CN117642423A - IL17 antibody and preparation method and application thereof - Google Patents

IL17 antibody and preparation method and application thereof Download PDF

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CN117642423A
CN117642423A CN202180100166.9A CN202180100166A CN117642423A CN 117642423 A CN117642423 A CN 117642423A CN 202180100166 A CN202180100166 A CN 202180100166A CN 117642423 A CN117642423 A CN 117642423A
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栗红建
陈亮
张继帅
王毅之
张一卓
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Shenzhen Huapu Pharmaceutical Research And Development Co ltd
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

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Abstract

The invention provides an IL17 antibody, a preparation method and application thereof, and belongs to the technical field of biological medicines. The IL17 antibody comprises a heavy chain variable region comprising a sequence as set forth in SEQ ID NO:1-4, the CDR1, sequence as set forth in any one of SEQ ID NOs: 5-8, and the sequence of which is set forth in SEQ ID NO: CDR3 as set forth in any one of claims 9-12. The IL17 antibody obtained by the invention can selectively bind one or more of human cytokines IL17A, IL F and IL17A/F with high affinity, and can specifically bind the cynomolgus monkey IL17A and IL17F with high affinity.

Description

IL17 antibody and preparation method and application thereof Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an IL17 antibody, a preparation method and application thereof.
Background
The cytokine IL17 can be derived from a variety of different immune cells, with Th17 helper T cell secretion as the primary source, CD8 + T cells, NK T cells, LLC3 (Type 3 innate lymphoid cells), γδ T cells, etc. can also secrete IL17 (Nat Rev immunol.2010,10,479). The cytokine IL17 protein family mainly comprises IL17A, IL17B, IL17C, IL17D, IL E (IL 25) and IL17F, wherein IL17A and IL17F have great sequence similarity and can be simultaneously expressed in Th17 cells. IL17A and IL17F may exist as homodimers or as IL17A/F heterodimers composed of IL17A and IL17F monomers (Cold Spring Harb Perspect biol.2018,10, a 028522). IL17 receptor proteins exist in a number of subunits, including five receptor subunits, IL17RA, IL17RB, IL17RC, IL17RD, and IL17RE, where IL17RA often serves as a common core subunit of the IL17 receptor, involved in binding of different IL17 molecules, such as IL17A, IL F and IL17A/F to the receptor complex composed of IL17RA/IL17RC, IL17C to the receptor complex composed of IL17RA/IL17RE, and IL17E to the receptor complex composed of IL17RA/IL17RB (Cytokine 2013,64,477;Trends Immunol.2017,38,310).
IL17RA is expressed in both hematopoietic and non-hematopoietic stem cells, but IL17RC is commonly expressed in non-hematopoietic stem cells, so dimers such as IL17A, IL F and IL17A/F activate non-hematopoietic stem cells, such as epithelial and stromal cells, by binding to the IL17RA/IL17RC receptor complex. After the cells are activated, a series of pro-inflammatory factors such as IL-1, IL-6, G-CSF, TNF and the like are produced, and the expression of chemokines such as CXCL1, CXCL2, CXCL5, CCL2, CCL7, CCL20, IL-8 and the like is induced, and the expression of matrix metalloproteinases MMP1, MMP3, MMP9, and MMP13 is promoted, and antibacterial peptides such as beta-deflectons, S-100 proteins and the like (Immunity 2011,34,149) are produced. Cytokine IL17 plays an important role in combating external bacterial, fungal infections, maintaining mucosal and epidermal integrity, promoting wound healing (Immunol rev.2008,226, 57). Conversely, once the activity of IL17 is not normally regulated, chronic inflammatory and autoimmune diseases may be triggered, possibly even inducing tumor formation.
Up-regulation of the expression levels of IL17A and related cytokines can trigger a range of autoimmune diseases including psoriasis (psoriasis), rheumatoid arthritis (rheumatoid arthritis), scleroderma (scleroderma), lupus (lupus), and the like. In the process of psoriasis formation, a DC cell (TIP-DC) capable of secreting TNF-alpha/iNOS plays an important role, the TIP-DC cell is activated by binding to TNF-alpha, the activated TIP-DC cell further secretes cytokines such as TNF-alpha, IL23, IL12 and the like, IL23 further activates Th17 cells, and the Th17 cells are promoted to secrete excessive cytokines such as IL17A, IL F, IL22 and the like, and the cytokines further drive keratinocytes to abnormally differentiate and proliferate, thereby forming psoriasis (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 efficacy in the treatment of psoriasis and related disorders. In addition, an increase in serum concentration of IL17A, IL F is positively correlated with the severity of atopic dermatitis (atopic dermatitis) in infants. Recently, there has also been a study showing that IL17A is also a potential therapeutic target for Sepsis (sepis) (Front immunol 2020,11,1558). In the field of neurological diseases, in addition to multiple sclerosis (multiple sclerosis), dysregulation of IL17 is also considered a causative factor for chronic neuritis, experimental autoimmune encephalomyelitis, alzheimer's disease and ischemic brain injury (Front immunol 2020,11,947).
The Th17 cell subset is involved in regulating inflammation of neutrophils, macrophages in the lung, and has been associated with severe neutrophilic asthma and chronic obstructive pulmonary disease (Nat.Rev.Immunol.2008, 8,183;Ann.Rev.Physiol.2010,72,495). Studies have shown that the expression level of IL17A, IL F correlates with the severity of asthma, as IL17 is effective in promoting expression of neutrophils growth factors, chemokines, such as IL-6, G-CSF, IL8, etc., thereby promoting neutrophil enrichment (Expert rev. Respir. Med.2014,8, 25). In patients with chronic obstructive pulmonary disease, bronchial asthma, and both chronic pulmonary obstructive pulmonary disease and asthma, the concentration of IL17A in serum increased and the extent of the increase in concentration correlated positively with the severity of the disease (Mediators inflamm 2020, 4652898). By studying biomarkers in a population of chronic obstructive pulmonary disease subtype, a group of populations insensitive to hormonal therapeutic response was found to be associated with IL 17-induced inflammation, meaning antibodies that bind IL17, and have clinical value in the treatment of such a population of chronic obstructive pulmonary disease subtype (J Clin invest.2019,129, 169).
In addition to autoimmune diseases, there is increasing evidence that IL17 dysregulation has a promoting effect on the early onset, middle and late progression of cancer. Tumor patients with higher IL17 concentrations were found to have poorer prognosis by testing the IL17 concentration in serum of a series of different solid tumor patients (Oncoimmunology 2015,4, e 984547). IL17 acts as a class of pro-inflammatory factors that play a role in some connection during inflammatory response, wound healing and tumor formation (J.Exp. Med.2019,217, e20190297; nat. Immunol.2019,20,1594). IL17 has been shown to promote the onset of a variety of 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 (Cancer 2018,155,210), liver Cancer (dig. Dis. Sci.2016,61,474), lung Cancer (Cell 2019,176,998), and myeloma (nat. Commun.2018,9,4832), among others. In summary, IL17 induces cytokines, chemokines to promote myeloid suppressors, thereby further promoting angiogenesis (Nat.Med.2013, 19,1114;PNAS 2014,111,5664), or enhancing the anti-tumor suppressive effect of tumor microenvironment, promoting tumor formation and development (J immunol.2010,184,2281; nature,2015,522,345;Adv Exp Med Biol.2020,1240,47). There is also evidence that activation of IL17 signals contributes to the development of tumor resistance to radiotherapy and chemotherapy, thereby reducing the therapeutic effect (J Exp med 2020,217, e 20190297). Based on the above research, the anti-IL 17 antibody has a great application prospect for anti-tumor treatment.
Disclosure of Invention
Psoriasis is an autoimmune disease with high incidence rate, the incidence rate of Europe and America is 1% -3%, the incidence rate of China is relatively low, and more than 600 ten thousand patients are estimated. For patients with moderate to severe psoriasis and/or psoriatic arthritis, who are not or poorly tolerated by conventional therapies, treatment may be by injection of biological agents; at present, three monoclonal antibody medicines (Cosentyx/Secukinumab, taltz/Ixekizumab and Netakimab) targeted to the IL17A target for treating psoriasis are approved in sequence for treating indications such as psoriasis, ankylosing spondylitis and the like, and the effect is well accepted. A new generation of multivalent monoclonal antibody drugs targeting IL17A, IL F, such as Bimekizumab, belgium better than company, showed good therapeutic effects on psoriasis in head-to-head clinical trials. The multivalent single domain antibodies Sonelokimab targeting IL17A, IL F and IL17A/F, in clinical phase IIb multi-center trials, a 120mg or less dose of Sonelokimab showed significant clinical benefit over placebo, with the characteristics of fast onset, durability, and acceptable safety (Lancet, 2021,397,1564). Meanwhile, multivalent antibodies targeting IL17A and other cytokines, such as TNF- α, BAFF, IL13, etc., are used for treating various indications such as chronic inflammation of the lung, and are also in different stages of development.
