis (multiple sclerosis), dysregulation of IL17 is also considered to be the cause of chronic neuritis, experimental autoimmune encephalomyelitis, Alzheimer's disease and ischemic brain injury. Disease factors (Front Immunol.2020, 11, 947).

Th17 cell subsets are involved in regulating the inflammation of neutrophils and macrophages in the lungs, and are associated with severe neutrophil asthma and chronic obstructive pulmonary disease (Nat.Rev.Immunol.2008,8,183; Ann.Rev. Physiol. 2010, 72, 495). Studies have shown that the expression levels of IL17A and IL17F are related to the severity of asthma. The correlation is because IL17 can effectively promote the expression of neutrophil growth factors and chemokines, such as IL-6, G-CSF, IL8, etc., thus Promote the enrichment of neutrophils (Expert Rev. Respir. Med. 2014,8,25). In patients with chronic obstructive pulmonary disease, bronchial asthma, and patients with both chronic obstructive pulmonary disease and asthma, the concentration of IL17A in serum is increased, and the degree of increased concentration is positively correlated with the severity of the disease (Mediators Inflamm.2020,2020, 4652898). By studying the biomarkers of subtypes of chronic obstructive pulmonary disease, it was found that a group of people who are insensitive to hormone therapy is associated with inflammation triggered by IL17, which means that antibodies that bind to IL17 are effective for this subtype of chronic obstructive pulmonary disease. The treatment of type 2 population has clinical value (J Clin Invest.2019, 129, 169).

In addition to autoimmune diseases, more and more evidences show that the dysregulation of IL17 can promote the early occurrence and the development of middle and late stages of cancer. By testing the concentration of IL17 in the serum of a series of different solid tumor patients, it was found that tumor patients with higher IL17 concentration had worse prognosis (Oncoimmunology 2015, 4, e984547). As a kind of pro-inflammatory factor, IL17 plays a certain connection role in the process of inflammatory response, wound healing and tumor formation (J. Exp. Med. 2019, 217, e20190297; Nat. Immunol. 2019, 20, 1594). Studies have shown that IL17 promotes the pathogenesis of various cancers, including colon cancer (Immunity, 2014, 41, 1052; J Immunol. 2017, 199, 3849), skin cancer (Cancer Res 2010, 70, 10112; J Exp. Med.2019,216,195), pancreatic cancer (Gastroenterology 2018,155,210), liver cancer (Dig.Dis.Sci.2016,61,474), lung cancer (Cell 2019,176,998) and myeloma (Nat.Commun.2018,9,4832) wait. In short, IL17 induces cytokines and chemokines to promote myeloid suppressor cells, thereby further promoting angiogenesis (Nat.Med.2013, 19, 1114; PNAS 2014, 111, 5664), or enhancing the tumor microenvironment to form an anti-tumor inhibitory effect , to promote the formation and development of tumors (J Immunol.2010, 184, 2281; Nature, 2015, 522, 345; Adv Exp Med Biol. 2020, 1240, 47). There is also research evidence that the activation of IL17 signaling helps to form tumor resistance to radiotherapy and chemotherapy, thereby reducing the therapeutic effect (J Exp Med.2020, 217, e20190297). Based on the above studies, anti-IL17 antibodies also have great application prospects for anti-tumor therapy.

Contents of the invention

Psoriasis is an autoimmune disease with a high incidence rate. The prevalence rate in Europe and the United States is 1%-3%. The prevalence rate in China is relatively low, and it is estimated that there are more than 6 million patients. For patients with moderate to severe psoriasis and/or psoriatic arthritis who are ineffective or poorly tolerated by traditional therapies, they can be treated by injecting biological agents; currently targeting IL17A is used to treat psoriasis. Three monoclonal antibody drugs for psoriasis (Cosentyx/Secukinumab, Taltz/Ixekizumab and Netakimab) have been approved for the treatment of psoriasis, ankylosing spondylitis and other indications, and the effect has been well recognized. A new generation of multivalent monoclonal antibody drugs targeting IL17A and IL17F, such as Bimekizumab from Belgium-based UCB, has shown good therapeutic effects on psoriasis in head-to-head clinical trials. Sonelokimab, a multivalent single-domain antibody targeting IL17A, IL17F and IL17A/F, in a clinical phase IIb multicenter trial, 120 mg or less dose of sonelokimab showed significant clinical benefits compared with placebo, with rapid onset of action , durability and acceptable safety (Lancet, 2021, 397, 1564). At the same time, multivalent antibodies targeting IL17A and other cytokines, such as TNF-α, BAFF, and IL13, are also in different development stages for the treatment of various indications such as chronic inflammation of the lung.

As a strict complete monomer, single domain antibody has the advantages of small molecular weight, high solubility, strong tissue permeability, high affinity and high degree of humanization. Its special structural characteristics and properties endow it with some other conventional antibodies or antibodies A property that fragments do not have (Annu Rev Biochem. 2013, 82, 775). V _H H single-domain antibodies have good druggability to soluble targets, membrane protein targets including GPCRs and ion channels (Biomolecules 2021, 11, 63), and are widely used in anti-tumor drugs and anti-viruses , autoimmune diseases, and CAR-T immune cell therapy have been widely used (BioDrugs 2020, 34, 11; Antib Ther 2020, 3, 257; Biomolecules 2021, 11, 238). Single-domain antibodies are easy to modify and are ideal building blocks for multivalent antibodies. They can be aggregated through short linker sequences (linker) to construct multivalent forms with higher antigen affinity than monovalent antibodies, or they can be used to combine multiple different antibodies at the same time. antigenic target. Due to the small molecular weight of the multivalent _VHH antibody, not only the traditional CHO expression production method can be used, but also the production system such as E.coli, P.pastoris, etc., and in the development of new dosage forms of biological drugs, including subcutaneous injection, inhalation And oral drugs, etc., which have advantages over traditional monoclonal antibodies (MAbs 2018, 10, 778; Mucosal Immunol. 2010, 3, 49).

Therefore, the development of single/multivalent single-domain antibodies targeting IL17A and IL17F has multiple potential applications, such as multivalent antibodies targeting multiple targets such as IL17A, IL17A/F and IL17F at the same time, for psoriasis and ankylosing spondylitis, also including indications such as plaque psoriasis, psoriatic arthritis, and nonradiographic axial spondyloarthritis (Expert Opin Biol Ther 2019,19,45); or, with anti-TNF- One or more of cytokine antibodies such as α, BAFF and IL13 form a multivalent antibody, which is used for the treatment of various autoimmune diseases including chronic lung diseases, rheumatoid diseases, and Sjögren's syndrome.

The technical problem to be overcome by the present invention is to provide an IL17 antibody and its preparation method and application in order to overcome the lack of effective single/multivalent single-domain antibodies targeting IL17A and IL17F in the prior art.

The present invention solves the above-mentioned technical problems through the following technical solutions.

The present invention provides an IL17 antibody comprising a heavy chain variable region, wherein:

The heavy chain variable region comprises a sequence such as CDR1 shown in any one of SEQ ID NOs: 1 to 24, a sequence such as CDR2 shown in any one of SEQ ID NOs: 25 to 47, and a sequence such as SEQ ID NO : CDR3 shown in any one of 48-71.