The single domain antibody, as a strictly complete monomer, has the advantages of small molecular weight, high solubility, strong tissue permeability, high affinity, high degree of humanization and the like, and the special structural characteristics and properties thereof endow the antibody with characteristics which some other conventional antibodies or antibody fragments do not (Annu Rev biochem.2013,82,775). V (V) H H single domain antibodies to soluble targets including GPCRs and ion channelsThe membrane protein target (biomacromolecules 2021,11,63) has good drug properties, and is widely applied to the aspects of anti-tumor drugs, antiviral drugs, autoimmune diseases, CAR-T immune cell therapy and the like (biomaugs 2020,34,11;Antib Ther 2020,3,257;Biomolecules 2021,11,238). Single domain antibodies are easy to engineer and are ideal building blocks for multivalent antibodies, which can be aggregated together by short linking sequences (linker) to construct multivalent forms with higher antigen affinity than monovalent antibodies, or used to bind multiple different antigen targets simultaneously. Due to polyvalent V H The H antibody has smaller molecular weight, can adopt a traditional CHO expression production mode, can also adopt E.coli, P.pastoris and other production systems, and has advantages (MAbs 2018,10,778;Mucosal Immunol.2010,3,49) superior to the traditional monoclonal antibody in the aspect of development of new biological medicine formulations including subcutaneous injection, inhalant, oral medicine and the like.
Therefore, developing single/multivalent single domain antibodies that target IL17A and IL17F has many potential applications, such as multivalent antibodies that target multiple targets of IL17A, IL a/F and IL17F simultaneously for psoriasis and ankylosing spondylitis, also including indications such as plaque psoriasis, psoriatic arthritis, and nonradioactive axial spondylitis (Expert Opin Biol Ther 2019,19,45); alternatively, multivalent antibodies are formed with one or more of the cytokine antibodies, such as anti-TNF- α, BAFF, and IL13, for the treatment of various autoimmune diseases, including chronic pulmonary diseases, rheumatoid diseases, and sjogren's syndrome.
The invention aims to overcome the technical problem that the prior art lacks an effective single/multivalent single domain antibody targeting IL17A and IL17F, and provides an IL17 antibody, a preparation method and application thereof.
The invention solves the technical problems through the following technical proposal.
The present invention provides an IL17 antibody comprising a heavy chain variable region, wherein:
the heavy chain variable region comprises a CDR1 with a sequence shown in any one of SEQ ID NO 1-24, a CDR2 with a sequence shown in any one of SEQ ID NO 25-47, and a CDR3 with a sequence shown in any one of SEQ ID NO 48-71.
Preferably, the heavy chain variable region of the invention:
(1) CDR1 comprises the sequence shown as SEQ ID NO. 1, CDR2 comprises the sequence shown as SEQ ID NO. 25, and CDR3 comprises the sequence shown as SEQ ID NO. 48; or,
(2) Wherein CDR1 comprises the sequence shown as SEQ ID NO. 2, CDR2 comprises the sequence shown as SEQ ID NO. 26, and CDR3 comprises the sequence shown as SEQ ID NO. 49; or,
(3) Wherein CDR1 comprises the sequence shown as SEQ ID NO. 3, CDR2 comprises the sequence shown as SEQ ID NO. 27, and CDR3 comprises the sequence shown as SEQ ID NO. 50; or,
(4) CDR1 comprises the sequence shown as SEQ ID NO. 4, CDR2 comprises the sequence shown as SEQ ID NO. 28, and CDR3 comprises the sequence shown as SEQ ID NO. 51; or,
(5) CDR1 comprises the sequence shown as SEQ ID NO. 5, CDR2 comprises the sequence shown as SEQ ID NO. 29, and CDR3 comprises the sequence shown as SEQ ID NO. 52; or,
(6) CDR1 comprises the sequence shown as SEQ ID NO. 6, CDR2 comprises the sequence shown as SEQ ID NO. 28, and CDR3 comprises the sequence shown as SEQ ID NO. 53; or,
(7) CDR1 comprises the sequence shown as SEQ ID NO. 7, CDR2 comprises the sequence shown as SEQ ID NO. 30, and CDR3 comprises the sequence shown as SEQ ID NO. 54; or,
(8) CDR1 comprises the sequence shown as SEQ ID NO. 8, CDR2 comprises the sequence shown as SEQ ID NO. 31, and CDR3 comprises the sequence shown as SEQ ID NO. 55; or,
(9) CDR1 comprises the sequence shown as SEQ ID NO. 9, CDR2 comprises the sequence shown as SEQ ID NO. 32, and CDR3 comprises the sequence shown as SEQ ID NO. 56; or,
(10) CDR1 comprises the sequence shown as SEQ ID NO. 10, CDR2 comprises the sequence shown as SEQ ID NO. 33, and CDR3 comprises the sequence shown as SEQ ID NO. 55; or,
(11) CDR1 comprises the sequence shown as SEQ ID NO. 11, CDR2 comprises the sequence shown as SEQ ID NO. 34, and CDR3 comprises the sequence shown as SEQ ID NO. 57; or,
(12) CDR1 comprises the sequence shown as SEQ ID NO. 12, CDR2 comprises the sequence shown as SEQ ID NO. 35, and CDR3 comprises the sequence shown as SEQ ID NO. 58; or,
(13) CDR1 comprises the sequence shown as SEQ ID NO. 13, CDR2 comprises the sequence shown as SEQ ID NO. 36, and CDR3 comprises the sequence shown as SEQ ID NO. 59; or,
(14) CDR1 comprises the sequence shown as SEQ ID NO. 14, CDR2 comprises the sequence shown as SEQ ID NO. 34, and CDR3 comprises the sequence shown as SEQ ID NO. 60; or,
(15) CDR1 comprises the sequence shown as SEQ ID NO. 15, CDR2 comprises the sequence shown as SEQ ID NO. 37, and CDR3 comprises the sequence shown as SEQ ID NO. 61; or,
(16) CDR1 comprises the sequence shown as SEQ ID NO. 9, CDR2 comprises the sequence shown as SEQ ID NO. 38, and CDR3 comprises the sequence shown as SEQ ID NO. 62; or,
(17) CDR1 comprises the sequence shown as SEQ ID NO. 14, CDR2 comprises the sequence shown as SEQ ID NO. 34, and CDR3 comprises the sequence shown as SEQ ID NO. 60; or,
(18) CDR1 comprises the sequence shown as SEQ ID NO. 15, CDR2 comprises the sequence shown as SEQ ID NO. 37, and CDR3 comprises the sequence shown as SEQ ID NO. 63; or,
(19) CDR1 comprises the sequence shown as SEQ ID NO. 16, CDR2 comprises the sequence shown as SEQ ID NO. 39, and CDR3 comprises the sequence shown as SEQ ID NO. 64; or,
(20) CDR1 comprises the sequence shown as SEQ ID NO. 17, CDR2 comprises the sequence shown as SEQ ID NO. 40, and CDR3 comprises the sequence shown as SEQ ID NO. 65; or,
(21) CDR1 comprises the sequence shown as SEQ ID NO. 18, CDR2 comprises the sequence shown as SEQ ID NO. 41, and CDR3 comprises the sequence shown as SEQ ID NO. 66; or,
(22) CDR1 comprises the sequence shown as SEQ ID NO. 19, CDR2 comprises the sequence shown as SEQ ID NO. 42, and CDR3 comprises the sequence shown as SEQ ID NO. 67; or,
(23) CDR1 comprises the sequence shown as SEQ ID NO. 20, CDR2 comprises the sequence shown as SEQ ID NO. 43, and CDR3 comprises the sequence shown as SEQ ID NO. 67; or,
(24) CDR1 comprises the sequence shown as SEQ ID NO. 21, CDR2 comprises the sequence shown as SEQ ID NO. 44, and CDR3 comprises the sequence shown as SEQ ID NO. 68; or,
(25) CDR1 comprises the sequence shown as SEQ ID NO. 22, CDR2 comprises the sequence shown as SEQ ID NO. 45, and CDR3 comprises the sequence shown as SEQ ID NO. 69; or,
(26) CDR1 comprises the sequence shown as SEQ ID NO. 23, CDR2 comprises the sequence shown as SEQ ID NO. 46, and CDR3 comprises the sequence shown as SEQ ID NO. 70; or,
(27) CDR1 comprises the sequence shown as SEQ ID NO. 24, CDR2 comprises the sequence shown as SEQ ID NO. 47, and CDR3 comprises the sequence shown as SEQ ID NO. 71.