Preferably, the heavy chain variable region of the present invention:

(1) its CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises the sequence shown in SEQ ID NO:25, and CDR3 comprises the sequence shown in SEQ ID NO:48; Or,

(2) its CDR1 comprises the sequence shown in SEQ ID NO:2, CDR2 comprises the sequence shown in SEQ ID NO:26, and CDR3 comprises the sequence shown in SEQ ID NO:49; Or,

(3) its CDR1 comprises the sequence shown in SEQ ID NO:3, CDR2 comprises the sequence shown in SEQ ID NO:27, and CDR3 comprises the sequence shown in SEQ ID NO:50; Or,

(4) Its CDR1 comprises the sequence shown in SEQ ID NO:4, CDR2 comprises the sequence shown in SEQ ID NO:28, and CDR3 comprises the sequence shown in SEQ ID NO:51; Or,

(5) its CDR1 comprises the sequence shown in SEQ ID NO:5, CDR2 comprises the sequence shown in SEQ ID NO:29, and CDR3 comprises the sequence shown in SEQ ID NO:52; or,

(6) Its CDR1 comprises the sequence shown in SEQ ID NO:6, CDR2 comprises the sequence shown in SEQ ID NO:28, and CDR3 comprises the sequence shown in SEQ ID NO:53; Or,

(7) Its CDR1 comprises the sequence shown in SEQ ID NO:7, CDR2 comprises the sequence shown in SEQ ID NO:30, and CDR3 comprises the sequence shown in SEQ ID NO:54; Or,

(8) Its CDR1 comprises the sequence shown in SEQ ID NO:8, CDR2 comprises the sequence shown in SEQ ID NO:31, and CDR3 comprises the sequence shown in SEQ ID NO:55; Or,

(9) Its CDR1 comprises the sequence shown in SEQ ID NO:9, CDR2 comprises the sequence shown in SEQ ID NO:32, and CDR3 comprises the sequence shown in SEQ ID NO:56; Or,

(10) its CDR1 comprises the sequence shown in SEQ ID NO:10, CDR2 comprises the sequence shown in SEQ ID NO:33, and CDR3 comprises the sequence shown in SEQ ID NO:55; Or,

(11) its CDR1 comprises the sequence shown in SEQ ID NO:11, CDR2 comprises the sequence shown in SEQ ID NO:34, and CDR3 comprises the sequence shown in SEQ ID NO:57; Or,

(12) Its CDR1 comprises the sequence shown in SEQ ID NO:12, CDR2 comprises the sequence shown in SEQ ID NO:35, and CDR3 comprises the sequence shown in SEQ ID NO:58; Or,

(13) Its CDR1 comprises the sequence shown in SEQ ID NO:13, CDR2 comprises the sequence shown in SEQ ID NO:36, and CDR3 comprises the sequence shown in SEQ ID NO:59; Or,

(14) Its CDR1 comprises the sequence shown in SEQ ID NO:14, CDR2 comprises the sequence shown in SEQ ID NO:34, and CDR3 comprises the sequence shown in SEQ ID NO:60; Or,

(15) Its CDR1 comprises the sequence shown in SEQ ID NO:15, CDR2 comprises the sequence shown in SEQ ID NO:37, and CDR3 comprises the sequence shown in SEQ ID NO:61; Or,

(16) Its CDR1 comprises the sequence shown in SEQ ID NO:9, CDR2 comprises the sequence shown in SEQ ID NO:38, and CDR3 comprises the sequence shown in SEQ ID NO:62; Or,

(17) Its CDR1 comprises the sequence shown in SEQ ID NO:14, CDR2 comprises the sequence shown in SEQ ID NO:34, and CDR3 comprises the sequence shown in SEQ ID NO:60; Or,

(18) Its CDR1 comprises the sequence shown in SEQ ID NO:15, CDR2 comprises the sequence shown in SEQ ID NO:37, and CDR3 comprises the sequence shown in SEQ ID NO:63; Or,

(19) Its CDR1 comprises the sequence shown in SEQ ID NO:16, CDR2 comprises the sequence shown in SEQ ID NO:39, and CDR3 comprises the sequence shown in SEQ ID NO:64; Or,

(20) Its CDR1 comprises the sequence shown in SEQ ID NO:17, CDR2 comprises the sequence shown in SEQ ID NO:40, and CDR3 comprises the sequence shown in SEQ ID NO:65; Or,

(21) Its CDR1 comprises the sequence shown in SEQ ID NO:18, CDR2 comprises the sequence shown in SEQ ID NO:41, and CDR3 comprises the sequence shown in SEQ ID NO:66; Or,

(22) Its CDR1 comprises the sequence shown in SEQ ID NO:19, CDR2 comprises the sequence shown in SEQ ID NO:42, and CDR3 comprises the sequence shown in SEQ ID NO:67; Or,

(23) Its CDR1 comprises the sequence shown in SEQ ID NO:20, CDR2 comprises the sequence shown in SEQ ID NO:43, and CDR3 comprises the sequence shown in SEQ ID NO:67; Or,

(24) Its CDR1 comprises the sequence shown in SEQ ID NO:21, CDR2 comprises the sequence shown in SEQ ID NO:44, and CDR3 comprises the sequence shown in SEQ ID NO:68; Or,

(25) Its CDR1 comprises the sequence shown in SEQ ID NO:22, CDR2 comprises the sequence shown in SEQ ID NO:45, and CDR3 comprises the sequence shown in SEQ ID NO:69; Or,

(26) Its CDR1 comprises the sequence shown in SEQ ID NO:23, CDR2 comprises the sequence shown in SEQ ID NO:46, and CDR3 comprises the sequence shown in SEQ ID NO:70; Or,

(27) Its CDR1 comprises the sequence shown in SEQ ID NO:24, CDR2 comprises the sequence shown in SEQ ID NO:47, and CDR3 comprises the sequence shown in SEQ ID NO:71.

In a preferred embodiment of the present invention, the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NO: 72-100.

Examples of antibodies in the present invention include, but are not limited to, full-length antibodies, heavy chain antibodies (HCAb), antigen-binding fragments (Fab, Fab', F(ab) ₂ , Fv fragments, F(ab') ₂ , scFv, di-scFv and/or dAb), immunoconjugates, multispecific antibodies (e.g. bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they exhibit the desired antigen binding activity; preferably a single domain antibody or a _VHH fragment thereof.

The present invention also provides a trivalent single-domain antibody, which comprises the above-mentioned IL17 antibody.

In the present invention, the trivalent single domain antibody preferably contains three _VHHs connected sequentially from the N-terminus _to the C _- terminus: _{VHH1 - VHH2} - _VHH3 .

_The above-mentioned _VHH2 is preferably an anti-HSA _VHH ; more preferably contains the sequence shown in SEQ ID NO:108.

The above-mentioned _{VHH 1} and _the above-mentioned _VHH3 are preferably IL17 antibodies in the form of single-domain antibodies; preferably:

_{The VHH1} contains the CDR combination as defined in any one of (1) to (6) above, and _{the VHH3} contains the CDR combination as defined in any one of the above (7) to (22); More preferably, the _VHH1 contains the sequence shown in any one of SEQ ID NO:72-79 _, and _{the VHH3} contains the sequence shown in any one of SEQ ID NO:80-100 ;

Or _{the VHH3} contains the CDR combination defined in any one of (1) to (6) above, and _{the VHH1} contains the CDR combination defined in any one of the above (7) to (22) more preferably, _{the VHH3} contains the sequence shown in any one of SEQ ID NO:72～79, and _{the VHH1} contains the sequence shown in any one of SEQ ID NO:80～100 sequence.

In a preferred embodiment of the present invention, _{the VHH1} or _VHH3 contains the CDR combination _as defined in _{( 2} ) above, and the _VHH3 or _VHH1 contains the above (7), ( 10) or the CDR combination defined in (26).

In a specific embodiment of the present invention, the _VHH1 contains the sequence shown in SEQ ID NO:73 _, and _{the VHH3} contains the sequence shown in SEQ ID NO:80, 83 or 99;

In another embodiment of the present invention, the _VHH1 contains the sequence shown in SEQ ID NO:80, 83 or 99, and _{the VHH3} contains the sequence shown in SEQ _ID NO:73.

The above different _VHHs are preferably operably connected through a linker; the linker preferably contains the sequence shown in SEQ ID NO:107.

In a specific embodiment of the present invention, the trivalent single domain antibody contains the amino acid sequence shown in any one of SEQ ID NO: 101-106.

The present invention also provides an isolated nucleic acid encoding the IL17 antibody as described above or the trivalent single domain antibody as described above.

The present invention also provides an expression vector comprising the isolated nucleic acid as described above.

The present invention also provides a host cell comprising the expression vector as described above; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

The present invention also provides a method for preparing an IL17 antibody or a trivalent single-domain antibody, which comprises culturing the above-mentioned host cells, and obtaining the antibody from the culture.