In a preferred embodiment of the invention, the heavy chain variable region comprises a sequence as set forth in any one of SEQ ID NOS.72 to 100.
Examples of antibodies described 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 fragment, F (ab') 2 scFv, di-scFv, and/or dAb), immunoconjugate, multispecific antibody (e.g., bispecific antibody), antibody fragment, antibody derivative, antibody analog, or fusion protein, etc., so long as they exhibit the desired antigen-binding activity; preferably a single domain antibody or V thereof H H fragment.
The invention also provides a trivalent single domain antibody comprising an IL17 antibody as described above.
In the present invention, the trivalent single domain antibody preferably contains three V's sequentially connected from N-terminus to C-terminus H H：V H H 1 -V H H 2 -V H H 3 。
V described above H H 2 Preferably anti-HSA V H H is formed; more preferably, it contains the sequence shown as SEQ ID NO. 108.
V described above H H 1 And said V H H 3 An IL17 antibody as described above, preferably in the form of a single domain antibody; preferably:
the V is H H 1 Comprising a CDR combination as defined in any one of (1) to (6) above, said V H H 3 A CDR combination comprising a CDR as defined in any one of (7) to (22) above; more preferably, the V H H 1 Contains a sequence shown in any one of SEQ ID NO 72-79, and V H H 3 Contains a sequence shown in any one of SEQ ID NO 80-100;
or said V H H 3 Comprising a CDR combination as defined in any one of (1) to (6) above, said V H H 1 A CDR combination comprising a CDR as defined in any one of (7) to (22) above; more preferably, the V H H 3 Contains a sequence shown in any one of SEQ ID NO 72-79, and V H H 1 Contains a sequence shown in any one of SEQ ID NO 80-100.
In a preferred embodiment of the invention, the V H H 1 Or V H H 3 Comprising a combination of CDRs as defined in (2) above, said V H H 3 Or V H H 1 Comprising a combination of CDRs as defined in (7), (10) or (26) above.
In a specific embodiment of the present invention, the V H H 1 Comprising the sequence shown as SEQ ID NO. 73, and V H H 3 Comprising the sequence shown as SEQ ID NO. 80, 83 or 99;
in another embodiment of the present invention, the V H H 1 Comprising the sequence shown as SEQ ID NO 80, 83 or 99, said V H H 3 Contains a sequence shown as SEQ ID NO. 73.
Above different V H H are preferably operatively connected by a linker; the linker preferably comprises the sequence shown in SEQ ID NO. 107.
In a specific embodiment of the invention, the trivalent single domain antibody contains the amino acid sequence shown in any one of SEQ ID NOS: 101-106.
The invention also provides an isolated nucleic acid encoding an IL17 antibody as described above or a trivalent single domain antibody as described above.
The invention also provides an expression vector comprising an isolated nucleic acid as described above.
The invention also provides a host cell comprising an expression vector as described above; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.
The invention also provides a method for producing an IL17 antibody or a trivalent single domain antibody comprising culturing a host cell as described above and obtaining the antibody from the culture.
The invention also provides a pharmaceutical composition comprising an IL17 antibody as described above or a trivalent single domain antibody as described above.
The invention also provides the use of an IL17 antibody as described above, a trivalent single domain antibody as described above, or a pharmaceutical composition as described above in the manufacture of a medicament for the prevention and treatment of an IL 17-related disease or disorder.
The disease or condition preferably includes chronic inflammation of the lung, rheumatoid arthritis, intestinal inflammation, dry eye, psoriasis, ankylosing spondylitis and various cancers including skin cancer, more preferably psoriasis, ankylosing spondylitis, chronic inflammation of the lung and rheumatoid arthritis; psoriasis, ankylosing spondylitis and rheumatoid arthritis are further preferred.
The invention also provides a chimeric antigen receptor comprising an IL17 antibody as described above or a trivalent single domain antibody as described above.
The invention also provides an antibody drug conjugate comprising a cytotoxic agent, and an IL17 antibody as described above or a trivalent single domain antibody as described above; preferably, the cytotoxic agent is MMAF or MMAE.
The invention also provides a kit comprising an IL17 antibody as described above, a trivalent single domain antibody as described above, a chimeric antigen receptor as described above, an antibody drug conjugate as described above and/or a pharmaceutical composition as described above;
preferably, the kit further comprises (i) means for administering an antibody or antigen binding fragment thereof or an antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
The invention also provides a kit comprising a kit a and a kit B, wherein:
the kit a contains an IL17 antibody as described above, a trivalent single domain antibody as described above, a chimeric antigen receptor as described above, an antibody drug conjugate as described above 9 and/or a pharmaceutical composition as described above;
the kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
The invention also provides a method of diagnosing, treating and/or preventing an IL 17-mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of an IL17 antibody as described above, a trivalent single domain antibody as described above, a chimeric antigen receptor as described above, an antibody drug conjugate as described above or a pharmaceutical composition as described above, or treating a patient in need thereof using a kit of parts as described above. Wherein the disease or condition is as defined above.
The invention also provides a method of immunodetection or determination of IL17 comprising using an IL17 antibody as described above, a trivalent single domain antibody as described above, a chimeric antigen receptor as described above, an antibody drug conjugate as described above or a pharmaceutical composition as described above, after incubation with a test substance; preferably, the detection or the assay is for non-diagnostic and/or therapeutic purposes.
The invention also provides a combination therapy comprising administering to a patient in need thereof an IL17 antibody as described above, a trivalent single domain antibody as described above, a chimeric antigen receptor as described above, an antibody drug conjugate as described above or a pharmaceutical composition as described above, respectively, and a second therapeutic agent; the second therapeutic agent preferably comprises a further anti-tumour antibody or a pharmaceutical composition comprising said further anti-tumour antibody, and/or one or more of the group consisting of a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of co-stimulatory molecules, an inhibitor of inhibitory molecules and a vaccine.
The invention uses IL17A homodimer, IL17F homodimer and IL17A/F heterodimer to respectively immunize alpaca for multiple times, separates mRNA of peripheral blood lymphocyte, synthesizes cDNA and constructs V H H antibody library, screening by phage display technique, ELISA technique, etc. to obtain multiple high affinity and specific binding of IL17A, IL F and IL17A/F V H H antibody, followed by V H Evaluation of H antibody expression purification, affinity, specificity, stability and the like; construction of multiple multivalent V by combination with Anti-HSA antibodies H H antibody, and the evaluation of expression purification, affinity, specificity, stability, and epi binding is completed. The results show that these monovalent and multivalent V H H antibodies have unique advantages, mainly expressed in the following areas:
1. these V' s H The H antibody can selectively and high-affinity bind to one or more of human cytokines IL17A, IL F and IL17A/F, and can high-affinity and specificity bind to cynomolgus monkey IL17A and IL17F;
2.V H the H antibody sequence has good thermal stability (T) m ≥65℃)；
3. Has brand new antigenic determinants when binding IL17A, IL F and IL 17A/F;
4. by combining different V H H antibody sequences are combined together, and the constructed multivalent antibody can be combined with IL17A, IL A/F and IL17F with high affinity and specificity;
5. The monovalent and multivalent antibodies can be efficiently expressed and correctly folded in the cell gap of E.coli, and can be prepared by affinity chromatography and ion exchange chromatography purification of Protein A tags;
6. cell activity tests show that the binding between IL17A, IL A/F and corresponding receptors can be effectively blocked;
7. monovalent, multivalent V H The H antibody is expected to be used for diseases caused by abnormal regulation of cytokines such as IL17A, IL F, IL17A/F and the like;
8.V H v of H antibody easy to other targets H The H single domain antibody is combined into multivalent antibody, and is used for treating tumor, lung diseases, autoimmune diseases, virus resistance, infection resistance and the like.
Drawings
FIG. 1A is V in FIG. 1 H The H-A19 affinity chromatography SDS-PAGE electrophoresis, the Superdex 75/300 chromatography is shown in FIG. 1B, and the protein SDS-PAGE electrophoresis after Superdex 75/10/300 chromatography is shown in FIG. 1C.
FIG. 2 is V H H-A19 with human IL17A affinity test pattern.
FIG. 3 is V H T of H-AF6 m Value test chart.
FIG. 4 is V H Paratope analysis of H-A8 interaction with IL 17A.
FIG. 5A shows a rProtein A sepharose FF purification pattern of PAF022, FIG. 5B shows a Hitrap Q column purification pattern thereof, and FIG. 5C shows a sample SDS-PAGE electrophoresis pattern after Hitrap Q column purification.
FIG. 6T of PAF022 m Test patterns.
In fig. 7: FIG. 7A is the neutralizing activity of PAF022, PAF025, and PAF041 on IL 17A; FIG. 7B is the neutralizing activity of PAF022, PAF025 and PAF041 on IL 17A/F.
FIG. 8 shows V H H is connected with anti-HSA through amino acid Linker (GGGGSGGGS).
Detailed Description
Example 1 alpaca immunization, potency detection and lymphocyte separation
Three alpaca were immunized by the immunization method described in literature such as procurement of IL17A (Sino Biological, 12047-HNAS), IL17A/F (Sino Biological, CT 047-HNAE) and IL7F (Sino Biological, 11855-HNAE), and reference to Methods (Mol Biol 2012,911,211;Curr Protoc Immunol 2013,103,2.17.1;Nat Protoc 2014,9,674).
Each immunization was injected separately on both sides of the alpaca neck muscle in an amount of 100 μg protein per alpaca, for a total of two injections. Complete Freund's adjuvant was used for the first time and was noted as day 0. On subsequent days 14, 28, 42, 56 and 70, second to sixth booster immunizations were performed with incomplete Freund's adjuvant. The effect of alpaca immunization is detected by using an ELISA method (J Vis Exp 2019,143), and the result shows that the titer of serum after five times of immunization is diluted 10000 times and is obviously higher than that of negative serum diluted 1000 times, thereby reaching the set titer requirement before immunization.
One week after the fifth immunization and one week after the sixth immunization, large-scale blood collection was performed, respectively, with a blood collection volume of 80mL. After a large amount of blood was collected, lymphocytes were isolated according to the experimental procedure described in the specification using QIAamp RNA Blood Mini Kit (QIAGEN, 52304), and RNA extraction was completed.
EXAMPLE 2 library construction and screening
Referring to the usual methods of single domain antibody discovery, antibody libraries were constructed and screened (Nat Protoc 2014,9,674;Nat Protoc 2007,2,3001) for high affinity sequences that bind specifically to antigens. In brief, the single domain antibody gene is first inserted into phage display vector, and then packed and displayed on phage, and corresponding screening condition is designed based on the experiment purpose to obtain the single domain antibody sequence. The specific experimental method is as follows:
lymphocyte RNA obtained in example 1 was subjected to PrimeScript TM II 1st Strand cDNA Synthesis Kit A cDNA was synthesized using the instructions of the kit (TAKARA, 6210A) and single domain antibodies (V) were obtained by nested PCR amplification using KOD-FX (TOYOBO, KFX-101) H H) Genes were digested simultaneously with BamHI-HF (NEB, #R3136L) and XhoI (NEB, #R0146L) overnight at 37 ℃. The pComb3XSS-GFP vector (modified from pComb3XSS (Biovector NTCC Inc.) was digested simultaneously with BamHI-HF (NEB, #R3136L) and XhoI (NEB, #R0146L), and the sequence between the sites SfiI was replaced with GFP gene, and BamHI and XhoI were added at both ends, so that the ligation sequence was 5'-OmpA signal peptide-BamHI-GFP-XhoI-gene III-stop codon-3'). Further double enzyme cutting V H The H fragment and pComb3xss-GFP vector were incubated with T4DNA Ligase (NEB, #M0202L) at 16℃overnight, and after further ion purification of the ligation product, TG1 competent cells (Lucigen, 60502-2) were transformed by electric shock, and E.coli cultured overnight was collected to complete library construction. In the experiment, 6.8 to 9.2 multiplied by 10 are respectively obtained for 3 alpacas 8 A cDNA library with fragment insertion rate of more than 95% and good sequence diversity.
The cDNA library is screened by a phage display technology and a liquid phase method, and the specific steps are as follows:
library bacteria were inoculated into 50mL of 2 XYT medium containing 100. Mu.g/mL of ampicillin and 2% glucose, OD after inoculation 600 Culture at 220rpm at 37℃until OD 600 =0.5. Helper phage M13KO7 (NEB, # N0315S) was added in a ratio of 20:1 (phage: bacteria), and the mixture was left standing at 37℃for 30 minutes; after centrifugation at 3000g for 10 min, the supernatant was discarded and resuspended in 50mL of 2 XYT medium plus 100. Mu.g/mL of ampicillin and 50. Mu.g/mL of kanamycin, at 26℃and 220 rpm.
The bacterial culture was centrifuged at 3000g at 4℃for 10 minutes, the supernatant was mixed with PEG/NaCl solution at a volume ratio of 4:1 and then ice-incubated for 1 hour, and the precipitate was collected by centrifugation at 3000g at 4℃for 20 minutes. Resuspension with 10mL PBS, centrifugation at 3000g for 20 min at 4 ℃, collection of supernatant and addition of 2.5mL PEG/NaCl solution, mixing well followed by ice bath for 30 min; centrifugation at 3000g for 30 min at 4℃and pellet re-suspended with 1mL PBS to obtain phage solution.
500 mu L of the abovePhage solution, 1% (w/v) BSA solution was added to block for 1 hour, 100nM biotinylated IL17A (ARCO Biosystems, ILA-H82Q 1) solution was added, incubated for 1 hour at room temperature with tumbling, 50. Mu.L streptavidin beads blocked with 1% BSA solution (ThermoFisher scientific, 11206D) were added, incubated for 30 minutes at room temperature with tumbling, magnetic beads were adsorbed by magnet, 0.1% PBST, PBS were washed 10 times respectively, triethylamine (Sigma, T0886) solution was added, mixed well, left at room temperature for 10 minutes, and a suitable amount of pre-chilled 1M Tris-HCl (pH 7.4) was added to mix well, and 10mL TG1 strain was infected with supernatant after magnet adsorption (preactivation, OD) 600 =0.5), plates containing 100 μg/mL ampicillin and 2% glucose were spread and incubated overnight at 32 ℃ inverted. All the thalli on the flat plate are collected and the strains are stored, and a proper amount of monoclonal on the small plates subjected to gradient dilution counting is picked up for sequencing analysis.
Reducing the IL17A concentration of antigen, repeating the above procedure with 10nM and 1nM, respectively, to complete the second and third rounds of screening, striving for high affinity V H H sequence.
Three rounds of screening of the library of antigens IL17A/F and IL17F were performed using the same method.
EXAMPLE 3 Positive Single-Domain antibody sequence acquisition
To further determine high affinity sequences, V obtained from the third round of screening H Cloning H gene onto expression vector pSJF2, ELISA detection to obtain positive clone, and sequencing the positive clone to obtain V H H sequence. The experimental method is as follows:
primers carrying BbsI-HF (NEB, # R3539L) and BamHI-HF (NEB, # R3136L) cleavage sites at both ends were designed, and V was obtained by PCR amplification from plasmids of the second and third rounds of screening sub-libraries H After completion of the cleavage of the H gene, bbsI-HF and BamHI-HF, the same digested pSJF2 vector was ligated using T4DNA ligase and transformed into TG1 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight at 37 ℃.
The overnight cultured monoclonals were picked up into 96-well deep well plates with 200. Mu.L of 2 XYT (100. Mu.g/mL of ampicillin) medium, after 4 hours of incubation at 37℃the supernatants were discarded by centrifugation and the induction medium (2 XYT+100. Mu.g/mL of ampicillin+1% Glycine+1% Triton X-100+0.5mM IPTG+5%sucrose) was replaced and incubated overnight at 27 ℃.