The present invention also provides a pharmaceutical composition, which comprises the above-mentioned IL17 antibody or the above-mentioned trivalent single-domain antibody.

The present invention also provides the application of the above-mentioned IL17 antibody, the above-mentioned trivalent single-domain antibody or the above-mentioned pharmaceutical composition in the preparation of drugs for preventing and treating IL17-related diseases or diseases.

The disease or condition preferably includes chronic inflammation of the lung, rheumatoid arthritis, intestinal inflammation, dry eye disease, psoriasis, ankylosing spondylitis and various cancers including skin cancer, more preferably psoriasis, ankylosing spondylitis, chronic inflammation of the lungs and rheumatoid arthritis; psoriasis, ankylosing spondylitis and rheumatoid arthritis are further preferred.

The present invention also provides a chimeric antigen receptor comprising the above-mentioned IL17 antibody or the above-mentioned trivalent single-domain antibody.

The present invention also provides an antibody-drug conjugate, which comprises a cytotoxic agent, and the aforementioned IL17 antibody or the aforementioned trivalent single-domain antibody; preferably, the cytotoxic agent is MMAF or MMAE.

The present invention also provides a kit comprising the above-mentioned IL17 antibody, the above-mentioned trivalent single domain antibody, the above-mentioned chimeric antigen receptor, the above-mentioned antibody drug conjugate and/or A pharmaceutical composition as described above;

Preferably, the kit further comprises (i) a device for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.

The present invention also provides a kit of medicines, which includes a medicine box A and a medicine box B, wherein:

The kit A contains the above-mentioned IL17 antibody, the above-mentioned trivalent single domain antibody, the above-mentioned chimeric antigen receptor, the above-mentioned antibody drug conjugate and/or the above-mentioned pharmaceutical composition;

The kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

The present invention also provides a method for diagnosing, treating and/or preventing IL17-mediated diseases or disorders, the method comprising administering to a patient in need a therapeutically effective amount of the above-mentioned IL17 antibody, the above-mentioned trivalent The single domain antibody, chimeric antigen receptor as above, antibody drug conjugate as above or pharmaceutical composition as above, or use the kit as above to treat patients in need. Wherein, with regard to said disease or disease as defined above.

The present invention also provides a method for immune detection or determination of IL17, which includes using the above-mentioned IL17 antibody, the above-mentioned trivalent single-domain antibody, the above-mentioned chimeric antigen receptor, and the above-mentioned antibody drug The conjugate or the pharmaceutical composition as described above is detected after incubation with the analyte; preferably, the detection or the determination is for non-diagnostic and/or therapeutic purposes.

The present invention also provides a combination therapy, which comprises separately administering the above-mentioned IL17 antibody, the above-mentioned trivalent single domain antibody, the above-mentioned chimeric antigen receptor, the above-mentioned antibody to patients in need A drug conjugate or a pharmaceutical composition as described above, and a second therapeutic agent; the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies, and/or consists of Hormonal agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccine components One or more of the groups.

The present invention uses IL17A homodimer, IL17F homodimer and IL17A/F heterodimer to immunize alpaca multiple times, isolates mRNA of peripheral blood lymphocytes, synthesizes cDNA and constructs V _H H Antibody library, using phage display technology, ELISA technology and other methods to screen and obtain multiple V H H antibodies with high affinity and specific binding to IL17A, IL17F and IL17A/F, followed by V _{H H} antibody expression purification, affinity, specificity and stability By combining with Anti-HSA antibody, construct multiple multivalent V _H H antibodies, and complete the evaluation of expression purification, affinity, specificity, stability and epitope binning. The results show that these monovalent and multivalent _VHH antibodies have unique advantages, mainly in the following aspects:

1. These V _H H antibodies can selectively and high-affinity bind to one or more of human cytokines IL17A, IL17F and IL17A/F, and can bind cynomolgus monkey IL17A and IL17F with high affinity and specificity;

2. The V _H H antibody sequence has good thermal stability (T _m ≥ 65°C);

3. When combined with IL17A, IL17F and IL17A/F, it has a new antigenic determinant;

4. The multivalent antibody constructed by combining different V _H H antibody sequences can simultaneously bind IL17A, IL17A/F and IL17F with high affinity and specificity;

5. Monovalent and multivalent antibodies can be efficiently expressed and folded correctly in the intercellular space of E.coli, and can be purified and prepared by protein A-labeled affinity chromatography and ion exchange chromatography;

6. Cell activity test shows that it can effectively block the binding of IL17A, IL17A/F and corresponding receptors;

7. Monovalent and multivalent V _H H antibodies are expected to be used in diseases caused by abnormal regulation of cytokines such as IL17A, IL17F and IL17A/F;

8. V _H H antibodies can be easily combined with V _H H single domain antibodies of other targets to form multivalent antibodies, which can be used for the treatment of anti-tumor, lung disease, autoimmune disease, anti-virus, anti-infection, etc.

Description of drawings

In Fig. 1, Fig. 1A is the SDS-PAGE electrophoresis diagram of V _H H-A19 affinity chromatography, 1B is the Superdex 75 10/300 chromatogram, and Fig. 1C is the protein SDS-PAGE electrophoresis diagram after Superdex 75 10/300 chromatography.

Fig. 2 is a diagram of the affinity test between V _H H-A19 and human IL17A.

Figure 3 is the test chart of T _m value of V _H H-AF6.

Fig. 4 is a Paratope analysis diagram of the interaction between V _H H-A8 and IL17A.

In Figure 5, 5A is the purification diagram of rProtein A sepharose FF of PAF022, 5B is the purification diagram of Hitrap Q column, and 5C is the SDS-PAGE electrophoresis diagram of sampling after Hitrap Q column purification.

Figure 6 is the T _m test chart of PAF022.

In Fig. 7: Fig. 7A shows the neutralizing activity of PAF022, PAF025 and PAF041 on IL17A; Fig. 7B shows the neutralizing activity of PAF022, PAF025 and PAF041 on IL17A/F.

Figure 8 shows the way V _H H is connected to anti-HSA through amino acid Linker (GGGGSGGGS).

Detailed ways

Example 1 Alpaca immunization, titer detection and lymphocyte separation

Purchase IL17A (Sino Biological, 12047-HNAS), IL17A/F (Sino Biological, CT047-HNAE) and IL7F (Sino Biological, 11855-HNAE) three proteins, refer to (Methods Mol Biol 2012, 911, 211; Curr Protoc Immunol 2013, 103, 2.17.1; Nat Protoc 2014, 9, 674) and other documents described in the immunization method, immunized three alpacas.

For each immunization, the amount of 100 μg protein/alpaca was injected on both sides of the neck muscle of the alpaca, and a total of two injections were injected. Complete Freund's adjuvant was used for the first time and recorded as day 0. On the subsequent 14th day, 28th day, 42th day, 56th day and 70th day, incomplete Freund's adjuvant was used for the second to sixth booster immunization. The immune effect of alpaca was tested by ELISA method (J Vis Exp 2019, 143). The results showed that the titer of the serum diluted 10,000 times after five immunizations was significantly higher than that of the negative serum diluted 1,000 times, reaching the level set before immunization. Determined potency requirements.

One week after the fifth immunization and one week after the sixth immunization, large-scale blood collection was carried out, and the blood collection volume was 80 mL. After a large amount of blood was collected, using the QIAamp RNA Blood Mini Kit (QIAGEN, 52304), the lymphocytes were separated according to the experimental procedures in the manual, and the RNA extraction was completed.