Antigen IL17A (Sino Biological, 12047-HNAS) was diluted to 1. Mu.g/mL with carbonate buffer, 100. Mu.L/well, added to ELISA plates (ThermoFisher scientific, 43954) and incubated overnight at 4 ℃. After three washes of ELISA plates with PBS, 380. Mu.L/well of 1% BSA was added and blocked at 37℃for 2 hours; PBST is washed 3 times, 100 mu L/hole of bacterial liquid supernatant is added, and the mixture is incubated for 1 hour at 37 ℃; PBST was washed 3 times, HRP-labeled His-tag antibody (Sino Biological,105327-MM 02T-H) diluted with 1% BSA was added, and incubated at 37℃for 1 hour; PBST was washed 5 times, TMB color development solution (Sigma, T8665) was added, 100. Mu.L/well, and reacted at room temperature for 10 minutes in the absence of light; 2M H 2 SO 4 Stop reaction, 50. Mu.L/well, detection of OD by ELISA 450 Numerical values. ELISA detection results are shown in Table 1: in accordance with the 96-well deep-hole plate, the first row A-H is the column number, the first row 1-12 is the horizontal row number, the cloning is numbered and positioned (A1-H12) by using the horizontal and vertical numbers as coordinates, and the cloning and OD in the deep-hole plate 450 The detection readings are corresponding; the readings of the 96 detection wells A1-H12 (except the first row and the first column) are highlighted with different gray scales, the larger the readings are, the darker the color is, the theoretical affinity is higher (the actual affinity cannot be in one-to-one correspondence with ELISA detection readings due to factors such as non-quantitative experiments and larger EILSA errors, and only reference is made), the negative clone reading is about 0.06 (G12 and H12), and the clone with the reading greater than 0.6 is regarded as positive clone in the detection. Sequencing 161 positive clones, and analyzing the sequencing result to obtain positive candidate V H H gene sequence.
TABLE 1 ELISA detection readings
Clones of antigen IL17A/F and IL17F were detected using the same method.
Example 4 monovalent single domain antibody characterization
1) Monovalent single domain antibody purification
According to ELISA positive sequence diversity analysis, 70 representative sequences are selected, and after E.coli codon optimization, the genes are synthesized again and cloned to pET28a (+) vector to construct expression plasmid. The expression sequence is OmpA signal peptide and V from N end to C end in sequence H H. linker (amino acid sequence GAA) and 6 xhis tag. The synthesized expression plasmid transformed BL21 (DE 3) competent cells, spread on plates and cultured overnight at 37 ℃.
Seed solution after overnight monoclonal culture was inoculated at 1:100 into 400mL of 2 XYT medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃and 250rpm to OD 600 =0.8, IPTG was added at a final concentration of 0.5mM and incubated at 16 ℃ for 16 hours. Collecting bacteria by low-speed centrifugation, adding 40mL Buffer A (10 mM/Tris-HCl, pH 8.0,1mM/EDTA,25% sucrose) to thoroughly resuspend the bacteria, and incubating at room temperature for 15 minutes with shaking; after high speed centrifugation, the supernatant was removed and 20mL of pre-chilled Buffer B (30 mM Tris-HCl, pH 8.0,5mM MgSO) 4 ) Re-suspending; after centrifugation again at high speed, the supernatant was taken and added with appropriate amounts of NaCl solution, imidazole solution and DTT solution to give final concentrations of 150mM, 20mM and 1mM, respectively.
200. Mu.L of Ni NTA Beads 6FF (Smart lifesciences, SA 005025) was added, the mixture was slowly mixed on a shaker for 30 minutes, transferred to a chromatographic column (Bio-Rad, 7311550), and Flow Through (FT) was collected; after washing with 5mL of 1 XPBS containing 40mM imidazole solution, the antibody was eluted with 0.5mL of 1 XPBS containing 300mM imidazole. In FIG. 1A is an SDS-PAGE electrophoresis of a sample during A19 affinity chromatography. Wherein S represents the supernatant after bacterial lysis by the "superosmosis" method; FT represents combined fluid penetration; 40. 80 represents buffer after washing Ni-Resin with 1 XPBS buffer containing 40mM, 80mM imidazole; e represents a sample eluted with 1 XPBS buffer containing 300mM imidazole; b represents the result of the eluted Ni-Resin boiling electrophoresis.
The eluted samples were concentrated and then subjected to molecular sieve chromatography using Superdex 75/300, followed by BCA (Shanghai Biyun Biotechnology Co., ltd., P0010) quantitative analysis and SDS-PAGE analysis. Superdex 75 10/300 molecular sieve chromatogram of A19 is shown in FIG. 1B, and C in FIG. 1SDS-PAGE electrophoresis of the protein product after A19 molecular sieve chromatography. It can be seen that high purity V is obtained by 2 purification steps H H sample.
2)V H H affinity test
Affinity testing of monovalent single domain antibodies was accomplished using Biacore T200, binding His-tag monoclonal antibodies to CM5 chips (GE Healthcare, BR-1005-30) for capturing His-tagged monovalent V, as described by the His-tag protein Capture kit (GE Healthcare, 28-9950-56) H The H antibody, human IL17A, IL A/F and IL17F are used as mobile phases for affinity detection.
FIG. 2 shows the binding and dissociation curves and fitting patterns of antibody A19 and IL17A, calculated and analyzed to obtain V H Affinity values of H antibodies to antigen. At 70V of inclusion characterization H In the H antibody, 29V with better expression and purification and higher affinity are obtained H The affinity data for the H antibodies are shown in table 3, where "x" represents no binding.
TABLE 2 monovalent V H Affinity test of H antibody with human IL17A, IL A/F and IL17F
3)V H H thermal stability analysis
Candidate V was subjected to fluorescence analysis (differential scanning fluorimetry, DSF) H H antibody molecules, performing preliminary screening of thermal stability. During the screening, the purified V was purified using SYPRO Orange Protein Gel Stain (Sigma, S5692) with stepwise heating by qPCR instrument H H antibody T m Preliminary test of values. In the preliminary screening, V H H antibody T m The value of V is greater than or equal to 55 ℃ as the screening condition, and V is listed in Table 2 H H antibodies all meet this requirement. FIG. 3 is a schematic diagram of the test T of antibody AF6 using DSF m Test chart of values, T m The value reached 74 ℃.
4)V H H-specific assay
As shown by the affinity test results in Table 2, V H The ability of H antibodies to bind with high affinity to one or more antigens in IL17A, IL A/F and/or IL17F, to further assess the ability of IL17 family proteins to specifically bind, the affinity of the antibodies to human IL17B (Prospec, CYT-753), IL17C (Acro Biosystems, ILC-H52H 7), IL17D (Atagnix, ATAP 01505) and IL17E (Acro Biosystems, IL 5-H4221) was tested according to the test method in step 2). 29 candidates V as shown in Table 2 H H antibody molecules can be combined with IL17A/F with stronger affinity, and IL17A/F is used as positive control molecule to test candidate V H Specific binding capacity of H antibodies to IL17B, IL17C, IL D and IL 17E. The test results showed 29 candidates V H H antibody molecules, the Ru value of the binding with IL17A/F is between 30 and 120, and the Ru absolute value of the binding with IL17B, IL17C, IL D and IL17E is mostly less than or equal to 10 and is far lower than the Ru absolute value with IL 17A/F. Therefore, these antibodies did not bind significantly to IL17B, IL17C, IL D and IL17E, and the specificity was satisfactory.
5)V H Affinity test of H for cynomolgus monkey (Cynomolgus monkey) IL17A, IL F
To further detect the V H H binding activity and preparation for animal experiments of the following antibody drugs in cynomolgus monkeys, and V detection H Affinity between H and cynomolgus monkey IL17A and IL17F, detection method is the same as step 2), V H H was coupled to CM5 chip via His tag as immobilization and IL17A and IL17F molecules of cynomolgus monkeys were detected as mobile phase. The results of the detection are shown in Table 3, wherein X represents unbound. Biacore test results show that other antibodies have certain binding capacity with cynomolgus monkey IL17A and IL17F except A29 which is not detected. Next, comparing the test results of tables 1 and 3, most of the candidate V H The binding affinity of the H antibody to human IL17A or IL17F, and to cynomolgus monkey IL17A or IL17F, were not orders of magnitude different.