Example 2 Library Construction and Screening

Refer to the common method of single domain antibody discovery, construct antibody library and screen (Nat Protoc 2014, 9, 674; Nat Protoc 2007, 2, 3001) to obtain high-affinity sequences that specifically bind to antigens. To put it simply, the single-domain antibody gene is first inserted into the phage display vector, then packaged and displayed on the phage, and the corresponding screening conditions are designed according to the purpose of the experiment, and finally the single-domain antibody sequence that meets the requirements is obtained. The specific experimental method is as follows:

The lymphocyte RNA obtained in Example 1 was synthesized into cDNA according to the instructions of the PrimeScript ^TM II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A), and amplified by nested PCR using KOD-FX (TOYOBO, KFX-101) The single domain antibody (V _H H) gene was obtained and double digested with BamHI-HF (NEB, #R3136L) and XhoI (NEB, #R0146L) overnight at 37°C. At the same time, BamHI-HF (NEB, #R3136L) and XhoI (NEB, #R0146L) were used to digest the pComb3XSS-GFP vector (transformed from pComb3XSS (BioVector NTCC Inc.), and the sequence between the enzyme cutting site SfiI was replaced with the GFP gene, At the same time, BamHI and XhoI were added at both ends, so that the connection sequence was 5'-OmpA signal peptide-BamHI-GFP-XhoI-gene III-stop codon-3'). Further, the double-digested V _H H fragment and the pComb3xss-GFP vector were incubated with T4DNA Ligase (NEB, #M0202L) at 16°C and ligated overnight. After further ion purification of the ligated product, TG1 competent cells (Lucigen, 60502- 2), collect the Escherichia coli cultured overnight to complete the library construction. In the experiment, 6.8-9.2×10 ⁸ cDNA libraries with a fragment insertion rate of more than 95% and good sequence diversity were obtained for three alpacas.

The cDNA library is screened by phage display technology and liquid phase method. Taking the screening of antigen IL17A as an example, the specific steps are as follows:

The library bacteria were inoculated into 50 mL of 2×YT medium containing 100 μg/mL ampicillin and 2% glucose, after inoculation, OD ₆₀₀ =0.1, and cultured at 37° C., 220 rpm until OD ₆₀₀ =0.5. Add helper phage M13KO7 (NEB, #N0315S) at a ratio of 20:1 (phage:bacteria), mix well and let stand at 37°C for 30 minutes; centrifuge at 3000g at room temperature for 10 minutes, discard the supernatant, and add 100μg/mL to 50mL Resuspend ampicillin and 50 μg/mL kanamycin in 2×YT medium, and culture overnight at 26°C and 220 rpm.

Bacterial culture solution was centrifuged at 3000g at 4°C for 10 minutes, the supernatant was mixed with PEG/NaCl solution at a volume ratio of 4:1, then cooled on ice for 1 hour, centrifuged at 3000g at 4°C for 20 minutes, and the precipitate was collected. Resuspend in 10mL PBS, centrifuge at 3000g at 4°C for 20 minutes, collect the supernatant and add 2.5mL PEG/NaCl solution, mix well and keep on ice for 30 minutes; centrifuge at 3000g at 4°C for 30 minutes, resuspend the pellet with 1mL PBS to obtain a phage solution .

Take 500 μL of the above phage solution, first add 500 μL of 1% (w/v) BSA solution to block for 1 hour, then add 100 nM biotinylated IL17A (ARCO Biosystems, ILA-H82Q1) solution, incubate at room temperature for 1 hour, then add 50 μL of streptavidin magnetic beads (ThermoFisher scientific, 11206D) blocked by 1% BSA solution, incubate at room temperature for 30 minutes with inversion, adsorb the magnetic beads with a magnet, wash 10 times with 0.1% PBST and PBS respectively, add triethylamine (Sigma, T0886) solution, after mixing, let it stand at room temperature for 10 minutes, add an appropriate amount of pre-cooled 1M Tris-HCl (pH7.4) and mix evenly, after magnet adsorption, take the supernatant to infect 10mL TG1 strain (pre-activated, OD ₆₀₀ = 0.5), spread a plate containing 100 μg/mL ampicillin and 2% glucose, and culture overnight at 32° C. upside down. Collect all the bacteria on the plate and save the strains, and pick an appropriate amount of single clones on the small plate for serial dilution and counting for sequencing analysis.

Reduce the concentration of the antigen IL17A, repeat the above process with 10nM and 1nM respectively to complete the second and third rounds of screening, and strive to obtain high-affinity _VHH sequences.

Libraries of antigens IL17A/F and IL17F were screened in three rounds using the same method.

Example 3 Acquisition of Positive Single Domain Antibody Sequence

In order to further determine the high-affinity sequence, the V _H H gene obtained in the third round of screening was cloned into the expression vector pSJF2, and the positive clone was obtained by ELISA detection method, and the V _H H sequence of the positive clone was obtained by sequencing analysis. The experimental method is as follows:

Primers carrying BbsI-HF (NEB, #R3539L) and BamHI-HF (NEB, #R3136L) restriction sites at both ends were designed to obtain V After digestion of _H H gene, BbsI-HF and BamHI-HF, use T4 DNA ligase to connect the same digested pSJF2 vector, and transform TG1 competent cells, spread LB plate containing 100 μg/mL ampicillin, and culture at 37°C overnight.

Pick the single clone cultured overnight to a 96-well deep-well plate with 200 μL of 2×YT (100 μg/mL ampicillin) medium, culture at 37°C for 4 hours, centrifuge to discard the supernatant, and replace the induction medium (2×YT +100μg/mL ampicillin+1%Glycine+1%TritonX-100+0.5mM IPTG+5%sucrose), cultivate overnight at 27°C.

The antigen IL17A (Sino Biological, 12047-HNAS) was diluted to 1 μg/mL with carbonate buffer, 100 μL/well, added to an ELISA plate (ThermoFisher scientific, 439454), and incubated overnight at 4°C. After washing the ELISA plate three times with PBS, add 380 μL/well of 1% BSA, and block for 2 hours at 37°C; wash 3 times with PBST, add 100 μL/well of the bacterial supernatant, and incubate at 37°C for 1 hour; wash 3 times with PBST, add 1 HRP-labeled His-tag antibody (Sino Biological, 105327-MM02T-H) diluted with %BSA, incubated at 37°C for 1 hour; washed 5 times with PBST, added to TMB chromogenic solution (Sigma, T8665), 100 μL/well, kept at room temperature Light reaction for 10 minutes; stop the reaction with 2M H ₂ SO ₄ , 50 μL/well, and detect the OD ₄₅₀ value with a microplate reader. The results of the ELISA test are shown in Table 1: consistent with the 96-well deep-well plate, the first column A to H is the column number, the first row 1-12 is the horizontal row number, and the clones are numbered and identified by using the horizontal and vertical numbers as coordinates. Positioning (A1～H12), corresponding to the clones in the deep well plate and the OD ₄₅₀ detection readings; the readings of the 96 detection wells (A1～H12 except the values in the first row and the first column) are highlighted with different gray levels, The larger the reading, the darker the color, and the theoretically higher affinity (due to factors such as non-quantitative experiments, large errors in EILSA, etc., the actual affinity and ELISA detection readings cannot be one-to-one correspondence, only for reference), and the reading of negative clones is about 0.06 ( G12 and H12), clones with reads greater than 0.6 were considered positive clones in this test. The 161 positive clones were sequenced and analyzed, and the sequence results were analyzed to finally obtain the positive candidate V _H H gene sequence.

Table 1 ELISA detection readings

The clones of antigens IL17A/F and IL17F were detected using the same method.

Example 4 Characterization of Monovalent Single Domain Antibody

1) Purification of monovalent single domain antibody

According to the ELISA positive sequence diversity analysis, 70 representative sequences were selected, and after E.coli codon optimization, the gene was resynthesized and cloned into the pET28a(+) vector to construct an expression plasmid. From the N-terminal to the C-terminal of the expression sequence, there are OmpA signal peptide, V _H H, linker (amino acid sequence is GAA) and 6×His tag. The synthesized expression plasmid was transformed into BL21(DE3) competent cells, and the plate was cultured overnight at 37°C.