Table 3V H Affinity of H with Cynomolgus monkey IL A and IL17F
6)V H Epitope binding analysis of H
The present invention uses SPR techniques on the above V H H was subjected to cluster analysis according to the epitop, as follows. Optimizing and synthesizing E.Coli codon preferred expression genes, so that the expression frame sequences are OmpA signal peptide and V in sequence H H. SASA (from US 2013/0129747 A1, which can bind BSA), his tag; the synthesized gene was subjected to SASA-V according to the monovalent single domain antibody purification method described in 1) H H expression and purification. Detection is performed by first coupling BSA to CM5 chip, followed by SASA-V H H(V H H 1 ) Through the flow path to bind with BSA and fix on the chip, and then IL17A/F is led through the flow path to V H H 1 Binding to form BSA-SASA-V H H(V H H 1 ) IL17A/F complex, finally V H H(V H H 2 ) The antibody passes through the flow path as a mobile phase, and a response value Ru is recorded. 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 With different epitopes, then V H H 2 Can pass through the flow path and be connected with BSA-SASA-V H H(V H H 1 ) IL17A/F complex binding, ru values will be higher; and vice versa.
Table 4 shows the results obtained by the detection method, and shows that part of antibodies have different epitopes, such as the combination of positive values and larger values of Ru, such as AF5, A8, AF10, A5 and the like; some antibodies have the same epitope, such as the combination of Ru near 0, such as A8 and A19, AF2 and A21.
TABLE 4 Epitope binding assay results
Antibody numbering 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 Using H/D exchange method
Hydrogen deuterium exchange mass spectrometry (HDX-MS) epitope localization can provide an efficient method for rapidly providing epitope structural integrity information. The principle is that hydrogen on protein amide bond can be controllably exchanged with deuterium in heavy water, protein is immersed in the heavy water, hydrogen on protein surface is closely contacted with the heavy water, and the exchange rate is fast; and the hydrogen inside the protein and forming hydrogen bonds is difficult to contact heavy water, so that the exchange rate is slow. And determining the deuteration number of the peptide sequences under different reaction time by utilizing mass spectrum, and further calculating the hydrogen deuterium exchange rate of each peptide sequence, so that the spatial structure information of the protein can be judged. This technique uses mass spectrometry to measure the solvent accessibility of amino acid residues in proteins to determine the interaction site of an antigen-antibody complex in its natural solution, and does not introduce any modifications to the antigen or antibody.
The experimental procedure was as follows: 5-10. Mu.M antigen, antibody, antigen-antibody complex were dissolved in 50mM HEPES,pH 7.4, 150mM NaCl,4mM TCEP solution at a molar ratio of 1:1 and left at 4℃for 1 hour, respectively, to ensure the formation of a stable antigen-antibody complex. After 1 hour of standing, 5. Mu.L of the different samples were diluted to 20. Mu. L D, respectively, at 4 ℃ 2 O (deuterium) and placed at different HDX time points (e.g., 0,10,60,300,900 seconds) by cooling with 25 μl ice-cold 100mM NaH 2 PO 4 The reaction was stopped by mixing 1M TCEP. Immediately after stopping the reaction, the sample tube was placed on dry ice until the sample was injected into the HDX LEAP PAL3.0 platform. After injection into a fully automated hydrogen-deuterium exchange platform, the sample was passed through a fixed pepsin column at a flow rate of 120 μl/min, and the enzymatically digested peptide fragments were captured and desalted by a 2.1mm×5cm c18 column (1.9 μm Hypersil Gold, thermo Fisher). The desalted peptide fragments were separated over 8 minutes with a linear gradient of 4-40% acetonitrile and 0.3% formic acid. During sample processing, both proteolysis and peptide fragment separation were performed at 4 ℃.
An Orbitrap mass spectrometer (Orbitrap Fusion was used TM Tribrid TM Mass Spectrometer, thermo Fisher) for analysis of eluted, isolated peptide fragments, the resolution of which was measured at 65,000 (m/z 400) to obtain hydrogen deuterium exchanged mass spectrometry data. Each sample had three HDX assays (triplets) at each time point. The mass spectrum peak intensity average m/z centroid value (generally10ppm accuracy) and then converted to deuterium incorporation percentages. The key amino acid sequences involved in the spatial epitope were calculated and the difference in% Delta D was determined by calculating the difference between the two samples (comparing the variation in percentage deuterium incorporation on the same peptide fragment). Differences in Delta% D outside-5 to 5% are considered significant differences. Furthermore, HDX Workbench detected statistical significance between samples at each time point by student's t test (p <0.05 A) the difference.
The invention uses the method to identify the Epitope/Paratope binding site. With A8 and AF5 two V H H As an example, according to the specific interaction pattern, AF5-IL17A, AF5-IL17A/F, AF5-IL17F, A8-IL17A and A8-IL17A/F total five interactions, H/D exchange experiments were performed. FIG. 4 is a Paratope analysis of the interaction of A8 with IL17A, showing that SGSIFNAHAMGW, CNADHTYYSDSAL is a potential Paratope, focusing primarily on CDR1 and CDR 3. AF5 has a strong binding affinity to both IL17A, IL17A/F and IL17F, but its binding pattern is not exactly the same from the Paratope involved in binding. The results of the measurements after finishing are shown in Table 5, A8 interacts with IL17A and IL17A/F mainly through CDR1 and CDR 3. AF5 binds IL17A primarily through partial sequences of CDR2, CDR 3; interaction with IL17A/F through CDR3 and part of the amino acids of frame-4; IL17F is bound by CDR 2.
TABLE 5 Paratope sequences of AF5, A8 and IL17A, IL A/F and IL17F
After obtaining the Paratope information for the antibody-antigen interactions, the Epitope information on IL17A, IL A/F and IL17F was further analyzed and the results are shown in Table 6.
TABLE 6 Epitope sequences of AF5, A8 and IL17A, IL A/F and IL17F
8) Candidate V H H sequence
Through the characterization of the steps, 29 single domain antibodies with high affinity, good specificity and good thermal stability are obtained, and the amino acid sequences of the single domain antibodies are shown in Table 7 (the bold part of each sequence is CDR1, CDR2 and CDR3 from front to back).
TABLE 7 candidate V H H amino acid sequence
Example 5 construction, characterization and functional validation of trivalent Single-domain antibodies
1) Trivalent single domain antibody construction
After the monovalent single domain antibody characterization analysis, 29 high-affinity V with better specificity and better thermal stability are obtained H H sequences, which are useful in the combinatorial construction of multivalent antibodies, achieve the goal of simultaneously binding IL17A, IL A/F and IL17F with high affinity. As shown in Table 1, in view of V of the A series H H has higher affinity with IL17A, whereas AF series V H H has higher affinity with IL17A/F and IL17F, and when combined, the AF series and the V of A series H H is each one by Anti-HSA (V H H from US 2007/0269422 a1, alb 11). Consider the steric hindrance pair V H Effect of H interaction with IL17A, IL A/F and IL17F Anti-HSA was placed in the middle, AF series, V of A series H H is respectively arranged at two ends, and V is constructed H Two combinations of H-position reversal: the first is the V of the AF series H V of the A series with H at the N-terminus H H at the C-terminus, the second being V of the A series H H is at N-terminal and AF is a series of V H H is at the C-terminus. V (V) H H constructs a different trivalent antibody by amino acid Linker (GGGGSGGGS) linkage to anti-HSA (shown in FIG. 8).
Anti-HSA(US 2007/0269422 A1,ALB11)：
With 4V of AF5, A8, A19 and A43 H For example, 6 different trivalent antibodies were constructed, wherein PAF022 consisted of AF5, anti-HSA and A8 connected in sequence, PAF025 consisted of AF5, anti-HSA and A19 connected in sequence, PAF041 consisted of AF5, anti-HSA and A43 connected in sequence, PA002 consisted of A8, anti-HSA and AF5 connected in sequence, PA026 consisted of A19, anti-HSA and AF5 connected in sequence, and PA154 consisted of A43, anti-HSA and AF5 connected in sequence. 6 trivalent single domain antibody amino acid sequences (see Table 8), ompA signal peptide and stop codon were added, and the genes were synthesized after codon optimization and cloned into pET28a (+).
Table 8 6 amino acid sequences of trivalent Single-Domain antibodies
2) Trivalent single domain antibody preparation
The expression method of the trivalent single domain antibody is the same as that of the monovalent single domain antibody, and the trivalent single domain antibody can be obtained with high purity after 2 steps of purification. The purification method is as follows: adding appropriate amount of precooled Lysis buffer (1× PBS,1mM DTT,1mM EDTA), ultrasonic crushing (400W, 3s on/3s off,15 min), centrifuging at high speed, and collecting supernatant to pretreated HiTrap TM MabSelect SuRe Column(Cytiva，11003493 1 XPBS equilibrated to zero at baseline A280, eluted with 100mM glycine-HCl (pH 3.0) at a flow rate of 1mL/min, and the peak of interest was collected; the eluted protein was pH-adjusted to 10.0, then subjected to Hitrap Q column (Cytiva, 17115401), the target protein was collected, concentrated in a 10K ultrafiltration tube, buffer-exchanged to 1 XPBS, and quantified by BCA method. FIG. 5A shows the affinity chromatography purification of protein A of antibody PAF022, FIG. 5B shows the purification of Hitrap Q column, and FIG. 5C shows the SDS-PAGE electrophoresis of samples after Hitrap Q column purification.
3) Trivalent single domain antibody affinity assay
Unlike monovalent single domain antibody affinity assays, trivalent single domain antibodies were first tested for affinity to human IL17A, IL A/F and IL17F by coupling the Anti-His in a His tag capture kit to a CM5 chip for capture of His tagged human IL17A (Acro Biosystems, ILA-H82Q 1), IL17A/F (Acro Biosystems, ILF-H52W 6) and IL17F (Sino Biosystems, 11855-H07H), as mobile phases. The detection results are shown in Table 9.
Table 9 trivalent antibody affinity assay
4) Thermal stability of trivalent Monomain antibodies
To test the thermal stability of the prepared trivalent single domain antibodies, the present invention performed thermal stability tests on seven trivalent antibodies in total of the prepared control antibodies MS3091 (US 2014/0314743 A1), PAF022, PAF025, PAF041, PA002, PA026 and PA154 using a differential scanning calorimeter (TA Instruments, discovery DSC 2500/250/25). The specific method is that 20 mu L of sample to be measured (2 mg/mL) is taken and added into a sample tray, the sample tray is sealed, the sample tray is put into an instrument, the sample tray is heated to 95 ℃ from 40 ℃ at a speed of 1 ℃/min, and the heat flow change between the sample tray and a reference tray is recorded. T of PA022 m At 76.7deg.C, FIG. 6 is a graph of the heat flow profile of PA 022. T of positive control m The values are 82.0deg.C, PAF025 and PAF041 of 79.4deg.C and 78.6deg.C, respectively, and PA002 of 76.0deg.C, both greater than 65deg.CThe heat stability is good.
5) Trivalent Single Domain antibody cell function evaluation
IL17 can stimulate epidermal cells (Hs 27 cell line, human epidermal fiber cells, ATCC#CRL-1634) to secrete GROα, if an antibody of IL17 is added, the effect of IL17 and a cell membrane surface receptor is blocked by combining with IL17 protein, so that the effect of inhibiting the epidermal cells from secreting GROα is achieved, and the IC50 value of a candidate antibody can be obtained by detecting the secretion amount of GROα.
The invention tests the activity of trivalent antibodies, and partial results are shown in fig. 7 and table 10, and the maximum inhibition rate of the tested 6 trivalent antibodies on IL17A is more than 83%; antibodies PAF022, PAF025 and PAF041 all had IC50 around 0.03nM, and were comparable in activity to the IC50 value of 0.04nM for MS3091, and antibodies PA002, PA026 and PA154 had IC50 10-20 times lower than MS 3091. The 6 trivalent antibodies tested all had greater than 90% inhibition of IL17A/F, and IC 50's between 0.6-2.7nM, differing from MS3091 only by a factor of 3-5.
TABLE 10 neutralizing Capacity of trivalent candidate antibodies against IL17A, IL17A/F
While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims (25)

  1. An IL17 antibody comprising a heavy chain variable region, wherein said heavy chain variable region comprises CDR1 as set forth in any one of SEQ ID NOs 1 to 24, CDR2 as set forth in any one of SEQ ID NOs 25 to 47, and CDR3 as set forth in any one of SEQ ID NOs 48 to 71.
  2. The IL17 antibody of claim 1,
    (1) CDR1 comprises the sequence shown as SEQ ID NO. 1, CDR2 comprises the sequence shown as SEQ ID NO. 25, and CDR3 comprises the sequence shown as SEQ ID NO. 48; or,
    (2) Wherein CDR1 comprises the sequence shown as SEQ ID NO. 2, CDR2 comprises the sequence shown as SEQ ID NO. 26, and CDR3 comprises the sequence shown as SEQ ID NO. 49; or,
    (3) Wherein CDR1 comprises the sequence shown as SEQ ID NO. 3, CDR2 comprises the sequence shown as SEQ ID NO. 27, and CDR3 comprises the sequence shown as SEQ ID NO. 50; or,
    (4) CDR1 comprises the sequence shown as SEQ ID NO. 4, CDR2 comprises the sequence shown as SEQ ID NO. 28, and CDR3 comprises the sequence shown as SEQ ID NO. 51; or,
    (5) CDR1 comprises the sequence shown as SEQ ID NO. 5, CDR2 comprises the sequence shown as SEQ ID NO. 29, and CDR3 comprises the sequence shown as SEQ ID NO. 52; or,
    (6) CDR1 comprises the sequence shown as SEQ ID NO. 6, CDR2 comprises the sequence shown as SEQ ID NO. 28, and CDR3 comprises the sequence shown as SEQ ID NO. 53; or,
    (7) CDR1 comprises the sequence shown as SEQ ID NO. 7, CDR2 comprises the sequence shown as SEQ ID NO. 30, and CDR3 comprises the sequence shown as SEQ ID NO. 54; or,
    (8) CDR1 comprises the sequence shown as SEQ ID NO. 8, CDR2 comprises the sequence shown as SEQ ID NO. 31, and CDR3 comprises the sequence shown as SEQ ID NO. 55; or,
    (9) CDR1 comprises the sequence shown as SEQ ID NO. 9, CDR2 comprises the sequence shown as SEQ ID NO. 32, and CDR3 comprises the sequence shown as SEQ ID NO. 56; or,
    (10) CDR1 comprises the sequence shown as SEQ ID NO. 10, CDR2 comprises the sequence shown as SEQ ID NO. 33, and CDR3 comprises the sequence shown as SEQ ID NO. 55; or,
    (11) CDR1 comprises the sequence shown as SEQ ID NO. 11, CDR2 comprises the sequence shown as SEQ ID NO. 34, and CDR3 comprises the sequence shown as SEQ ID NO. 57; or,
    (12) CDR1 comprises the sequence shown as SEQ ID NO. 12, CDR2 comprises the sequence shown as SEQ ID NO. 35, and CDR3 comprises the sequence shown as SEQ ID NO. 58; or,
    (13) CDR1 comprises the sequence shown as SEQ ID NO. 13, CDR2 comprises the sequence shown as SEQ ID NO. 36, and CDR3 comprises the sequence shown as SEQ ID NO. 59; or,
    (14) CDR1 comprises the sequence shown as SEQ ID NO. 14, CDR2 comprises the sequence shown as SEQ ID NO. 34, and CDR3 comprises the sequence shown as SEQ ID NO. 60; or,
    (15) CDR1 comprises the sequence shown as SEQ ID NO. 15, CDR2 comprises the sequence shown as SEQ ID NO. 37, and CDR3 comprises the sequence shown as SEQ ID NO. 61; or,
    (16) CDR1 comprises the sequence shown as SEQ ID NO. 9, CDR2 comprises the sequence shown as SEQ ID NO. 38, and CDR3 comprises the sequence shown as SEQ ID NO. 62; or,
    (17) CDR1 comprises the sequence shown as SEQ ID NO. 14, CDR2 comprises the sequence shown as SEQ ID NO. 34, and CDR3 comprises the sequence shown as SEQ ID NO. 60; or,
    (18) CDR1 comprises the sequence shown as SEQ ID NO. 15, CDR2 comprises the sequence shown as SEQ ID NO. 37, and CDR3 comprises the sequence shown as SEQ ID NO. 63; or,
    (19) CDR1 comprises the sequence shown as SEQ ID NO. 16, CDR2 comprises the sequence shown as SEQ ID NO. 39, and CDR3 comprises the sequence shown as SEQ ID NO. 64; or,
    (20) CDR1 comprises the sequence shown as SEQ ID NO. 17, CDR2 comprises the sequence shown as SEQ ID NO. 40, and CDR3 comprises the sequence shown as SEQ ID NO. 65; or,
    (21) CDR1 comprises the sequence shown as SEQ ID NO. 18, CDR2 comprises the sequence shown as SEQ ID NO. 41, and CDR3 comprises the sequence shown as SEQ ID NO. 66; or,
    (22) CDR1 comprises the sequence shown as SEQ ID NO. 19, CDR2 comprises the sequence shown as SEQ ID NO. 42, and CDR3 comprises the sequence shown as SEQ ID NO. 67; or,
    (23) CDR1 comprises the sequence shown as SEQ ID NO. 20, CDR2 comprises the sequence shown as SEQ ID NO. 43, and CDR3 comprises the sequence shown as SEQ ID NO. 67; or,
    (24) CDR1 comprises the sequence shown as SEQ ID NO. 21, CDR2 comprises the sequence shown as SEQ ID NO. 44, and CDR3 comprises the sequence shown as SEQ ID NO. 68; or,
    (25) CDR1 comprises the sequence shown as SEQ ID NO. 22, CDR2 comprises the sequence shown as SEQ ID NO. 45, and CDR3 comprises the sequence shown as SEQ ID NO. 69; or,
    (26) CDR1 comprises the sequence shown as SEQ ID NO. 23, CDR2 comprises the sequence shown as SEQ ID NO. 46, and CDR3 comprises the sequence shown as SEQ ID NO. 70; or,
    (27) CDR1 comprises the sequence shown as SEQ ID NO. 24, CDR2 comprises the sequence shown as SEQ ID NO. 47, and CDR3 comprises the sequence shown as SEQ ID NO. 71.