Inoculate 400 mL of 2×YT medium containing 50 μg/mL kanamycin at a ratio of 1:100 to 400 mL of 2×YT medium containing 50 μg/mL kanamycin after monoclonal cultivation overnight, and cultivate to OD ₆₀₀ = 0.8 at 250 rpm at 37°C, add IPTG with a final concentration of 0.5mM, and cultivate at 16°C 16 hours. Collect the bacteria by low-speed centrifugation, add 40mL of Buffer A (10mM/Tris-HCl, pH 8.0, 1mM/EDTA, 25% sucrose) to thoroughly resuspend the bacteria, shake and incubate at room temperature for 15 minutes; remove the supernatant after high-speed centrifugation, and pre-cool 20mL Buffer B (30mM Tris-HCl, pH 8.0, 5mM MgSO ₄ ) was resuspended; the supernatant was taken after high-speed centrifugation again, and an appropriate amount of NaCl solution, imidazole solution and DTT solution were added to make the final concentrations respectively 150mM, 20mM and 1mM.

Add 200 μL Ni NTA Beads 6FF (Smart lifesciences, SA005025), shake the shaker slowly for 30 minutes, transfer the mixture to the chromatography column (Bio-Rad, 7311550), collect the flow through (flow through, FT); After washing with 1×PBS containing 40 mM imidazole, the antibody was eluted with 0.5 mL of 1×PBS containing 300 mM imidazole. A in Fig. 1 is the SDS-PAGE electrophoresis image of the sample during A19 affinity chromatography. Among them, S represents the supernatant after lysing bacteria by "superosmotic method"; FT represents the combined flow-through; 40 and 80 represent the buffer after washing Ni-Resin with 1×PBS buffer containing 40mM and 80mM imidazole; E represents Samples eluted with 1×PBS buffer containing 300mM imidazole; B represents the electrophoresis results of Ni-Resin boiled samples after elution.

After concentration, the eluted samples were subjected to molecular sieve chromatography using Superdex 75 10/300, followed by BCA (Shanghai Biyuntian Biotechnology Co., Ltd., P0010) method for quantitative analysis and SDS-PAGE analysis. B in Figure 1 is the Superdex 75 10/300 molecular sieve chromatogram of A19, and C in Figure 1 is the SDS-PAGE electrophoresis of the finished protein product after A19 molecular sieve chromatography. It can be seen that a high-purity _VHH sample was obtained after two steps of purification.

2) V _H H affinity test

The affinity test of the monovalent single-domain antibody was completed using Biacore T200, and the His-tag monoclonal antibody was bound to the CM5 chip (GE Healthcare, BR- 1005-30), used to capture monovalent V _H H antibody with His tag, human IL17A, IL17A/F and IL17F were used as mobile phase for affinity detection.

Figure 2 is the binding and dissociation curves and fitting diagrams of antibody A19 and IL17A, and the affinity value between V _H H antibody and antigen was obtained through calculation and analysis. Among the 70 _VHH antibodies included in the characterization, 29 _VHH antibodies with better expression and purification and higher affinity were obtained. The affinity data are shown in Table 3, where "×" means no binding.

Table 2 Affinity test of monovalent V _H H antibody and human IL17A, IL17A/F and IL17F

3) V _H H thermal stability analysis

The thermal stability of candidate _VHH antibody molecules was initially screened by differential scanning fluorimetry (DSF). During the screening process, using SYPRO Orange Protein Gel Stain (Sigma, S5692) and gradually heating with a qPCR instrument, a preliminary test of the _Tm value of the purified _VHH antibody was performed. In the initial screening, the T _m value of _VHH antibody is ≥ 55°C as the screening condition, and the _VHH antibodies listed in Table 2 all meet this requirement. Fig. 3 is a test chart of antibody AF6 using DSF to test the T _m value, and its T _m value reaches 74°C.

4) V _H H specificity analysis

As shown in the affinity test results in Table 2, the ability of V _H H antibody to bind one or more antigens in IL17A, IL17A/F or/and IL17F with high affinity, in order to further evaluate the ability of specific binding of IL17 family proteins, follow the steps 2) In the test method, the affinity between the antibody and human IL17B (Prospec, CYT-753), IL17C (Acro Biosystems, ILC-H52H7), IL17D (Atagenix, ATAP01505) and IL17E (Acro Biosystems, IL5-H4221) was detected . As shown in Table 2, the 29 candidate _VHH antibody molecules can all bind IL17A/F with strong affinity, and IL17A/F is used as a positive control molecule to test the candidate _VHH antibody and IL17B, IL17C, IL17D and IL17E specific binding capacity. The test results showed that the binding Ru values of 29 candidate V _H H antibody molecules to IL17A/F ranged from 30 to 120, while most of the binding Ru values to IL17B, IL17C, IL17D and IL17E were ≤10, far from The absolute value of Ru is lower than that of IL17A/F. Therefore, these antibodies have no obvious binding to IL17B, IL17C, IL17D and IL17E, and the specificity meets the requirements.

5) Affinity test between V _H H and Cynomolgus monkey (Cynomolgus monkey) IL17A, IL17F

In order to further detect the binding activity of the above-mentioned _VHH , and to prepare for the subsequent animal experiments of antibody drugs in cynomolgus monkeys, it is necessary to detect the affinity between _VHH and cynomolgus monkey IL17A and IL17F, and the detection method is the same as step 2), V _H H was coupled to a CM5 chip through a His tag as immobilization, and IL17A and IL17F molecules of cynomolgus monkey were used as mobile phase for detection. The detection results are shown in Table 3, where × represents no binding. The results of Biacore test showed that except for A29 which was not detected, all the other antibodies had certain binding ability to IL17A and IL17F in cynomolgus monkeys. Secondly, comparing the test results in Table 1 and Table 3, there is no order of magnitude difference between the binding affinity of most candidate V _H H antibodies to human IL17A or IL17F, and the binding affinity to cynomolgus monkey IL17A or IL17F.

Table 3 Affinity between V _H H and Cynomolgus monkey IL17A and IL17F

6) Epitope binning analysis of V _H H

The present invention uses SPR technology to perform cluster analysis on the above V _H H according to the difference of Epitope, and the method is as follows. Optimize and synthesize the expressed gene of E.Coli codon preference, so that the sequence of the expression frame is OmpA signal peptide, V _H H, SASA (from US 2013/0129727 A1, which can be combined with BSA), and His tag; the synthesized gene follows 1) Expression and purification of SASA-V _H H was performed using the monovalent single domain antibody purification method described in . During the detection, BSA is first coupled to the CM5 chip, then SASA-V _H H (V _H H ₁ ) is combined with BSA through the flow path to be immobilized on the chip, and then IL17A/F is combined with V _H H ₁ through the flow path to form BSA-SASA-V _H H (V _H H ₁ )-IL17A/F complex, and finally the V _H H (V _H H ₂ ) antibody passed through the flow path as the mobile phase, and the response value Ru was recorded at this time. Judging whether two antibodies have the same epitope according to the change of Ru value, if V _H H ₁ and V _H H ₂ have different epitopes, then V _H H ₂ can be compared with BSA-SASA-V _H H ( V _H H ₁ )-IL17A/F complex binding, the Ru value will be higher; otherwise, it will be low.

Table 4 shows the results obtained by using this method. It can be seen that some antibodies have different epitopes, such as combinations of AF5 and A8, AF10 and A5, etc., where Ru is positive and has a large value; some antibodies have the same epitope, Such as the combination of A8 and A19, AF2 and A21 with Ru around 0.

Table 4 Epitope binning detection results

Antibody number AF2 AF5 AF10 A10 A19 A21 A29 A46 SASA-AF3 -10.2 138.8 -15.0 36.0 50.5 35.4 48.4 100.1 SASA-AF6 35.3 90.3 107.0 101.1 140.9 77.1 100.5 122.0 SASA-AF10 -5.3 256.3 -7.0 107.3 138.7 106.3 170.3 233.1 SASA-A5 42.4 94.0 108.2 -15.5 -2.2 -6.6 -4.3 -99.4 SASA-A8 18.7 31.2 57.2 42.2 3.3 56.1 62.5 -14.3 SASA-A10 -18.7 32.5 12.7 -37.5 46.6 -40.1 -28.6 17.7 SASA-A21 0.8 31.7 30.6 -8.3 -4.1 -2.2 8.0 -19.9 SASA-A23 -1.6 -7.5 16.7 -28.8 -29.7 -19.2 -32.6 -58.6 SASA-A29 -16.2 -124.7 31.5 -63.4 114.8 -64.8 -30.3 -47.8 SASA-A33 31.7 41.1 60.4 37.8 29.9 24.2 24.9 17.7 SASA-A34 55.7 87.4 79.9 75.2 118.9 68.1 87.1 87.0 SASA-A36 8.2 40.8 62.2 -42.1 -10.8 -34.7 -69.2 -24.4 SASA-A45 16.7 70.3 90.9 -19.0 24.6 -23.5 -18.3 70.8 SASA-A46 22.9 98.7 94.2 -5.8 -19.0 -10.9 -15.6 89.0 SASA-A59 -9.5 189.5 -10.2 75.1 104.2 77.8 135.8 191.2 SASA-A53 -28.9 21.2 31.3 -40.6 31.5 0.9 33.5 74.3 SASA-A39 -36.2 -61.2 -18.4 -61.6 -45.7 -67.8 -23.5 58.2

7) Identification of Epitope/Paratope binding sites by H/D exchange method

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) epitope mapping is an effective method that can quickly provide complete information on epitope structure. The principle is that the hydrogen on the protein amide bond can exchange controllably with the deuterium in heavy water. When the protein is immersed in heavy water, the hydrogen on the surface of the protein is in close contact with the heavy water, and the exchange rate is fast; while the hydrogen inside the protein and the hydrogen that forms the hydrogen bond are difficult to contact. Heavy water, slow exchange rate. Using mass spectrometry to measure the number of deuterated peptide sequences under different reaction times, and then calculate the hydrogen-deuterium exchange rate of each peptide sequence, the spatial structure information of the protein can be judged. The technique uses mass spectrometry to measure the solvent accessibility of amino acid residues in proteins to determine the interaction sites of antigen-antibody complexes in their native solution without introducing any modification to the antigen or antibody.

The experimental steps are as follows: Dissolve 5-10μM antigen, antibody, and antigen-antibody complex in 50mM HEPES, pH 7.4, 150mM NaCl, 4mM TCEP solution at a molar ratio of 1:1, and place them at 4°C for 1 hour to ensure Form a stable antigen-antibody complex. After standing for 1 hour, at 4°C, dilute 5 μL of different samples into 20 μL _D2O (deuterium) respectively, and place them at different HDX time points (for example, 0, 10, 60, 300, 900 seconds), by cooling with 25 μL 100mM NaH ₂ PO ₄ mixed with 1M TCEP to stop the reaction. Place the sample tube on dry ice immediately after stopping the reaction until the sample is injected into the HDX LEAP PAL3.0 platform. After injection into the fully automatic hydrogen-deuterium exchange platform, the sample passed through the immobilized pepsin column at a flow rate of 120 μL/min, and the enzymatically digested peptides were captured and desalted by a 2.1 mm×5 cm C18 column (1.9 μm Hypersil Gold, Thermo Fisher). The desalted peptides were separated with a linear gradient of 4-40% acetonitrile and 0.3% formic acid over 8 minutes. During sample processing, protein digestion and peptide separation were performed at 4°C.

The eluted and separated peptides were analyzed using an Orbitrap mass spectrometer (Orbitrap Fusion ^TM Tribrid ^TM Mass Spectrometer, Thermo Fisher), with a measurement resolution of 65,000 (m/z 400), and hydrogen-deuterium exchanged mass spectrometry data were obtained. Each sample has three HDX assays (triplicates) at each time point. The average m/z centroid value of the mass spectrum peak intensity of each enzymatic peptide was calculated by HDX Workbench software (generally, the accuracy is 10ppm), and then converted into the percentage of deuterium incorporation. The key amino acid sequences involved in the spatial epitope are calculated, and the difference in Delta%D (comparing the change in the percentage of deuterium incorporation on the same peptide) is determined by calculating the difference between the two samples. Differences in Delta %D outside -5 to 5% were considered significant. In addition, HDX Workbench detects statistically significant (p<0.05) differences between samples at each time point by student's t test.

The present invention uses the above method to identify the binding site of Epitope/Paratope. Taking the two V _H Hs of A8 and AF5 as an example, according to the specific interaction mode, a total of five interactions, AF5-IL17A, AF5-IL17A/F, AF5-IL17F, A8-IL17A and A8-IL17A/F, were carried out. Experiment with the /D swap. Figure 4 is the Paratope analysis of the interaction between A8 and IL17A. The results show that SGSIFNAHAMGW and CNADHTYYSDSAL are potential Paratopes, mainly focusing on CDR1 and CDR3. AF5 has strong binding affinity with IL17A, IL17A/F and IL17F, but the binding mode of AF5 is not exactly the same according to the Paratope involved in the binding. The collated detection results are shown in Table 5. A8 mainly interacts with IL17A and IL17A/F through CDR1 and CDR3. AF5 mainly binds to IL17A through partial sequences of CDR2 and CDR3; interacts with IL17A/F through partial amino acids of CDR3 and Framework-4; binds to IL17F through CDR2.

Table 5 Paratope sequences of AF5, A8 and IL17A, IL17A/F and IL17F

After obtaining the Paratope information of the antibody-antigen interaction, the Epitope information on IL17A, IL17A/F and IL17F was further analyzed, and the results are shown in Table 6.

Table 6 Epitope sequences of AF5, A8 and IL17A, IL17A/F and IL17F

8) Candidate V _H H sequences

After the characterization of the above multiple steps, 29 single-domain antibodies with high affinity, good specificity, and good thermal stability were obtained, and their amino acid sequences are shown in Table 7 (the bold parts in each sequence are CDR1, CDR2 from front to back) and CDR3).

Table 7 Candidate V _H H Amino Acid Sequences

Example 5 Construction, Characterization and Functional Verification of Trivalent Single Domain Antibody

1) Construction of trivalent single domain antibody

After characterization and analysis of monovalent single-domain antibodies, the present invention has obtained 29 high-affinity _VHH sequences with good specificity and good thermal stability, and these antibodies can be used to combine to construct multivalent antibodies to achieve simultaneous high-affinity binding to IL17A , IL17A/F and IL17F purposes. As shown in Table 1, in view of the higher affinity between A series V _H H and IL17A, and the higher affinity between AF series V _H H and IL17A/F and IL17F, when combining AF series, A series One for each _VHH , combined by Anti-HSA ( _VHH , from US 2007/0269422 A1, ALB11). Considering the influence of steric hindrance on the interaction between V _H H and IL17A, IL17A/F and IL17F, Anti-HSA was placed in the middle, and V _H H of AF series and A series were placed at both ends respectively, and the V _H H positions were constructed Two reversed combinations: the first one is the V _H H of the AF series at the N terminal and the V _H H of the A series is at the C terminal, the second is the V _H H of the A series at the N terminal and the V _H H of the AF series at the C end. V _H H is connected to anti-HSA through amino acid Linker (GGGGSGGGS) (as shown in Figure 8) to construct different trivalent antibodies.

Anti-HSA (US 2007/0269422 A1, ALB11):

Taking the four V _H Hs of AF5, A8, A19 and A43 as an example, six different trivalent antibodies were constructed, among which PAF022 was composed of AF5, Anti-HSA and A8 in sequence, and PAF025 was composed of AF5, Anti-HSA and A19 in sequence PAF041 is composed of AF5, Anti-HSA and A43, PA002 is composed of A8, Anti-HSA and AF5, PA026 is composed of A19, Anti-HSA and AF5, and PA154 is composed of A43, Anti-HSA and AF5 is sequentially connected to form. Amino acid sequences of 6 trivalent single domain antibodies (see Table 8), OmpA signal peptide and stop codon were added, codons were optimized and the gene was synthesized and cloned into pET28a(+).