  3. The IL17 antibody of claim 2, wherein the heavy chain variable region comprises a sequence as set forth in any one of SEQ ID NOs 72 to 100.
  4. The IL17 antibody of any one of claims 1-3, which is a single domain antibody or V thereof H H fragment.
  5. A trivalent single domain antibody, characterized in that it comprises an IL17 antibody according to any one of claims 1-4.
  6. The trivalent single domain antibody of claim 5, comprising three V linked in sequence from N-terminus to C-terminus H H：V H H 1 -V H H 2 -V H H 3 。
  7. The trivalent single domain antibody of claim 6, wherein V H H 2 Is H-resistantV of SA H H is formed; preferably comprising the sequence shown as SEQ ID NO. 108.
  8. The trivalent single domain antibody of claim 7, wherein V H H 1 Comprising a CDR combination as defined in any one of claims (1) to (6) of claim 2, said V H H 3 A CDR combination comprising a CDR as defined in any one of claims 2 (7) to (22); preferably, the V H H 1 Contains a sequence shown in any one of SEQ ID NO 72-79, and V H H 3 Contains a sequence shown in any one of SEQ ID NO 80-100;
    or said V H H 3 Comprising a CDR combination as defined in any one of claims (1) to (6) of claim 2, said V H H 1 A CDR combination comprising a CDR as defined in any one of claims 2 (7) to (22); preferably, the V H H 3 Contains a sequence shown in any one of SEQ ID NO 72-79, and V H H 1 Contains a sequence shown in any one of SEQ ID NO 80-100.
  9. The trivalent single domain antibody of claim 8, wherein V H H 1 Or V H H 3 Comprising a combination of CDRs as defined in claim 2 (2), said V H H 3 Or V H H 1 A CDR combination comprising a CDR as defined in claim 2 (7), (10) or (26); preferably, the method comprises the steps of,
    the V is H H 1 Comprising the sequence shown as SEQ ID NO. 73, as described V H H 3 Comprising the sequence shown as SEQ ID NO. 80, 83 or 99;
    alternatively, the V H H 1 Comprising the sequence shown as SEQ ID NO. 80, 83 or 99, The V is H H 3 Contains a sequence shown as SEQ ID NO. 73.
  10. The trivalent single domain antibody according to any one of claims 5-9, characterized by different V H H are operatively connected by a joint; the linker preferably comprises the sequence shown in SEQ ID NO. 107.
  11. The trivalent single domain antibody according to claim 10, characterized in that it contains an amino acid sequence as shown in any one of SEQ ID NOs 101 to 106.
  12. An isolated nucleic acid encoding the IL17 antibody of any one of claims 1-4 or the trivalent single domain antibody of any one of claims 5-11.
  13. An expression vector comprising the isolated nucleic acid of claim 12.
  14. A host cell comprising the expression vector of claim 13; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.
  15. A method of producing an IL17 antibody or a trivalent single domain antibody comprising culturing the host cell of claim 14, and obtaining the antibody from the culture.
  16. A pharmaceutical composition comprising the IL17 antibody of any one of claims 1-4 or the trivalent single domain antibody of any one of claims 5-11.
  17. Use of an IL17 antibody according to any one of claims 1 to 4, a trivalent single domain antibody according to any one of claims 5 to 11 or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the prevention and treatment of an IL 17-related disease or disorder;
    the disease or condition preferably includes chronic inflammation of the lung, rheumatoid arthritis, intestinal inflammation, dry eye, psoriasis, ankylosing spondylitis and various cancers including skin cancer, more preferably psoriasis, ankylosing spondylitis, chronic inflammation of the lung and rheumatoid arthritis; psoriasis, ankylosing spondylitis and rheumatoid arthritis are further preferred.
  18. A chimeric antigen receptor comprising the IL17 antibody of any one of claims 1-4 or the trivalent single domain antibody of any one of claims 5-11.
  19. An antibody drug conjugate comprising a cytotoxic agent and an IL17 antibody of any one of claims 1-4 or a trivalent single domain antibody of any one of claims 5-11; preferably, the cytotoxic agent is MMAF or MMAE.
  20. A kit comprising an IL17 antibody according to any one of claims 1-4, a trivalent single domain antibody according to any one of claims 5-11, a chimeric antigen receptor according to claim 18, an antibody drug conjugate according to claim 19 and/or a pharmaceutical composition according to claim 16;
    Preferably, the kit further comprises (i) means for administering an antibody or antigen binding fragment thereof or an antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
  21. A kit of parts comprising a kit a and a kit B, wherein:
    the kit a comprises an IL17 antibody according to any one of claims 1 to 4, a trivalent single domain antibody according to any one of claims 5 to 11, a chimeric antigen receptor according to claim 18, an antibody drug conjugate according to claim 19 and/or a pharmaceutical composition according to claim 16;
    the kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  22. A method of diagnosing, treating and/or preventing an IL 17-mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of the IL17 antibody of any one of claims 1-4, the trivalent single domain antibody of any one of claims 5-11, the chimeric antigen receptor of claim 18, the antibody drug conjugate of claim 19 or the pharmaceutical composition of claim 16, or treating a patient in need thereof using the kit of parts of claim 21.
  23. A method according to claim 22, wherein the disease or condition is as defined in claim 17.
  24. A method of immunodetection or determination of IL17 comprising detecting after incubation with a test substance using the IL17 antibody of any one of claims 1-4, the trivalent single domain antibody of any one of claims 5-11, the chimeric antigen receptor of claim 18, the antibody drug conjugate of claim 19, or the pharmaceutical composition of claim 16; preferably, the detection or the assay is for non-diagnostic and/or therapeutic purposes.
  25. A combination therapy comprising administering to a patient in need thereof an IL17 antibody of any one of claims 1-4, a trivalent single domain antibody of any one of claims 5-11, a chimeric antigen receptor of claim 18, an antibody drug conjugate of claim 19 or a pharmaceutical composition of claim 16, respectively, and a second therapeutic agent; the second therapeutic agent preferably comprises a further anti-tumour antibody or a pharmaceutical composition comprising said further anti-tumour antibody, and/or one or more of the group consisting of a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of co-stimulatory molecules, an inhibitor of inhibitory molecules and a vaccine.
CN202180100166.9A 2021-09-13 2021-09-13 IL17 antibody and preparation method and application thereof Pending CN117642423A (en)

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UA117218C2 (en) * 2011-05-05 2018-07-10 Мерк Патент Гмбх Amino acid sequences directed against il-17a, il-17f and/or il17-a/f and polypeptides comprising the same
CU24300B1 (en) * 2013-02-08 2017-12-08 Novartis Ag ANTI-IL-17A ANTIBODIES USEFUL IN THE TREATMENT OF AUTOIMMUNE AND INFLAMMATORY DISORDERS
GB201522391D0 (en) * 2015-12-18 2016-02-03 Ucb Biopharma Sprl Antibody molecules
WO2019154420A1 (en) * 2018-02-12 2019-08-15 上海原能细胞医学技术有限公司 Il17 antibody and application thereof
CN110003329B (en) * 2019-04-12 2022-09-27 深圳普瑞金生物药业股份有限公司 Polypeptide, IL17A/F single domain antibody, nucleotide sequence and kit

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