Table 8 Amino acid sequences of 6 trivalent single domain antibodies

2) Preparation of trivalent single domain antibody

The expression method of the trivalent single-domain antibody is the same as that of the monovalent single-domain antibody. After two steps of purification, a high-purity trivalent single-domain antibody can be obtained. The purification method is as follows: After expressing the trivalent single-domain antibody, add an appropriate amount of pre-cooled Lysis buffer (1×PBS, 1mM DTT, 1mM EDTA), ultrasonicate (400W, 3s on/3s off, 15min) and then centrifuge at a high speed , load the supernatant to the pretreated HiTrap ^TM MabSelect SuRe Column (Cytiva, 11003493), equilibrate with 1×PBS until the A280 baseline goes flat to zero, wash with 100mM glycine-hydrochloric acid (PH3.0) at a flow rate of 1mL/min Remove and collect the target peak; after adjusting the pH of the eluted protein to 10.0, pass through the Hitrap Q column (Cytiva, 17115401) to collect the target protein, concentrate in a 10K ultrafiltration tube, change the buffer to 1×PBS, and quantify using the BCA method. A in Figure 5 is the protein A affinity chromatography purification diagram of the antibody PAF022, B in Figure 5 is the purification diagram of the Hitrap Q column, and C in Figure 5 is the SDS-PAGE electrophoresis diagram of the Hitrap Q column after purification.

3) Trivalent single domain antibody affinity test

Different from the affinity test of monovalent single-domain antibodies, when testing the affinity of trivalent single-domain antibodies with human IL17A, IL17A/F and IL17F, the Anti-His in the His tag capture kit was first coupled to the CM5 chip for Capture human IL17A (Acro Biosystems, ILA-H82Q1), IL17A/F (Acro Biosystems, ILF-H52W6) and IL17F (Sino Biological, 11855-H07H) with His-tagged, trivalent single domain antibody as mobile phase for testing . The test results are shown in Table 9.

Table 9 Trivalent antibody affinity test

4) Thermal stability of trivalent single domain antibody

In order to detect the thermal stability of the prepared trivalent single domain antibody, the present invention uses a differential scanning calorimeter (TA Instruments, Discovery DSC2500/250/25) to test the prepared control antibody MS3091 (US 2014/0314743 A1), A total of seven trivalent antibodies, PAF022, PAF025, PAF041, PA002, PA026 and PA154, were tested for thermal stability. The specific method is to take 20 μL of the sample to be tested (2mg/mL) and add it to the sample plate, seal it, put it into the instrument, heat it from 40°C to 95°C at a speed of 1°C/min, and record the heat flow between the sample plate and the reference plate Variety. The T _m of PA022 is 76.7°C, and Figure 6 shows the heat flow curve of PA022. The T _m value of the positive control is 82.0°C, PAF025 and PAF041 are 79.4°C and 78.6°C, respectively, and PA002 is 76.0°C, all of which are greater than 65°C, showing good thermal stability.

5) Cell function evaluation of trivalent single domain antibody

IL17 can stimulate epidermal cells (Hs27 cell line, human epidermal fibroblasts, ATCC#CRL-1634) to secrete GROα. If IL17 antibody is added, it will block the interaction between IL17 and cell membrane surface receptors by binding to IL17 protein, thereby achieving Inhibiting the effect of epidermal cells secreting GROα, by detecting the amount of GROα secretion, the IC50 value of the candidate antibody can be obtained.

The present invention has tested the activity of trivalent antibodies, and some results are shown in Figure 7 and Table 10. The maximum inhibitory rates of the six tested trivalent antibodies to IL17A were all greater than 83%; the IC50 of antibodies PAF022, PAF025 and PAF041 were all In the vicinity of 0.03nM, compared with the IC50 value of MS3091 of 0.04nM, the activity was comparable, and the IC50 of antibodies PA002, PA026 and PA154 were 10-20 times lower than MS3091. The six trivalent antibodies tested had a maximum inhibitory rate of more than 90% on IL17A/F, and their IC50 were all between 0.6-2.7nM, which was only 3-5 times different from MS3091.

Table 10 Trivalent Candidate Antibodies Neutralizing Ability to IL17A and IL17A/F

Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (25)

An IL17 antibody comprising a heavy chain variable region, characterized in that the heavy chain variable region comprises a sequence such as CDR1 shown in any one of SEQ ID NO: 1-24, a sequence such as SEQ ID NO: 25 The CDR2 shown in any one of ~47, and the CDR3 whose sequence is shown in any one of SEQ ID NO:48~71. IL17 antibody as claimed in claim 1, is characterized in that, (1) its CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises the sequence shown in SEQ ID NO:25, and CDR3 comprises the sequence shown in SEQ ID NO:48; Or, (2) its CDR1 comprises the sequence shown in SEQ ID NO:2, CDR2 comprises the sequence shown in SEQ ID NO:26, and CDR3 comprises the sequence shown in SEQ ID NO:49; Or, (3) its CDR1 comprises the sequence shown in SEQ ID NO:3, CDR2 comprises the sequence shown in SEQ ID NO:27, and CDR3 comprises the sequence shown in SEQ ID NO:50; Or, (4) Its CDR1 comprises the sequence shown in SEQ ID NO:4, CDR2 comprises the sequence shown in SEQ ID NO:28, and CDR3 comprises the sequence shown in SEQ ID NO:51; Or, (5) its CDR1 comprises the sequence shown in SEQ ID NO:5, CDR2 comprises the sequence shown in SEQ ID NO:29, and CDR3 comprises the sequence shown in SEQ ID NO:52; or, (6) Its CDR1 comprises the sequence shown in SEQ ID NO:6, CDR2 comprises the sequence shown in SEQ ID NO:28, and CDR3 comprises the sequence shown in SEQ ID NO:53; Or, (7) Its CDR1 comprises the sequence shown in SEQ ID NO:7, CDR2 comprises the sequence shown in SEQ ID NO:30, and CDR3 comprises the sequence shown in SEQ ID NO:54; Or, (8) Its CDR1 comprises the sequence shown in SEQ ID NO:8, CDR2 comprises the sequence shown in SEQ ID NO:31, and CDR3 comprises the sequence shown in SEQ ID NO:55; Or, (9) Its CDR1 comprises the sequence shown in SEQ ID NO:9, CDR2 comprises the sequence shown in SEQ ID NO:32, and CDR3 comprises the sequence shown in SEQ ID NO:56; Or, (10) its CDR1 comprises the sequence shown in SEQ ID NO:10, CDR2 comprises the sequence shown in SEQ ID NO:33, and CDR3 comprises the sequence shown in SEQ ID NO:55; Or, (11) its CDR1 comprises the sequence shown in SEQ ID NO:11, CDR2 comprises the sequence shown in SEQ ID NO:34, and CDR3 comprises the sequence shown in SEQ ID NO:57; Or, (12) Its CDR1 comprises the sequence shown in SEQ ID NO:12, CDR2 comprises the sequence shown in SEQ ID NO:35, and CDR3 comprises the sequence shown in SEQ ID NO:58; Or, (13) its CDR1 comprises the sequence shown in SEQ ID NO:13, CDR2 comprises the sequence shown in SEQ ID NO:36, and CDR3 comprises the sequence shown in SEQ ID NO:59; Or, (14) Its CDR1 comprises the sequence shown in SEQ ID NO:14, CDR2 comprises the sequence shown in SEQ ID NO:34, and CDR3 comprises the sequence shown in SEQ ID NO:60; Or, (15) Its CDR1 comprises the sequence shown in SEQ ID NO:15, CDR2 comprises the sequence shown in SEQ ID NO:37, and CDR3 comprises the sequence shown in SEQ ID NO:61; Or, (16) Its CDR1 comprises the sequence shown in SEQ ID NO:9, CDR2 comprises the sequence shown in SEQ ID NO:38, and CDR3 comprises the sequence shown in SEQ ID NO:62; Or, (17) Its CDR1 comprises the sequence shown in SEQ ID NO:14, CDR2 comprises the sequence shown in SEQ ID NO:34, and CDR3 comprises the sequence shown in SEQ ID NO:60; Or, (18) Its CDR1 comprises the sequence shown in SEQ ID NO:15, CDR2 comprises the sequence shown in SEQ ID NO:37, and CDR3 comprises the sequence shown in SEQ ID NO:63; Or, (19) Its CDR1 comprises the sequence shown in SEQ ID NO:16, CDR2 comprises the sequence shown in SEQ ID NO:39, and CDR3 comprises the sequence shown in SEQ ID NO:64; Or, (20) Its CDR1 comprises the sequence shown in SEQ ID NO:17, CDR2 comprises the sequence shown in SEQ ID NO:40, and CDR3 comprises the sequence shown in SEQ ID NO:65; Or, (21) Its CDR1 comprises the sequence shown in SEQ ID NO:18, CDR2 comprises the sequence shown in SEQ ID NO:41, and CDR3 comprises the sequence shown in SEQ ID NO:66; Or, (22) Its CDR1 comprises the sequence shown in SEQ ID NO:19, CDR2 comprises the sequence shown in SEQ ID NO:42, and CDR3 comprises the sequence shown in SEQ ID NO:67; Or, (23) Its CDR1 comprises the sequence shown in SEQ ID NO:20, CDR2 comprises the sequence shown in SEQ ID NO:43, and CDR3 comprises the sequence shown in SEQ ID NO:67; Or, (24) Its CDR1 comprises the sequence shown in SEQ ID NO:21, CDR2 comprises the sequence shown in SEQ ID NO:44, and CDR3 comprises the sequence shown in SEQ ID NO:68; Or, (25) Its CDR1 comprises the sequence shown in SEQ ID NO:22, CDR2 comprises the sequence shown in SEQ ID NO:45, and CDR3 comprises the sequence shown in SEQ ID NO:69; Or, (26) Its CDR1 comprises the sequence shown in SEQ ID NO:23, CDR2 comprises the sequence shown in SEQ ID NO:46, and CDR3 comprises the sequence shown in SEQ ID NO:70; Or, (27) Its CDR1 comprises the sequence shown in SEQ ID NO:24, CDR2 comprises the sequence shown in SEQ ID NO:47, and CDR3 comprises the sequence shown in SEQ ID NO:71. The IL17 antibody according to claim 2, wherein the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 72-100. The IL17 antibody according to any one of claims 1 to 3, which is a single domain antibody or a _VHH fragment thereof. A trivalent single-domain antibody, characterized in that it comprises the IL17 antibody according to any one of claims 1-4. The trivalent single-domain antibody according to claim 5, wherein the trivalent single-domain antibody contains three _VHHs sequentially connected from the N _- terminus to the C-terminus: _{VHH1 - VHH2-} V _H H ₃ . The trivalent single domain antibody according to claim 6, wherein _{the VHH2} is an anti-HSA _VHH ; preferably containing the sequence shown in SEQ ID NO:108. The trivalent single domain antibody according to claim 7, wherein _{the VHH1} contains the CDR combination as defined in any one of (1) to (6) in claim 2, and the _VH H ₃ contains the CDR combination as defined in any one of (7)-(22) in claim 2; preferably, the V _H H ₁ contains the CDR combination shown in any one of SEQ ID NO:72-79 sequence, and _{the VHH3} contains a sequence as shown in any one of SEQ ID NO: 80-100; Or _{the VHH3} contains the CDR combination as defined in any one of (1)-(6) in claim 2, and _{the VHH1} contains the CDR combination as defined in (7)-(22) in claim 2 Any one of the CDR combinations defined; preferably, the _VHH3 contains the sequence shown in any one of SEQ ID NO:72-79, and _{the VHH1} contains the sequence shown in SEQ _ID NO:80 The sequence shown in any one of ~100. The trivalent single domain antibody according _to claim 8, wherein the _VHH1 or _VHH3 contains the CDR combination as defined in claim 2 (2) _, and the _{VHH3 or} V _H H ₁ contains the CDR combination as defined in claim 2 (7), (10) or (26); preferably, _{The VHH1} contains the sequence shown in SEQ ID NO:73, such as _{the VHH3} contains the sequence shown in SEQ ID NO:80, 83 or 99; Alternatively, _{the VHH1} contains the sequence shown in SEQ ID NO:80, 83 or 99, and _{the VHH3} contains the sequence shown in SEQ ID NO:73. The trivalent single-domain antibody according to any one of claims 5 to 9, wherein different V _H Hs are operably connected through a linker; the linker preferably contains the sequence. The trivalent single domain antibody according to claim 10, wherein the trivalent single domain antibody contains the amino acid sequence shown in any one of SEQ ID NO: 101-106. An isolated nucleic acid encoding the IL17 antibody according to any one of claims 1-4 or the trivalent single domain antibody according to any one of claims 5-11. An expression vector comprising the isolated nucleic acid of claim 12. A host cell comprising the expression vector according to claim 13; preferably, the host cell is a prokaryotic cell or a eukaryotic cell. A method for preparing an IL17 antibody or a trivalent single-domain antibody, comprising culturing the host cell according to claim 14, and obtaining the antibody from the culture. A pharmaceutical composition comprising the IL17 antibody according to any one of claims 1-4 or the trivalent single-domain antibody according to any one of claims 5-11. The IL17 antibody according to any one of claims 1 to 4, the trivalent single domain antibody according to any one of claims 5 to 11, or the pharmaceutical composition according to claim 16 in the preparation and prevention of IL17-related diseases or the application of medicines for diseases; The disease or condition preferably includes chronic inflammation of the lung, rheumatoid arthritis, intestinal inflammation, dry eye disease, psoriasis, ankylosing spondylitis and various cancers including skin cancer, more preferably psoriasis, ankylosing spondylitis, chronic inflammation of the lungs and rheumatoid arthritis; psoriasis, ankylosing spondylitis and rheumatoid arthritis are further preferred. A chimeric antigen receptor comprising the IL17 antibody according to any one of claims 1-4 or the trivalent single-domain antibody according to any one of claims 5-11. An antibody-drug conjugate comprising a cytotoxic agent, and the IL17 antibody according to any one of claims 1-4 or the trivalent single-domain antibody according to any one of claims 5-11; preferably , the cytotoxic agent is MMAF or MMAE. A kit comprising the IL17 antibody according to any one of claims 1 to 4, the trivalent single domain antibody according to any one of claims 5 to 11, and the chimeric antigen receptor according to claim 18 . The antibody drug conjugate as claimed in claim 19 and/or the pharmaceutical composition as claimed in claim 16; Preferably, the kit further comprises (i) a device for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use. A kind of set medicine box, it comprises medicine box A and medicine box B, wherein: The kit A contains the IL17 antibody according to any one of claims 1-4, the trivalent single-domain antibody according to any one of claims 5-11, the chimeric antigen receptor according to claim 18, body, the antibody drug conjugate as claimed in claim 19 and/or the pharmaceutical composition as claimed in claim 16; The kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines. A method for diagnosing, treating and/or preventing IL17-mediated diseases or disorders, the method comprising administering a therapeutically effective amount of the IL17 antibody according to any one of claims 1 to 4, or The trivalent single domain antibody according to any one of claims 5-11, the chimeric antigen receptor according to claim 18, the antibody drug conjugate according to claim 19 or the drug according to claim 16 Composition, or use the kit as claimed in claim 21 to treat patients in need. The method of claim 22, wherein the disease or condition is as defined in claim 17. A method for immunologically detecting or measuring IL17, comprising using the IL17 antibody according to any one of claims 1-4, the trivalent single-domain antibody according to any one of claims 5-11, or the trivalent single-domain antibody according to claim 18 The chimeric antigen receptor, the antibody drug conjugate as claimed in claim 19 or the pharmaceutical composition as claimed in claim 16 is detected after incubation with the analyte; preferably, the detection or the determination For non-diagnostic and/or therapeutic purposes. A combination therapy, which comprises administering the IL17 antibody according to any one of claims 1 to 4, the trivalent single domain antibody according to any one of claims 5 to 11, and the The chimeric antigen receptor as described in 18, the antibody drug conjugate as claimed in claim 19 or the pharmaceutical composition as claimed in claim 16, and a second therapeutic agent; the second therapeutic agent preferably comprises Other anti-tumor antibodies or pharmaceutical compositions containing said other anti-tumor antibodies, and/or induced by hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic One or more of the group consisting of agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

Images